More than 20 years after the discovery of the first anelloviruses (AVs), they still remain a mysterious group of viruses. To date, there is no clear understanding of their impact on the host organism, although information is emerging about their participation in various pathologies. However, it is well known that AVs are an important component of the human virome. In the current study we assessed the abundance of AVs in viromes from four nasal and pharyngeal samples from children with respiratory symptoms using a metagenomic approach. It is worth noting that three patients had known respiratory viruses and one did not have any. Regarding AVs, they were detected in all samples, namely Torque teno mini virus (TTMV). The greatest abundance and diversity of AVs (Torque teno virus (TTV), TTMV and Torque teno midi virus (TTMDV)) were shown in a diseased patient with an absence of respiratory viruses, which may indicate the potential involvement of AVs in the maintenance of respiratory symptoms. An activated immune system to combat the main pathogens, against the background of which the number of AVs decreases, is discussed as a possible reason for the reduced diversity of anellome in other patients. We also compared the results of the metagenomic approach and qPCR for major respiratory viruses, which were in agreement. In addition, metagenomic sequencing made it possible to obtain additional data on viral genomes in order to establish their taxonomic identification and characterize individual viral profiles in patients from the Siberian part of Russia.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity is needed to exploit innovative solutions against these hazards. Lately, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs, was evaluated against the non-enveloped RNA viruses, Hepatitis A virus (HAV), and Murine Norovirus (MNV), a surrogate for human Norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. Results showed that almost all peptides displayed virucidal effects with about 90% of infectivity reduction on HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles or inhibition of the viral bonding to the cell receptors. Hence, RiLK1 could represent a versatile ‘antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

A relevant aspect in the epidemiology of Tegumentary Leishmaniasis (TL) is the Leishmania parasites carrying a viral endosymbiont Leishmania RNA Virus 1 (LRV1), a dsRNA virus. Leishmania parasites carrying LRV1 are prone to causing more severe TL symptoms, increasing the likelihood of unfavorable clinical outcomes. LRV1 has been observed in cultured strains of five L. (Viannia) species, and host specificity was suggested when studying LRV1 from L. braziliensis and L. guyanensis strains. The coevolution hypothesis of LRV1 and Leishmania was based on phylogenetic analyses, implying an association between LRV1 genotypes, Leishmania species, and their geographic origins. This study aimed to investigate LRV1 specificity to Leishmania (Viannia) species hosts by analyzing LRV1 from L. (Viannia) species. To this end, LRV1 was screened in L. (Viannia) species other than L. braziliensis or L. guyanensis, and it was detected in 11 out of 15 L. naiffi and in 2 out of 4 L. shawi. Phylogenetic analyses based on partial LRV1 genomic sequencing supported the hypothesis of host specificity, as LRV1 clustered according to their respective Leishmania species hosts. These findings underscore the importance of investigating Leishmania and LRV1 coevolution and its impact on Leishmania (Viannia) species dispersion and pathogenesis in the American Continent.

Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed potentiating or inhibiting viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells.

Leishmaniasis are neglected diseases that affect regions such as South Asia, South Africa and Latin America, less developed regions. Current treatment, for cutaneous, mucocutaneous and visceral leishmaniosis, which are the main forms of the disease, is based on the administration of pentavalent antimonial and amphotericin B. However, these drugs have low efficacy and high toxicity. For this reason, the search for new treatments for this disease is necessary, based on studies of new molecules and their conformations. The research proposed the conformational study of brominated guanidine compounds with potential antileishmanial activity using Nuclear Magnetic Resonance (NMR) and X-ray diffraction (XRD) techniques. The present study involves the bro-minated molecules, LQOF-G2, LQOF-G30, LQOF-G35 and LQOF-G35-Br. The later was synthesized by the reaction of LQOF-G35 with NBS under IR irradiation at 120 Watts of potency and dichloro-methane as solvent by 12-h of exposition. The obtained results demonstrated the efficiency of the bromination method, since two bromines atoms entered the molecule. Furthermore, NMR analysis showed the occurrence of a conformational change from Z to E when compound LQOF-G35 was brominated to LQOF-G35-Br. This feature was well confirmed by comparative DRX study of the LQOF-G35 and LQOF-G35-Br compounds. The antileishmanial activity of LQOF-G2 e LQOF-G35 motivated the synthesis of new brominated compounds LQOF-G30 e LQOF-G35-Br.

Leishmanial skin lesions are characterized by inflammatory hypoxia alongside the activation of hypoxia inducible factors, HIF-1a and HIF-2a, and subsequent expression of the HIF-a target VEGF-A during Leishmania major infection. However, the factors responsible for HIF-a activation are not known. We hypothesize hypoxia and pro-inflammatory stimuli contribute to HIF-a activation during infection. RNASeq on leishmanial lesions found transcripts associated with HIF-1a signaling are induced. To determine whether hypoxia contributes to HIF-a activation, we followed the fate of myeloid cells infiltrating from the blood and into hypoxic lesions. Recruited myeloid cells experience hypoxia when they enter inflamed lesions, and the length of time in lesions increases their hypoxic signature. To determine whether pro-inflammatory stimuli in the inflamed tissue can also influence HIF-a activation, we subjected macrophages to various pro-inflammatory stimuli and measured VEGF-A. While parasites alone did not induce VEGF-A, and pro-inflammatory stimuli only modestly induce VEGF-A, HIF- stabilization increases VEGF-A during infection. HIF-a stabilization does not impact parasite entry, growth or killing. Alternatively, the absence of ARNT/HIF- signaling enhances parasite internalization. Altogether, these findings suggest HIF-a is active during infection, and while macrophage HIF-a activation promotes lymphatic remodeling through VEGF-A production, HIF-a activation does not impact parasite internalization or control.

Leishmania spp. is the aetiologic agent of leishmaniasis, a disease endemic in several developing countries. The parasite expresses and secretes several virulence factors that subvert macrophage function and immune response. Extracellular vesicles (EVs) can carry molecules of the parasites that show immunomodulatory effects on macrophage activation and disease progression. In the present work, we detected a significantly higher expression of lpg3 and gp63 genes in Leishmania amazonensis promastigotes recovered after successive experimental infections (IVD-P) compared to those cultured for a long period (LT-P). In addition, we observed a significantly higher percentage of infection and internalized parasites in groups of macrophages infected with IVD-P. Macrophages previously treated with EVs from LT-P showed higher percentages of infection and production of inflammatory cytokines after challenge with the parasite compared to untreated ones. However, macrophages infected with parasites and treated with EVs did not reduce the parasite load. In addition, no synergistic effects were observed in infected macrophages treated with EVs and reference drugs. In conclusion, parasites cultured for a long period in vitro and recovered from animals’ infection differently affect the macrophage response. Furthermore, EVs produced by these parasites affected macrophage response in the early infection of these cells.

Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like modulation of host immune system, inflammatory response, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in stimulation of the humoral and cellular immune responses of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resuspended in RPMI medium and incubated for 2 to 24h, at 25ºC or 37°C to release Leish-EVs. This dynamic was evaluated by transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37°C released more Leish-EVs than those incubated at 25ºC. In comparison to the control group, infected THP-1 cells produced a significantly higher concentration of EVs (THP-1 cells-EVs). Similar results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data suggest that the concentration of host-EVs-Can distinguish between infected and uninfected cells. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to produce IFN-γ. These same cells produced IL-10, which inhibited TNF and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. These findings indicate that the increased levels of miR-21-5p and miR-146a-5p observed in Leish-EVs-treated-THP-1 cells lead to immunosuppression and the secretion of pro-inflammatory cytokines.

Leishmania parasites present astonishing adaptative abilities that represent a matter of life or death within disparate environments during the heteroxenous parasite life cycle. From an evolutionary perspective, organisms develop methods of overcoming such challenges. Strategies that extend beyond the genetic diversity have been discussed and include variability between parasite cells during the infections of their hosts. The occurrence of Leishmania subpopulation fluctuations with variable structural genomic contents demonstrates that a single strain might shelter the variability required to overcome inconsistent environments. Such intrastrain variability provides parasites with an extraordinary ability to adapt and thus survive and propagate. However, different perspectives on this evolution have been proposed. Strains or species living in the same environment can cooperate but also compete. These interactions might increase the replication rate of some parasites but cause the loss of more aggressive competitors for others. Adaptive responses to intra- and interspecific competition can evolve as a fixed strategy (replication is adapted to the average genetic complexity of infections) or an optional strategy (replication varies according to the genetic complexity of the current infection). This review highlights the complexity of interspecies and intrastrain interactions among Leishmania parasites as well as the different factors that influence this interplay.

This review presents an updated overview of research concerning Leishmania protein structures, primarily sourced from the Protein Data Bank (PDB), that play a role in the metabolic pathways of the Leishmania parasite. Furthermore, we assess the current progress in the identification and development of bioactive chemical agents aimed at addressing this substantially overlooked tropical disease. We have analyzed experimental data obtained from in vitro, in vivo and in silico sources. This data has been categorized into four main areas: a) vector taxonomy and geographic distribution; b) parasite taxonomy and geographic distribution; c) enzymatic functions of proteins engaged in parasite/host interactions throughout various developmental stages (such as oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and cytokines); and d) established and experimental treatments employing bioactive chemical compounds. Our objective is to establish a foundational point of reference for research efforts concentrating on the elucidation of interaction mechanisms and the processes of ligand-protein activation/inactivation, specifically linked to Leishmania infections. Consequently, we emphasize enzymes recognized for their involvement in the biochemical pathways incited during Leishmania infection episodes. This review encapsulates the current understanding, offering insights to inform and direct future explorations aimed at targeting proteins and pathways to enhance the management of Leishmania-related diseases.

Canine leishmaniosis (CanL) is a chronic, systemic and often severe disease. The main causative agent of CanL is a protozoan parasite, Leishmania infantum, with phlebotomine sand flies acting as vectors. In Europe and other continents, L. infantum is also responsible for leishmaniosis in other animals, such as cats, horses and humans. In Portugal, animal and human leishmanioses are endemic, and high prevalence levels of infection and disease have been reported in dogs. There is a prejudice against stray animals and also those housed in shelters, assuming they have higher levels of infection with vector-borne pathogens, including L. infantum, when compared to domestic animals. In north-eastern Portugal, serum samples were obtained from March to May 2022 in three shelters (n = 179) and 13 veterinary clinics (n = 164), resulting in 343 dogs being analysed for antibodies to Leishmania spp. by the direct agglutination test (DAT). The overall seroprevalence was 9.9%, with 15.2% seroprevalence in domestic dogs and 5.0% in the shelter ones (p = 0.003). The fact that shelter dogs had a lower seroprevalence could be explained by more regular veterinary care provided in shelters regarding preventive measures, including insecticides with an antifeeding effect, in comparison with domestic dogs.

Leishmania parasites are a group of kinetoplastid pathogens that cause a variety of clinical forms while maintaining cell communication by secreting extracellular vesicles. Emerging technologies have been adapted for the studies of Leishmania-host-cell interactions to enable broad scale analysis of parasite extracellular vesicles. Leishmania extracellular vesicles (LEVS) are naturally released spheroidal nanoparticles of polydispersed suspensions surrounded by a lipid layer of membrane. Although LEVs have increasingly gained in importance, much is still unexplained, including bioavailability and function in the complex molecular mechanisms of pathogenesis. Considering the importance of LEVs in the parasite-host interaction and in the parasite-parasite relationships emerged during evolution, the current review aims at giving an overview of Leishmania summarizing knowledge and formulating guidelines for LEVs research. In the end, we report, direct methods for specific isolation of LEVs from promastigotes and amastigotes culture supernatant suitable for a range of different downstream applications increasing the compatibility and reproducibility to establish optimal and comparable isolation conditions and full LEVs characterization, and crucial immunomodulatory events triggered by this important group of parasites.

After decades of research, oncolytic virotherapy has recently advanced to clinical application, and currently a multitude of novel agents and combination treatments are being evaluated for cancer therapy. Oncolytic agents preferentially replicate in tumor cells, inducing tumor cell lysis and complex anti-tumor effects, such as innate and adaptive immune responses and the destruction of tumor vasculature. With the availability of different vector platforms and the potential of both genetic engineering and combination regimens to enhance particular aspects of safety and efficacy, the identification of optimal treatments for patient subpopulations or even individual patients becomes a top priority. Mathematical modeling can provide support in this arena by making use of experimental and clinical data to generate hypotheses about the mechanisms underlying complex biology and, ultimately, predict optimal treatment protocols. Increasingly complex models can be applied to account for therapeutically relevant parameters such as components of the immune system. In this review, we describe current developments in oncolytic virotherapy and mathematical modeling to discuss the benefit of integrating different modeling approaches into biological and clinical experimentation. Conclusively, we propose a mutual combination of these fields of research for more efficient development and effective treatments.

To date, viruses closely related to SARS-CoV-2 have been reported in four bat species: Rhinolophus acuminatus, Rhinolophus affinis, Rhinolophus malayanus, and Rhinolophus shameli. Here, we analysed 343 sequences of the mitochondrial cytochrome c oxidase subunit 1 gene (CO1) from georeferenced bats of the four Rhinolophus species identified as reservoirs of SARS-CoV-2-like viruses. Haplotype networks were constructed in order to investigate patterns of genetic diversity among bat populations of Southeast Asia. No strong geographic structure was found for the four Rhinolophus species, suggesting high dispersal capacity. The ecological niche of SARS-CoV-2 like viruses was predicted using the four localities of bat SARS-CoV-2-like viruses and the localities where bats showed identical or very similar CO1 haplotypes than virus-positive bats. The ecological niche of SARS-CoV-like viruses was deduced from the localities where bat SARS-CoV-like viruses were previously detected. The results show that the ecological niche of SARS-CoV2-like viruses includes several regions of mainland Southeast Asia whereas that of SARS-CoV-like viruses is mainly restricted to China. In agreement with these results, human populations in Laos, Vietnam, Cambodia, and Thailand appear to be much less affected by the Covid-19 pandemic than other countries of Southeast Asia. In the climatic transitional zone between the two ecological niches (southern Yunnan, northern Laos, northern Vietnam, and possibly Hainan and Taiwan), genomic recombination between highly divergent viruses is more likely to occur. Since recombinant viruses can threaten the benefit of vaccination campaigns, these regions should be under surveillance.

The extension of virology beyond its traditional medical, veterinary or agricultural applications, now called environmental virology, has shown that viruses are both the most numerous and diverse biological entities on earth. In particular, virus isolation studies involving unicellular eukaryotic hosts (heterotrophic and photosynthetic protozoans) revealed numerous viral types previously unexpected in terms of virion structure and morphology, genome size and gene content, or mode of replication. Complemented by large-scale metagenomic analyzes, these discoveries have rekindled interest in the enigma of the evolutionary origin of viruses, for which no simple definition encompassing all of their diversity is still unanimous. Several laboratories have repeatedly tackled the deep reconstruction of the evolutionary history of viruses, using various methods of molecular phylogeny applied to the few shared genes detected in certain virus groups (e.g. the Nucleocytoviricota). Beyond the practical difficulties of establishing reliable homology relationships from extremely divergent sequences, I present here purely conceptual arguments highlighting several fundamental limitations plaguing the reconstruction of the deep evolutionary history of viruses, and even more the identification of their unique of multiple origin (s). Those limitations are direct consequences of the particularly random mechanisms which govern the reductive evolution of obligate intracellular parasites.

Viruses are the most prevalent infectious agents, populating almost every ecosystem on earth. Most viruses carry only a handful of genes supporting their replication and the production of capsids. It came as a great surprise in 2003 when the first giant virus was discovered and found to have a >1Mbp genome encoding almost a thousand proteins. Following this first discovery, dozens of giant virus strains across several viral families have been reported. Here, we provide an updated quantitative and qualitative view on giant viruses and elaborate on their shared and variable features. We review the complexity of giant virus proteomes, which include functions traditionally associated only with cellular organisms. These unprecedented functions include components of the translation machinery, DNA maintenance, and metabolic enzymes. We discuss the possible underlying evolutionary processes and mechanisms that might have shaped the diversity of giant viruses and their genomes, highlighting their remarkable capacity to hijack genes and genomic sequences from their hosts and environments. This leads us to examine prominent theories regarding the origin of giant viruses. Finally, we present the emerging ecological view of giant viruses, found across widespread habitats and ecological systems, with respect to the environment and human health.

Ever since the virus was discovered in the 80s of the 19th century, plant viruses had a great impression within the field of plant pathology. In this book we trace the growth in the research on plant viruses beginning from traditional era (1882-1900) in which a new pathogen was discovered other than fungi and bacteria, later known as virus; biological era (1900-2000) in which the mode of spread and relationship of the virus with its vector was understood; biochemical/biophysical era (1930-1968) that marked the basis of virus purification and chemical characterization; molecular era of plant virology (1943-1995) in which establishment, progression & transmission of plant viruses was described within the host at molecular details ; era of viral molecular genetics (1980–2000), in which a significant role was played by the plant virologists to understand the position and function of genes, methods of viral gene cloning, gene silencing and other viral genome associated properties ; era of plant viral diagnostics (1980 – 2021) which led to rapid, specific and sensitive detection of plant viruses.

Shrews (Soricidae) are common small wild mammals with a significant overlap in their habitats with humans and domestic animals. Currently, viruses from 24 families have been identified in shrews, including Adenoviridae, Arenaviridae, Arteriviridae, Astroviridae, Bornaviridae, Caliciviridae, Circoviridae, Coronaviridae, Filoviridae, Flaviviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Nairoviridae, Orthoherpesviridae, Paramyxoviridae, Parvoviridae, Phenuiviridae, Picornaviridae, Polyomaviridae, Poxviridae, Rhabdoviridae, Sedoreoviridae, and Spinareoviridae, which pose potential threats to humans and domestic animals. This review compiled fundamental information about shrews and provided a comprehensive summary of the viruses that have been detected in shrews, with the aim to facilitating a deeper understanding of shrews and the diversity and risks of their viruses for researchers and the general public.

Transporter proteins, P-glycoprotein (P-gp) and P4ATPase-CDC50, are responsible for the transport of Miltefosine drug across cell membrane of a protozoan parasite Leishmania major. Mutations or change in activity of these proteins may lead to emergence of resistance in the parasite. Owing to the structural and functional importance of these transporter proteins, in this ppaper, we have tried to decipher the evolutionary divergence of these Miltefosine transporter proteins across different forms of life including Protists, Fungi, Plants and Animals. We retrieved 96, 207, and 189 sequences of P-gp, P4ATPase and CDC50 proteins respectively, across diverse variety of organisms for the conserved analysis. Phylogenetic trees were constructed for these three transporter proteins based on Bayesian posterior probability inference. The evolutionary analysis concluded that these proteins remain highly conserved throughout the species diversity but still substantial differences in the proteins for host (Homo sapiens) and parasite (L. major) were observed which have led in targeting these Miltefosine transporter proteins in a parasite specific manner.

Viruses were classically named after the very same Latin word virus, originally meaning poison or venom. Public understanding of viruses reinforces their “malign” aspects, especially nowadays under the COVID-19 global pandemic. It is our aim here to propose a new way to view viruses and understand their origins and evolution. First, viruses are the most abundant biological systems found on Earth. They can be found almost everywhere and form a subtle biological layer named virosphere. Second, viruses are probably the most important drivers of molecular evolution and they are active agents of ecosystems maintenance and homeostasis, allowing and driving their dynamic modification. A significant number of eukaryotic genomes are composed by genome elements similar to viruses and these endogenous viruses are continuously acting for our equilibrium and fitness. They are responsible for the origin of species-specific orphan genes that allow adaptation through the development of specific traits in separate lineages of eukaryotes. Accumulated evidence indicate that a viral infection was responsible to create the eukaryotic nucleus and, also, it is a syncytium structure caused by viral replication that allows the formation of the placenta. Therefore, viruses were fundamental for the evolutionary fate of eukaryotes and mammals. The presence of virus-specific genes that are absent in cellular organisms indicates that viruses existed before cells. Besides, such as progenotes, viruses are simply ribonucleoproteic entities and their capsids are orders of magnitude simpler than proteolipidic membranes. Here, we (i) propose a complete scenario to describe the major transitions in prebiotic evolution, (ii) present the possibility that viruses emerged before LUCA, and (iii) suggest that viruses originated at the age of progenotes. However, viruses do not form a monophyletic clade. They should be seen as an evolutionary stable strategy recurrently achieved by biological systems to survive. We propose that the word “VIRUS”, known as venom, is historically mistaken and introduce a new interpretation for their name as an acronym for “Very Important Replicator Unit and Symbiont”. But more than being “very important”, viruses are of “Utmost” relevance for the maintenance of life in biosphere, by which reason we suggest referring to them as “UIRUS” to reinforce their incredible role in symbiosis and their beneficial characteristics over the infectious ones.

Our main objective in this work was to examine the possibility of non-intrusive, label-free, detection of whole Zika viruses using terahertz signals with or without a targeting/binding oligonucleotide (aptamers). We report for the first time the use of terahertz electromagnetic waves (0.75 THz – 1.1 THz) to detect Zika viruses. The Zika/aptamer complexes showed a reproducible terahertz reflection coefficient minimum at 1.064 THz while the Zika virus’s reflection minimum was at 1.073 THz. Of different substrates we examined, the polyester petri dish provided a very low loss and excellent terahertz transmission. To increase the interaction between the terahertz signal and the sample we also used polyester microbeads coated with aptamers. We then measured the terahertz reflection from the microbeads as a function of Zika concentration. The resulting terahertz Zika sensor had sensitivity of 63 Hz/Zika and minimum detectable signal of ~ 16x10³ Zika. Other substrates such as Graphene on polyethylene terephthalate (PET), 50 nm-thick gold film on polycarbonate, thin (30 um-thick) glass slide and Teflon were also examined. Graphene substrate enabled direct detection of the Zika without any aptamers.

High Resolution Melting Analysis (HRM) has been pointed as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to the parasite load-dependent accuracy. In the light of these findings and considering the importance of parasite load determination for clinical and research purposes we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Leishmania species are parasitic protozoans which cause leishmaniasis affecting millions of people worldwide. Sterols are important components of plasma and organellar membranes and serve as precursors for the synthesis of signaling molecules. In contrast to animals, Leishmania do not produce cholesterol but instead synthesize ergostane-based sterols. C14-demethylase is a key enzyme involved in the biosynthesis of sterols in Leishmania parasites and an important drug target. Its inactivation leads to multiple defects including increased plasma membrane fluidity, mitochondrion dysfunction, hypersensitivity to stress and reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance of RNA stability and translation. Sterol alteration in C14-demethylase knockout mutant leads to increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol biosynthesis plays an unexpected role in global gene regulation in Leishmania parasites.

Immune responses to influenza (flu) antigens reflect memory to prior infections or vaccinations, which might influence immunity to new flu antigens. Memory of past antigens has been termed “original antigenic sin” or more recently “immune imprinting” and “seniority”. We have researched a comparison between the immune response to live flu infections and inactivated flu vaccina-tions. A brief history of antibody generation theories is presented culminating in new findings about the immune-network theory suggesting that a network of clones exists between an-ti-idiotypic antibodies and T cell receptors. Findings regarding the 2009 pandemic flu strain and immune responses to it are presented, including memory B cells and conserved regions within the hemagglutinin protein. The importance of CD4+ memory T cells and cytotoxic CD8+ T cells responding to both infections and vaccinations are discussed and compared. Innate immune cells like natural killer (NK) cells and macrophages are discussed regarding their roles in adaptive immune responses. Antigen presentation via macroautophagy processes are described. New vaccines in development are mentioned along with the results of some clinical trials. The manu-script concludes with how repeated vaccinations are impacting the immune system and a sketch of what might be behind the imprinting phenomenon, including future research directions.

Non-ulcerated cutaneous leishmaniasis (NUCL) is an atypical and peculiar clinical form of the disease that was first described by Ponce et al. (1988) in Honduras (Central America), where iso-lated or disseminated popular and/or nodular skin lesions are commonly seen in adolescents and young adults. Leishmania (L.) chagasi was then incriminated as the causal agent of the disease, as well as of American visceral leishmaniasis (AVL), although NUCL has been shown to be more prevalent than AVL in the region. Considering, however, the dubious taxonomic character of the causal agent of NUCL, as there is no evidence of this form of disease associated with L. (L.) cha-gasi in South America, this study aimed to carry out a retrospective taxonomic analysis on a se-ries of studies on the diagnostic approach to the Honduran parasite in loco (El Tigre island, Amapala municipality, west coast of Honduras), where taxonomic characters of a biological (in vitro and in vivo), epidemiological (vector and canine reservoir), clinical-immunological (im-munopathological features of in situ skin lesion and systemic infection) and molecular (ge-nomic/phylogenetic analyses) nature were considered. The findings, particularly those of mo-lecular nature, such as: the Honduran parasite showed to be more ancestral (molecular clock analysis of the DNA polymerase alpha subunit gene) than all viscerotropic ones [L. (L.) chagasi, L. (L.) donovani and L. (L.) infantum], as well as presenting an absolutely unprecedented structural variation on chromosome 17 and the highest frequency of genomic SNPs (more than twice the number seen in the Brazilian parasite), in addition to representing a different phylogenetic lin-eage (mainly in the NJ - RNA Pol II tree) from those viscerotropic ones, have pointed to the characterization of a new leishmanine parasite, Leishmania (Leishmania) poncei n. sp. (Kinetoplas-tea: Trypanosomatidae), in honor of Professor Ponce, who was the first researcher to describe NUCL in Honduras.

Today, we are all threatened by an unprecedented pandemic: COVID-19. How different is it from other coronaviruses? Will it be attenuated or become more virulent? Which animals may be its original host? In this study, we analyzed 377 publicly available complete genome sequences for the COVID-19 virus, the previously known flu-causing coronaviruses (HCov-229E, HCov-OC43, HCov-NL63 and HCov-HKU1) and the lethal, pathogenic P3/P4 viruses, SARS, MERS, Victoria, Lassa, Yamagata, Ebola, and Dengue. We found strong similarities between the current circulating COVID-19 and SARS and MERS, as well as COVID-19 in rhinolophines and pangolins. On the contrary, COVID-19 shares little similarity with the flu-causing coronaviruses and the other P3/P4 viruses. Strikingly, we observed divergence of COVID-19 strains isolated from human hosts has steadily increased from December 2019 to March 2020, suggesting COVID-19 is actively evolving in human hosts. From all existing human COVID-19 genome sequences, we calculated the first common model that represents the shared sequences of the human COVID-19 strains, which provides important information for vaccine and antibody development. Geographic and time-course analysis of the evolutionary trees of the human COVID-19 reveals possibly heterogeneous evolutional paths among strains from 21 countries. This finding has important implications to the management of COVID-19 and the development of vaccines.

Influenza A (IAV) is a major human respiratory pathogen, causing illness, hospitalizations, and mortality annually worldwide. IAV is also a zoonotic pathogen with a multitude of hosts, allowing for interspecies transmission, reassortment events, and the emergence of novel pandemics, as was seen in 2009 with the emergence of a swine origin H1N1 (pdmH1N1) virus into humans, causing the first influenza pandemic of the 21st century. While the 2009 pandemic was considered to have high morbidity and low mortality, studies have linked the pdmH1N1 virus and its gene segments to increased disease in humans and animal models. Genetic components of the pdmH1N1 virus currently circulate in the swine population, reassorting with endemic swine viruses that co-circulate and occasionally spillover into humans. This is evidenced by the regular detection of variant swine IAVs in humans associated with state fairs and other intersections of humans and swine. Defining genetic changes that support species adaptation, virulence, and cross-species transmission, as well as mutations that enhance or attenuate these features will improve our understanding of influenza biology, aid in surveillance and virus risk assessment, and guide the establishment of counter measures for emerging viruses. Here, we review current understanding of determinants of specific IAV phenotypes, focusing on the fitness, transmission, and virulence determinants that have been identified in swine IAVs and/or in relation to the 2009 pdmH1N1 virus.

Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract Orthoquin^TM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner, across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or pre-mature release of viral genomes as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins.

Dogs play an important role in the transmission of Leishmania infantum but epidemiologic and clinical studies of canine tegumentary leishmaniasis (CTL) are scarce. In an endemic area of human American Tegumentary Leishmaniasis (ATL) caused by Leishmania braziliensis, we determine the prevalence and incidence of both CTL and subclinical (SC) L. braziliensis infection in dogs and evaluated if the presence of dogs with the CTL or the SC L. braziliensis infection is associated with the occurrence of human ATL. SC infection in healthy animals and CTL in animals with ulcers was determined by PCR on biopsied healthy skin or on the ulcer or detecting antibodies against soluble leishmania antigen. We compared the occurrence of human ATL in homes with dogs with CTL or SC infection with control homes without dogs or with dogs without CTL or SC infection. The prevalence of SC infection was 35% and of CTL 31%. The incidence of SC infection in dogs was 4.6% and of CTL was 9.3%. The frequency of ATL in humans was 50% in homes with infected dogs and 13% in homes without L. braziliensis infection in dogs. CTL and SC infection is highly prevalent, and dogs may participate in the transmission chain of L. braziliensis.

Leishmaniasis is a tropical disease caused by a protozoan parasite Leishmania that is transmitted via infected female sandflies. At present, leishmaniasis treatment mainly counts on chemotherapy. The currently available drugs against leishmaniasis are costly, toxic, with multiple side effects, and limitations in the administration route. The rapid emergence of drug resistance has severely reduced the potency of anti-leishmanial drugs. As a result, there is a pressing need for the development of novel anti-leishmanial drugs with high potency, low cost, acceptable toxicity, and good pharmacokinetics features. Due to the availability of preclinical data, drug repurposing is a valuable approach for speeding up the development of effective antileishmanial through pointing to new drug targets in less time having low costs and risk. Metabolic pathways of this parasite play a crucial role in the growth and proliferation of Leishmania species during the various stages of their life cycle. Based on available genomics/proteomics information, known pathways-based (sterol biosynthetic pathway, purine salvage pathway, glycolysis, GPI biosynthesis, hypusine, polyamine biosynthesis) Leishmania-specific proteins could be targeted with known drugs that were used in other diseases, resulting in finding new promising anti-leishmanial therapeutics. The present review discusses various metabolic pathways of the Leishmania parasite and some drug candidates targeting these pathways effectively that could be potent drugs against leishmaniasis in the future.

Leishmania infantum is causative of visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, lethal if untreated. Few years ago, re-sequencing and de novo assembling of the L. infantum (strain JPCM5) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we have performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2,352 proteins based on the search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We have detected many proteins belonging to organelles such as glycosomes, mitochondria or flagellum, as well as many metabolic enzymes, and a large number of putative RNA binding proteins and molecular chaperones. Moreover, we listed the proteins presenting post-translational modifications, such as phosphorylations, acetylations and methylations, among others. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding has allowed to correct annotations, leading to N-terminal extension of protein sequences, and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics and transcriptomics has resulted in a powerful combination for improving the L. infantum reference genome annotation.

For over a century, viruses have left a long trail of evidence implicating them as frequent suspects in the development of type 1 diabetes. Through vigorous interrogation of viral infections in individuals with islet autoimmunity and type 1 diabetes using serological and molecular virus detection methods, and mechanistic studies of virus infected human pancreatic β-cells, the prime suspects have been narrowed down to predominantly human enteroviruses. Here we provide a comprehensive overview of evidence supporting the hypothesised role of enteroviruses in the development of islet autoimmunity and type 1 diabetes. We also discuss concerns over the historical focus and investigation bias toward enteroviruses, and summarise current unbiased efforts aimed at characterising the complete population of viruses (the “virome”) contributing early in life to the development of islet autoimmunity and type 1 diabetes. Finally, we review the range of vaccine and antiviral drug candidates currently being evaluated in clinical trials for the prevention and potential treatment of type 1 diabetes.

Treatment of neurological disease is hampered by the lack of validated specific and sensitive biomarkers, resulting in delayed diagnosis and nonspecific disease modifying therapies. For example, in ALS the lack of a sensitive and specific biomarker impedes the ability to administer a treatment prior to or at the onset of motor neuron dysfunction. Although viral or other infectious etiologies have been proposed as a contributing factor to neurodegeneration, it can be argued that these are casual and not causal associations. In the case of ALS, evidence for direct causality would require in vivo validation of specific nucleotide sequences of microbial origin which are known to be neuroinvasive with tropism for motor neurons, such as polio and non-polio. Several viral enteropathogens recapitulate the pathological events in sporadic ALS, including the ability to cleave TDP-43 resulting in accumulation of misfolded proteins in the cytoplasm. This review provides supporting evidence that motor neuron disease is causally related to specific enteroviruses and argues that prospective validation is imperative for effective prevention and treatment.

What is life, what is the difference between something that is alive and something that is not, are viruses living beings, or what would life be like elsewhere in the universe, are questions that still do not have clear-cut answers fully accepted by the scientific community. Based on the fundamental attributes of all living things, I define life as a process that takes place in very ordered organic structures and is characterized by being automatic, interactive and evolutionary. I also define a living being as an organic, highly ordered, automatic, interacting and evolutionary system, and a robot as an ordered automatic and interacting system. Based on this definition and what we know about the biology of viruses, I maintain that they should be considered as living entities. Finally, I explain why if there were life elsewhere in the universe, it would be very similar to what we know on our planet.

The serendipitous discovery of antiparasitic drugs, such as quinine and artemisinin from plant origin reveals that the search for new chemical pharmacophores from medicinal plants is valuable. The present study sought to explore the antiplasmodial, antileishmanial, and antitrypanosomal activities of extracts from Lippia adoensis. Crude extracts of L. adoensis leaves and twigs, which were obtained by extraction using 70% ethanol in water, were assayed for antiplasmodial activity against P. falciparum 3D7 and Dd2 through the SYBR green I-based fluorescence assay; and for antileishmanial, antitrypanosomal, and cytotoxic effects on Leishmania donovani, Trypanosoma brucei brucei, and Vero cells, respectively, using the resazurin colorimetric assays. The phytochemical analysis of the extracts was performed using a liquid chromatography-mass spectrometry (LC-MS) feature-based detection and molecular networking flow on Global Natural Product Social (GNPS). As a result, the crude extracts from L. adoensis inhibited growth of P. falciparum (3D7 and Dd2) (IC50s; (3D7): 10.008 and 97.467 μg/mL; (Dd2): 29.48 and 26.96 μg/mL), L. Donovani (IC50s: 22.879-10.522 μg/mL), and T. brucei brucei (IC50s: 2.3085-55.06 μg/mL). The extracts were found to be non-cytotoxic to Vero cells, thus yielding median cytotoxic concentrations (CC50s) above 100 μg/mL. The LC-MS tandem molecular networking flow predicted that the extracts contain valsafungin A and bacillamidin on the first cluster, and fatty acids, ketone and aldehyde derivatives on the second cluster. Overall, the present study demonstrated the antiparasitic effects of L. adoensis extracts, thus justifying the use of this plant in the traditional treatment of fever and malaria conditions. Nevertheless, detailed metabolomic studies and antiparasitic mechanisms of action of the extracts are expected to unveil the potential antiparasitic hit compounds.

Mast cells (MCs) play a crucial role during infections with Leishmania, that is transmitted through the bite of an infected sand fly that injects saliva together with the parasite. Sand fly saliva is a complex fluid that modulates the host immune response. In addition, hormonal factors modulate the host immune response, impacting the susceptibility to infections. Thus, to assess the impact of androgens and salivary proteins of sand fly vectors on the mast cell (MC) response to Leishmania infections, we infected orchiectomized male mice with the parasite in the presence or absence of sand fly salivary proteins and analyzed the inflammatory response of MCs. Our results showed a differential MC response to the parasite and to vector salivary proteins in mice deprived of gonadal hormones, as compared to sham-operated mice. Orchidectomy induced a different pattern of activation in MC of animals infected with Leishmania and vector-salivary proteins. Our results show that during Leishmania infection, androgens modulate the innate immunity response against the parasite and salivary proteins of the sand fly vector.

The description of the genus Leishmania as causative agents of leishmaniasis occurred during this modern age. But evolutionary studies suggest that the origin of Leishmania can be traced back to the Mesozoic era. Subsequently, during its evolutionary process, it sustained a worldwide dispersion predating the breakup of the Gondwana supercontinent. It is assumed that this parasite evolved from monoxenic Trypanosomatidae. Phylogenetic studies locate the dixenous Leishmania in a well-supported clade, recently named subfamily Leishmaniinae, which includes also monoxenous trypanosomatids. Virus-Like Particles were reported in many species of this family. So far, several Leishmania species have been reported as infected by Leishmania RNA Virus (LRV) and Leishbunyavirus (LBV). Since the first descriptions of LRV decades ago, differences in its genomic structure have been highlighted, leading to the designation of a LRV1 in L. (Viannia) species and a LRV2 in other L. (Leishmania) species. There are strong indications of virus infecting Leishmania spp. ability to enhance parasitic survival both in human and experimental infections, through highly complex and specialized mechanisms. Phylogenetic analyzes of these viruses have shown that their genomic differences correlate with the infected parasite species, suggesting a coevolutionary process. Herein, we will present unpublished results regarding the relationship Leishmania – endosymbiotic Leishmania viruses and will explore what has been described in the literature, and what is known about this association that could contribute to discussions about the worldwide dispersion of Leishmania.

Yearly, 1,500,000 cases of leishmaniasis are diagnosed, causing thousands of deaths. To advance in its therapy, we present an interdisciplinary protocol that unifies ethnobotanical knowledge of natural compounds and the latest bioinformatics advances to respond to an orphan disease such as leishmaniasis and specifically the one caused by Leishmania amazonensis. The use of ethnobotanical information serves as a basis for the development of new drugs, a field in which computer-aided drug design (CADD) has been a revolution. Taking this information from Amazonian communities, located in the area with the highest prevalence of this disease, a protocol has been designed to verify new leads. Moreover, a method has been developed that allows the evaluation of lead molecules, and the improvement of their affinity and specificity against therapeutic targets. Through this approach, deguelin has been identified as a good lead to treat the infection due to its potential as an ornithine decarboxylase (ODC) inhibitor, a key enzyme in Leishmania development. Using an in silico-generated combinatorial library followed by docking approaches, we have found deguelin derivatives with better affinity and specificity against ODC than the original compound, suggesting that this approach could be adapted for developing new drugs against leishmaniasis.

Nuclear bodies (NBs) are dynamic structures present in eukaryotic cell nuclei. They are not bounded by membranes and are often considered biomolecular condensates, defined structurally and functionally by the localisation of core components. Nuclear architecture can be reorganised during normal cellular processes such as the cell cycle as well as in response to cellular stress. Many plant and animal viruses target their proteins to NBs, in some cases triggering their structural disruption and redistribution. Although not all such interactions have been well characterised, subversion of NBs and their functions may form a key part of the life cycle of eukaryotic viruses that require the nucleus for their replication. This review will focus on Cajal bodies (CBs) and the viruses that target them. Since CBs are dynamic structures, other NBs (principally nucleoli and promyelocytic leukaemia, PML, bodies), whose components interact with CBs, will also be considered. As well as providing important insights into key virus: host cell interactions, studies on Cajal and associated NBs may identify novel cellular targets for development of anti-viral compounds.

Acute respiratory viruses (ARVs) are leading cause of diseases in human worldwide. High risk individuals including children and elderly could potentially develop severe illness that could result in hospitalization or death in the worst case. Most common ARVs are Human respiratory syncytial virus, Human Metapneumovirus, Human Parainfluenza Virus, rhinovirus, coronaviruses (including SARS and MERS CoV), adenoviruses, Human Bocavirus, enterovirus (-D68 and 71), and influenza viruses. The olfactory deficits due to ARVs infection is a common symptom among patients. This mini review provides an overview of the role of SARS-CoV-2 and other common ARVs in the development of human olfactory pathophysiology. We highlight the critical need for understanding the signaling underlying the olfactory dysfunction and the development of therapeutics for this wide-ranging category of AVRs to restore the altered or loss of smell in affected patients.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer care and shown remarkable efficacy clinically. This efficacy is, however, limited to subsets of patients with significant infiltration of lymphocytes into the tumor microenvironment. To extend their efficacy to patients who fail to respond or achieve durable responses, it is now becoming evident that complex combinations of immunomodulatory agents may be required to extend efficacy to patients with immunologically “cold” tumours. Oncolytic viruses (OVs) have the capacity to selectively replicate within and kill tumour cells resulting in the induction of immunogenic cell death and the augmentation of anti-tumour immunity and have emerged as a promising modality for combination therapy to overcome the limitations seen with ICIs. Pre-clinical and clinical data has demonstrated that OVs can increase immune cell infiltration into the tumour and induce anti-tumour immunity, thus changing a “cold” tumour microenvironment that is commonly associated with poor response to ICIs, to a “hot” microenvironment which can render patients more susceptible to ICIs. Here, we review the major viral vector platforms used in OV clinical trials, their success when used as a monotherapy and when combined with adjuvant ICIs, as well as pre-clinical studies looking at the effectiveness of encoding OVs to deliver ICIs locally to the tumour microenvironment through transgene expression.

Shrimps are under the influence of several environmental factors such as fluctuation of physical and chemical parameters of the water affected by variations in rainfall, temperature, salinity, and pH. These factors have also been identified as risk factors for shrimp disease outbreaks. Despite the high levels of production, shrimp producers suffered significant economic losses in years, mainly due to the presence of diseases that now plague the industry. In particular, viral diseases have had and will continue to have profound impact on industry growth. In response to stress such environmental or pathophysiological, cells are able to up regulating selectively the expression of a protein group known as Heat Shock Proteins (HSPs). In a recent search at the literature we observed a close relation between HSP70 with apoptotic proteins and others stress proteins such HSP60 and HSP90. Moreover, the response of shrimp to viral stress was examined, some of which are correlated to the reactions of HSP70. Thus, the aim of this review is to describe the current knowledge on the status of stress responses in shrimps, particularly HSP70 responses, triggered by viruses.

This paper studies pandemic viruses that spread during the period (1759–2020) according to solar activity cycles. Our findings and results include the following: (1) The severity of a pandemic correlates negatively with the strength of solar activity; (2) Pandemic viruses are classified into three types based on their compatibility with solar activity associations. Most of them spread through the quiet Sun, where viruses survive better in cold and rainy weather, and in stable geomagnetic fields without strong disturbances; (3) The emergence of new strains of influenza viruses was manifested in two ways. First, the annual epidemics due to antigenic drift. Second, pandemics recur every 1–12 solar cycles (about 11–120 years) due to viral reassortment of new subtypes, which results in antigenic shifts; (4) Pandemic viruses have two groups according to their recurring period: first, recurring in nine solar cycles; second, recurring in twelve solar cycles. Furthermore, we reassort pandemic viruses from their previous spread in the same periodic classification. Moreover, we derive a periodicity formula for each subtype of the pandemic virus as a spread date.

The successful advancement of xenotransplantation has led to the development of highly sensitive detection systems for the screening for potentially zoonotic viruses in donor pigs and preventing their transmission to the recipient. To validate these methods, genetically modified pigs generated for xenotransplantation, numerous minipig and other pig breeds have been tested, thereby increasing our knowledge concerning the pig virome and the distribution of pig viruses. Of particular importance are the porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV) and the hepatitis E virus genotype 3 (HEV3). PCMV/PRV has been shown to reduce the survival time of pig transplants in non-human primates and was also transmitted in the first pig heart transplantation to a human patient. Here, we determined the sensitivities of our methods to detect PCMV/PRV, HEV3, porcine lymphotropic herpesvirus - 1 (PLHV-1), PLHV-2, PLHV-3, porcine circovirus 2 (PCV2), PCV3, PCV4 and porcine parvovirus 1 (PPV1) efficiently and used the methods to screen indigenous Greek black pigs. Since porcine endogenous retroviruses (PERVs) of type A and B are integrated in the genome of all pigs, but PERV-C is not, the animals were screened for PERV-C and PERV-A/C. Our detection methods were sensitive and detected PCMV/PRV, PLHV-1, PLHV-1, PLHV-3, PVC3 and PERV-C in most animals. PPV1, HEV3, PCV4 and PERV-A/C were not detected. These data are of great interest since the animals are healthy and very resistant to diseases.

Wastewater surveillance, initially conceived in the early 20th century during typhoid fever outbreaks, has evolved into a powerful tool for monitoring public health-relevant analytes. Recent applications in tracking SARS-CoV-2 infection highlight its potential. Beyond humans, it can be extended to livestock populations due to the increasing demand for livestock products. Livestock intensification poses risks of zoonotic disease emergence. Wastewater surveillance offers non-invasive, cost-effective means to detect potential outbreaks. This approach aligns with the "One Health" paradigm, emphasizing the interconnectedness of human, animal, and ecosystem health. By monitoring viruses in livestock wastewater, early detection, prevention, and control strategies can be employed, safeguarding both animal and human health, economic stability, and international trade. This integrated One Health approach enhances collaboration and a comprehensive understanding of disease dynamics, supporting proactive measures in the Anthropocene era where animal and human diseases are on the rise.

The idea of using the lytic power of viruses against the malignant cells has been entertained for many decades. However, oncolytic viruses (OV) gained broad attention as an emerging anti-cancer therapy only recently with the successful implementation of the oncolytic herpesvirus to treat advanced melanoma. OVs offer an attractive therapeutic combination of tumor-specific cell lysis together with immune stimulation, yet the latter effect is less well studied. Nevertheless, OVs can be envisaged as potential in situ tumor vaccines. The therapeutic potential of OVs can be instigated further by using the molecular biological and biotechnological tools to modify the existing viruses for their optimal tumor selectivity and enhanced immune stimulation. Furthermore, OVs can be readily combined with other therapeutic agents to increase the efficacy of the existing therapeutic schemes. In this review, we discuss biotechnological advances in the development of therapeutic applications of OVs in Russia. Particular emphasis is made on the OV-mediated treatment of glioblastoma. In addition, we highlight the challenges of oncolytic virotherapy, and describe the strategies to optimize current approaches to improve clinical outcomes.

Leishmaniosis is a zoonoses caused by Leishmania spp., an intracellular protozoan parasite. This parasite is transmitted by sandflies and the disease is endemic in Mediterranean basin. In the last years, the number of species which could be reservoir of parasite is increased. One of the most relevant is the horses, due to contact with humans and its ability to control the disease, being a possible silent reservoir. In this study, we have analyzed the prevalence and factors related to L. infantum infection in healthy horses in Mediterranean region. Epidemiological data and serum samples were obtained to 167 apparently healthy horses and presence of L. infantum was evaluated by ELISA method and qPCR. The results show 27,5% of prevalence and the main factors related to infection are equine breed, outdoor living, use and season. In conclusion, the prevalence of L. infantum infection in apparently horses from Eastern Spain (Mediterranean basin) is elevated. To control this zoonosis, it would be advisable to carry out more studies on this and other species that could be silent reservoirs of the parasite, as well as carry out measures such as the use of repellents on a regular basis.

Background: Visceral leishmaniasis affects approximately 12 million people worldwide. Current existing drugs cause several side effects and resistance, the identification of new therapeutic targets for the development of new drugs is necessary. The vesicular trafficking in the parasite flagellar pocket is essential for parasite survival and proliferation, but little is known about the proteins involved in the endocytic/secretory pathways. In this work, we are describing the characterization of a transporter cargo protein SFT-2/Got-1 like in Leishmania infantum. Material and Methods: Using bioinformatics tools, the putative Got-1 protein A4HSK3 was characterized structurally and phylogenetically. Molecular biology was used to clone and express the protein and confocal fluorescence to determine its subcellular localization. Results: The Got-1 like protein presents four alpha helix TM conserved domains (aa38-aa147) characteristics of the Got-1/SFT-2 family proteins and an important Pen-2 conserved motif LGDYXXF located in the extracellular loop. Confocal microscopy suggests that Got-1/SFT-2 like is localized in the flagellar pocket, and is partially co-localized with the L. infantum presenilin-like protein. Phylogenetically, the Leishmania sp Got-1 like is contained in a subgroup of proteins clearly detached from the other Trypanosomatidae, such as T. cruzi and T. brucei. Conclusions: Our data show the presence of an important transported proteins, and its colocalization with the presenilin aspartyl protease in the flagellar pocket of the L. infantum open new perspectives to identify novel targets, which can be an alternative therapeutic strategy against leishmaniasis.

Leishmaniasis is a vector-borne parasitic infection caused by the bite of female Phlebotomine sandflies. World Health Organization (WHO) estimates 100,000 cases to be reported annually on a global scale, moreover, 13 million people were infected between 2010 and 2015 worldwide. Treatment of leishmaniasis by conventional synthetic compounds is met by challenges pertaining to adverse effects which call for the discovery of newer anti-leishmanial molecules. This study was performed to evaluate the effect and modes of action of a sesquiterpene alcoholic molecule Farnesol on Leishmania major, the causative agent of Zoonotic Cutaneous leishmaniasis. The cytotoxic effect of Farnesol against L. major promastigotes, amastigotes and macrophages was assessed by MTT test and counting. The IC50 on promastigotes by Farnesol on L. major was evaluated by flow cytometry. In the findings, Promastigotes were reduced at 167 µM/mL & the mean numbers of L. major amastigotes in macrophages were significantly decreased in exposure to Farnesol at 172 µM/ml. In addition, Farnesol demonstrated no cytotoxicity on macrophages as the IC50 value was 945 µM/ml; it induced significant apoptosis dose-dependent on L. major promastigotes. In silico protein-ligand binding analyses indicated the effect of Farnesol in perturbation of the ergosterol synthesis pathway of Leishmania with attributes suggesting inhibition of Lanosterol-α-demethylase, the terminal enzyme of ergosterol synthesis machinery. Findings from flow cytometry reveal the role of farnesol in apoptosis-induced killing in promastigotes. Farnesol was effective at very lower concentrations when compared to Paromomycin. Further studies are crucial to evaluate the therapeutic potentials of Farnesol alone and in combination with other conventional drugs using clinical settings.

Protein synthesis has been a very rich target for developing classes of drugs to control prokaryotic and eukaryotic pathogens. Despite the development of new drug formulations, treating human cutaneous and visceral Leishmaniasis still needs significant improvement due to considerable side effects and low adherence to the usual treatment regimen. In this work, we show that the di-substituted urea-derived compound I-17 is effective in inhibiting the promastigote forms and intracellular amastigotes of the Leishmania (L.) amazonensis and L. infantum species, in addition to exhibiting low macrophage cytotoxicity. We also show a potential immunomodulatory effect of I-17 in infected macrophages, which exhibited increased expression of inducible Nitric Oxide Synthase (NOS2) and Nitric Oxide (NO) production. Our data suggest that I-17 and new derivatives of this compound may be helpful in developing new drugs for treating leishmaniasis.

Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6 and H11N9 subtypes isolated from wild mallard duck and gull habitants of a pond in Moscow city, Russia were sequenced. The sequences were closely related to those of avian IAVs isolated in Sweden and the Netherlands. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in the Netherlands. Our results revealed a significant difference in diversity and rates of reassorment of IAVs in ducks and gulls.

It remains poorly understood how the life history strategies and transmission ecologies of viruses of plants, arthropods, and vertebrates are interrelated. The present analysis hinges on the virus transmission success. Virus transmission reflects where in the host-body viruses are retained or replicating. Plants, arthropods, and vertebrates share a protective outer-layer, a circulatory system, and reproductive organs. The latter enables vertical virus transmission and associates with virus-host mutualism. Two broadly opposing virus life history strategies are considered. Acute viruses tend to be replicative and are swiftly transmitted to the next host. Instead, persistent viruses keep virus replicating costs and host damage to a minimum. The intertwined life histories and transmission ecologies are accordingly pieced together, based on the virus mono- or instead dual-host tropism, the location of virus retention or replication on or in the host-body, the presence of cyclical or mechanical transmission by arthropods, and of horizontal and vertical host-to-host transmission modes. It is shown that in the arthropod and in the vertebrate animal host, virus circulation in the hemocoel or blood circulation goes hand-in-hand with vertical transmission. Instead, plant phloem viruses do not transmit via seed. The latter is the rule for the plant-only viruses. The risk management implications are discussed in brief.

Virus taxonomy uses a Linnaean-like subsumption hierarchy to classify viruses into taxonomic units at species and higher rank levels. Virus species are considered monophyletic groups of mobile genetic elements (MGEs) often delimited by phylogenetic analysis of aligned genomic or metagenomic sequences. Taxonomic units are assumed independent organizational, functional and evolutionary units that follow a ‘natural’ historical rationale. Here, I use phylogenomic and other arguments to show that viruses are not self-standing genetically-driven biological systems. Instead, they are crucial components of holobionts, units of biological organization that dynamically integrate the genetics, epigenetic, physiological and functional properties of their co-evolving members. Remarkably, phylogenomic analyses show viruses share protein domain and loop structures with cells throughout history via massive processes of reticulate evolution, helping spread evolutionary innovations across a wider taxonomic spectrum. Thus, viruses are not merely MGEs or microbes. Instead, their genomes and proteomes conduct cellularly integrated processes akin to those cataloged by the GO Consortium. This prompts the generation of compositional hierarchies that replace the ‘is-a-kind-of’ by a ‘is-a-part-of’ logic to better describe the mereology of integrated cellular and viral makeup. My analysis demands a new paradigm that integrates virus taxonomy into a modern evolutionarily centered taxonomy of organisms.

Chronic Fatigue Syndrome (CFS) can be triggered by different factors and create a complex health situation. In the last decades incidence has been increasing. This situation is a clear example of how humans, viruses, and the environment are all connected. In the 90s cases related to CFS, com-plaints about a feeling of chronic fatigue, inability for everyday tasks, dull pain, cephalalgia, de-pression, anxiety, poor concentration. Clinical tests for EBV, HHV, CMV, IgG, IgM, T4 and T8 subsets were tested, along with hormones and hemogram tests. Most of the cases were women. The timeline of the medical history showed also myomas, breast lumps, premenstrual syndrome previously to CFS development. The nature of these conditions promoted the idea of a possible common link among them and CFS. Some cases also suffered from allergies, food intolerances, candidiasis, intestinal impairment, thyroid implications, endometriosis. As an initial working hypothesis, The Liver Link (TLL) was proposed in order to understand those different conditions affecting body, mind and emotional wellbeing. Considering liver implication can make a differ-ence in treatment and recovery. Low grade inflammatory conditions are related to Th2 predom-inance and liver functions. Functional disharmonies are very important because they usually still do not appear in any conventional tests. In 2002, TLL was presented as a framework to explain the concomitance of CFS and other conditions and the relationship with some viruses such as EBV, HHV, CMV, as a lecture in a congress at the University of Westminster (London). When SARS-CoV-2 outbroke, TLL helped to warn about the post-covid syndrome more likely to occur in specific individuals.

Amid the deepening imbalance in the supply and demand of allogeneic organs, xenotransplanta-tion can be a practical alternative because it makes an unlimited supply of organs possible. How-ever, to perform xenotransplantation on patients, the source animals to be used must be free from infectious agents. This requires breeding of animals using assisted reproductive techniques, such as somatic cell nuclear transfer, embryo transfer, and cesarean-section, without colostrum de-rived in designated pathogen-free (DPF) facilities. Most infectious agents can be removed from animals produced by these methods, but several viruses known to pass through the placenta are not easy to remove, even with these methods. Therefore, in this review, we look at the characteris-tics of several viruses known to cross the placenta and determine how they can be detected. This review is intended to help maintain DPF facilities by preventing animals infected with the virus from entering DPF facilities and to help select pigs suitable for xenotransplantation.

Alphaviruses, flaviviruses, measles viruses and rhabdoviruses are enveloped single-stranded RNA viruses, which have been engineered as expression vector systems for recombinant protein expression and vaccine development. Due to the presence of non-structural genes encoding the replicase complex, a 200,000-fold amplification of viral RNA occurs in the cytoplasm of infected cells providing extreme transgene expression levels, which is why they are named self-replicating RNA viruses. Expression of surface proteins of pathogens causing infectious disease and tumor antigens provide the basis for vaccine development against infectious diseases and cancer. The self-replicating RNA viral vectors can be administered as replicon RNA, recombinant viral particles, or layered DNA/RNA replicons. Self-replicating RNA viral vectors have been applied for vaccine development against influenza virus, HIV, hepatitis B virus, human papilloma virus, Ebola virus and recently coronaviruses, especially SARS-CoV-2 the causative agent of the COVID-19 pandemic. Measles virus and rhabdovirus vector-based SARS-CoV-2 vaccine candidates have been subjected to clinical trials. Moreover, RNA vaccine candidates based on self-amplifying alphaviruses have also been evaluated in clinical settings. Various cancers such as brain, breast, lung, ovarian, prostate cancer and melanoma have also been targeted for vaccine development. Robust immune responses and protection have been demonstrated in animal models. Clinical trials have shown good safety and target-specific immune responses. Ervebo, the VSV-based vaccine against Ebola virus disease has been approved for human use.

This tutorial is organized into three major sections—viruses, vaccines and the race for a Covid-19 vaccine. The goal is to provide enough background on viruses, history of vaccines, and the science of vaccinology founded on the principles of immunity. The hope is that this will enable us to understand the challenges, methods and prospects for developing a safe and effective vaccine against SARS-CoV-2. Many important viruses such as smallpox, HIV, HCV and SARS-CoV-2 which is responsible for causing the Coronavirus disease 2019 (Covid-19) are presented in detail, which is then followed by a description of different vaccine development methods and strategies. The tutorial then discusses different candidate SARS-CoV-2 vaccines and provides specific details of many of the prospective vaccines on the leader-board which are undergoing clinical trials. The tutorial concludes with a realistic projection for a safe and effective vaccine against SARS-CoV-2 based on the historical scientific record.

Hepatitis-C is one of the most common viral diseases caused by hepatitis C virus (HCV). It is responsible for millions of deaths each year in the developing world. The common dissemination paths of HCV include the use of contaminated water and transfusion of infected blood. Control of this virus has become a challenge for scientists and health professionals due to its versatility and adaptability in different host environments. Along with other problems, lack of efficient diagnosis, quantification and genotyping of viral strains are the major hindrances in a management of this notorious epidemic. The knowledge of HCV genotype and an amount of virus in patient’s blood are pre-requisites to determine the duration and method of treatment. In this review, we discuss the implications of HCV molecular diagnostic methods and their clinical applications. We conclude that while, several commercial and home-brewed methods are available for this purpose, and there is a visible vacuum for cost effective, robust, sensitive assays that can detect multiple viral genotypes in a single reaction. We are of the view that the level of sensitivity offered by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique is unequivocal as compared to other techniques. Therefore, researchers may explore further possibilities using this technique in the management of HCV.

Dengue virus (DENV) and chikungunya virus (CHIKV) are reemergent arboviruses that are transmitted by mosquitoes of the Aedes genus. During the last several decades, these viruses have been responsible for millions of cases of infection and thousands of deaths worldwide. Therefore, several investigations were conducted over the past few years to find antiviral compounds for the treatment of DENV and CHIKV infections. One attractive strategy is the screening of compounds that target enzymes involved in the replication of both DENV and CHIKV. In this review, we describe advances in the evaluation of natural products targeting the enzymes involved in the replication of these viruses.

Viruses are obligate intracellular parasites that are dependent on host factors for their replication. One such host protein, p97 or the valosin-containing protein (VCP), is a highly conserved AAA ATPase that facilitates replication of diverse RNA- and DNA-containing viruses. The wide range of cellular functions attributed to this ATPase is consistent with its participation in multiple steps of the virus life cycle from entry and uncoating to viral egress. Studies of VCP/p97 interactions with viruses will provide important information about host processes and cell biology, but also viral strategies that take advantage of these host functions. The critical role of p97 in viral replication might be exploited as a target for development of pan-antiviral drugs that exceed the capability of virus-specific vaccines or therapeutics.

Influenza A viruses (IAV) are widespread viruses affecting avian and mammalian species worldwide. Outbreaks of IAV in poultry are usually associated with substantial morbidity and mortality, significantly affecting the poultry industry and food security. IAVs from avian species can be transmitted to mammals including humans and, thus, they are of inherent pandemic concern. Most of the efforts to understand the pathogenicity and transmission of avian origin IAVs have been focused on H5 and H7 subtypes due to their highly pathogenic phenotype in poultry. However, IAV of the H9 subtype that circulate endemically in poultry flocks in some regions of the world have also been associated with cases of zoonotic infections. As a result, the World Health Organization includes avian origin H9N2 IAV among the top in the list of IAVs of pandemic concern. In this review, we discuss the interspecies transmission of H9N2 between avian and mammalian species and the molecular factors that are thought relevant for this spillover. Additionally, we discuss factors that have been associated with the ability of these viruses to transmit through the respiratory route in mammalian species.

Pathogen fitness landscapes change when transmission cycles establish in non-native environments or spill over into new vectors and hosts. The introduction of Leishmania infantum in the Americas into the Neotropics during European colonization represents a unique case study to investigate mechanisms of ecological adaptation of this important parasite. Defining the evolutionary trajectories that drive L. infantum fitness in this new environment are of great public health importance as they will allow unique insight into pathways of host/pathogen co-evolution and their consequences for region-specific changes in disease manifestation. This review summarizes current knowledge on L. infantum genetic and phenotypic diversity in the Americas and its possible role in the unique epidemiology of VL in the New World. We highlight the importance of appreciating adaptive molecular mechanisms in L. infantum to understand the parasites’ successful establishment on the continent.

No system nor standardized analytical procedures at commercial laboratories exist to facilitate and accurately measure potential viable virus contamination in feed ingredients and complete feeds globally. As a result, there is high uncertainty of the extent of swine virus contamination in global feed supply chains. Many knowledge gaps need to be addressed to improve our ability to prevent virus contamination and transmission in swine feed. This review summarizes the current state of knowledge involving: 1) the need for biosecurity protocols to identify production, processing, storage, and transportation conditions that may cause virus contamination of feed ingredients and complete feed, 2) challenges of measuring virus inactivation, 3) virus survival in feed ingredients during transportation and storage, 4) minimum infectious doses, 5) differences between using a Food Safety Objective versus a Performance Objective as potential approaches for risk assessment in swine feed, 6) swine virus inactivation from thermal and irradiation processes, and chemical mitigants in feed ingredients and complete feed, 7) efficacy of virus decontamination strategies in feed mills, 9) benefits of functional ingredients, nutrients, and commercial feed additives in pig diets during a viral health challenge, and 10) considerations for improved risk assessment models of virus contamination in feed supply chains.

: Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology, and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop hand-held ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid, and simple molecular diagnostic test for con-comitant detection and identification of the main Leishmania parasites encountered in Tunisia and the Old World. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica group of species DNAs, respectively. We optimized the experimental conditions of a du-plex ultra-fast PCR. The amplification is performed by a portable Palm convection PCR machine within 18 min and the products are detected by a LF cassette within 10 minutes. The test allows the identification of the infecting species according to the position and number of test lines re-vealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N=37), the ultra-fast duplex PCR-LF showed consistent, stable, and reproducible results. The analytical limit of detection of the test was 0.4pg for L. major, 4pg for L. infantum and 40pg for L. tropica.

Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and validation by PCR and RT-PCR were used. Single and mixed infections with Tomato mosaic virus (ToMV), Tomato golden mosaic virus (ToGMoV), and Pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR of Mexican ToMV isolates suggest the need to evaluate known resistance genes against these viral variants. The precise identification and characterization of viruses that naturally infect tomato germplasm could be used as a strategy for selecting the appropriate disease management and guiding breeding efforts.

Our understanding of the divergence of bacteria, archaea, viruses, and mobile elements from the last universal common ancestor (LUCA) has not improved substantially since the crystallization hypothesis (explaining transition from cellular machinery to cellular life) was proposed by Woese. Here, we propose a hypothesis for the simultaneous emergence of bacteria, archaea, viruses, and mobile elements by sequential and concrete biochemical and cellular pathways. According to the hypothesis, the LUCA was a non-free-living pool of single operon type genomes composed of single-stranded (ss) RNA, double-stranded (ds) RNA, RNA/DNA hybrid, ssDNA, or dsDNA at an ancient submarine alkaline vent. Each dsDNA operon was transcribed by different systems in σ, TFIIB, or TBP genomes. Upon the fusion of multiple dsDNA operons by recombinase, the transcription system (in the σ, TFIIB, or TBP genome) was preferentially selected, leading to the first genetic linkage (described by Morgan). Vertical inheritance eventually led to Bacteria (σ genome) and Archaea (TBP genome). Eigen’s paradox (error catastrophe) can be overcome by the parallel gain of DNA replication and DNA repair mechanisms in both genomes. Enlarged DNA enabled efficient local biochemical reactions. Both genomes independently recruited lipids to facilitate reactions by forming coacervates (liquid droplets) at the chamber of the vent, leading to the lipid divide. Bilayer lipid membrane formation, proto-cell formation with a permeable membrane, proto-cell division, and the evolution of membrane-associated biochemistry are presented in detail. Simultaneous crystallization of systems in non-free-living bacteria and non-free-living archaea triggered the co-crystallization of primitive viruses and mobile elements. An arms race between non-free-living cells and primitive viruses finally led to free-living cells with a cell wall and mature viruses at the original vent. Cells and viruses spread to all vents on the planet, explaining the universality of the genetic code on Earth. The proposed scenario provides a plausible explanation for the origin of diverse taxa from the LUCA vertical and horizontal gene transfer.

Virus infection is one of the most common etiologies of hearing loss. Hearing loss associated with viral infection can be unilateral or bilateral, mild or severe, sudden or progressive, and permanent or recoverable. Many viruses cause hearing loss in adults and children; however, the pathogenesis of hearing loss caused by viral infection is not fully understood. This review describes cytomegalovirus, the most common virus causing hearing loss, and other reported hearing loss-related viruses, along with their pathogenic characteristics and research progress on their pathology, hearing phenotypes, possible associated mechanisms, treatment and prevention measures, aiming to provide diagnostic and treatment assistance to clinical workers.

The critical enzyme Dihydrofolate Reductase-Thymidylate Synthase in Leishmania major (LmDHFR-TS) serves a dual-purpose role and is essential for DNA synthesis, a cornerstone of the parasite's reproductive processes. Consequently, the development of inhibitors against LmDHFR-TS is crucial for the creation of novel anti-Leishmania chemotherapies. In this study, we employed an in-house database containing 314 secondary metabolites derived from cinnamic acid that occurred in the Asteraceae family. We conducted a combined ligand/structure-based virtual screening to identify potential inhibitors against LmDHFR-TS. By consensus analysis of both approaches, we identified three compounds, i.e., lithospermic acid (237), diarctigenin (306), and isolappaol A (308), that exhibited a high probability of being inhibitors according to both approaches and were consequently classified as promising hits. Subsequently, we expanded the binding mode examination of these compounds within the active site of the test enzyme through molecular dynamics simulations, revealing a high degree of structural stability and minimal fluctuations in its tertiary structure. The in-silico predictions were then validated through in-vitro assays to examine the inhibitory capacity of the top-ranked naturally-occurring compounds against LmDHFR-TS recombinant protein. The test compounds effectively inhibited the enzyme with IC50 values ranging from 6.1 to 10.1 μM. In contrast, other common cinnamic acid derivatives (i.e., flavonoid glycosides) from the Asteraceae family, such as hesperidin, isovitexin 4′-O-glucoside, and rutin, exhibited low activity against this target. The selective index (SI) for all tested compounds was determined using HsDHFR, with moderate inhibitory effect. Among these hits, lignans 306 and 308 demonstrated the highest selectivity, displaying superior SI values compared to methotrexate, the reference inhibitor of DHFR-TS. Therefore, continued research into the anti-leishmanial potential of these C6C3-hybrid butyrolactone lignans may offer a brighter outlook for combating this neglected tropical disease.

Background: Leishmaniasis is a neglected disease with a global spread that affects both domestic and wild animals in addition to people. Leishmania donovani is the suspected anthroponotic cause of VL in India, where it is an endemic disease. The reservoir hosts play a crucial role in the life cycle of the Leishmania parasite. The complicated connection between the pathogen, vector, and reservoir exhibits geographical and temporal diversity. Human-to-human and, to a lesser extent, human-to-animal transmission is the principal mechanism for the maintenance of anthroponotic diseases. Scope and approach: A deliberate, systematic search was conducted on PubMed, Science Direct, and Google Scholar using keywords such as "Leishmania donovani," "zoonotic visceral leishmaniasis," and "wild animal reservoir for leishmania donovani." 530 potentially significant references were obtained from these 507 were disallowed due to copy avoidance, irrelevant titles, research publications from nations other than India, or modified compositions. The remaining 20 investigations were later rejected because they did not meet the criteria for inclusion. Finally 3 research papers with 867 goats, 161 cattles, 106 chickens, 26 sheep, 3 buffaloes, 406 dogs and 309 rats were reported. Conclusion: According to the review, goats are the epidemic's primary host and possible reservoir in several regions of India. In the endemic regions of the disease, some species of rodents along with the canines appear to be maintaining the L. donovani transmission cycle.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions have been reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation has been improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regards, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

This study reports the design of a dielectric resonator (DR) metasurface-based terahertz biosensor for rapid and accurate detection of different kinds of viruses (H1N1, SARA-COV-2, etc.). The characterization of the sensor is done by analyzing its absorbance properties with and without the presence of the H1N1 virus layer of different concentrations and electrical properties in the frequency range of 2.4 THz to 3 THz. A full-wave EM model of the sensor is designed using CST MWS software. The geometrical parameters of the proposed biosensor, as well as its dielectric constant of the virus swab, are varied to achieve good performance with an absorption level close to 1 (100%). A significant change in the resonating pick-up frequency owing to the variation in the effective refractive index of the realized sensor ensures the detection and classification of the different concentrations of the same or different viruses. The presented design could be used for the rapid detection of viruses with the added advantages of contactless, label-free, and accurate identification of different concentrations of viruses.

Biothermodynamics of viruses is among the youngest, but rapidly developing scientific disciplines. During the COVID-19 pandemic, it has closely followed the results published by molecular biologists. Empirical formulas were published for 50 viruses and thermodynamic properties for multiple viruses and virus variants, including all variants of concern of SARS-CoV-2, SARS-CoV, MERS-COV, Ebola virus, Vaccinia and Monkeypox virus. A review of development of biothermodynamics of viruses during the last several decades and intense development during the last 3 years has been described in this paper.

Systematic reviews of 591 primary studies of the modes of transmission for SARS-CoV-2 show significant methodological shortcomings and heterogeneity in the design, conduct, testing and reporting of SARS-CoV-2 transmission. While this is partly understandable at the outset of a pandemic, evidence rules of proof for assessing the transmission of this virus are needed for pre-sent and future pandemics of viral respiratory pathogens. We review the history of causality as-sessment related to microbial etiologies with a focus on respiratory viruses and suggest a hierar-chy of evidence to integrate clinical, epidemiologic, molecular and laboratory perspectives on transmission. The hierarchy, if applied to future studies, should narrow the uncertainty over the twin concepts of causality and transmission of human respiratory viruses. We attempt to address the translational gap between the current research evidence and the assessment of causality in the transmission of respiratory viruses with a focus on SARS-CoV-2. Experimentation, consistency and independent replication of research alongside our proposed framework provide a chain of evidence that can reduce the uncertainty over the transmission of respiratory viruses and increase the level of confidence in specific modes of transmission, informing the measures that should be undertaken to prevent transmission.

Glioblastoma is one of the most difficult tumor types to treat with conventional therapy options like tumor debulking, chemo and radiotherapy. Immunotherapeutic agents like oncolytic viruses, immune checkpoint inhibitors and chimeric antigen receptor T cells have revolutionized cancer therapy, but their success in glioblastoma remains limited and further optimization of immunotherapies is needed. Several oncolytic viruses have demonstrated ability to infect tumors and trigger anti-tumor immune responses in malignant glioma patients. Leading the pack, oncolytic herpesvirus, first in its class, awaits an approval for treating malignant glioma from MHLW, the federal authority of Japan. Nevertheless, some major hurdles like the blood brain barrier, immunosuppressive tumor microenvironment, and tumor heterogeneity can engender suboptimal efficacy in malignant glioma. In this review, we discuss the current status of malignant glioma therapies with a focus on oncolytic viruses in clinical trials. Furthermore, we discuss the obstacles faced by oncolytic viruses in malignant glioma patients and strategies that are being used to overcome these limitations to 1) optimize delivery of oncolytic viruses beyond the blood brain barrier; 2) trigger inflammatory immune responses in and around tumors; and 3) use of multimodal therapies in combination to tackle tumor heterogeneity, with an end goal of optimizing the therapeutic outcome of oncolytic virotherapy.

Since its introduction, the Triangulation number has been the most successful and ubiquitous scheme for classifying spherical viruses. However, despite its many successes, it fails to describe the relative angular orientations of proteins, as well as their radial mass distribution within the capsid. It also fails to provide any insight into critical sites of stability, modifications or possible mutations. We show how classifying spherical viruses using icosahedral point arrays, introduced by Keef and Twarock, unveils new geometric rules and constraints for understanding virus stability and key locations for exterior and interior modifications. We present a modified fitness measure which classifies viruses in an unambiguous and rigorous manner, irrespective of local surface chemistry, steric hinderance, solvent accessibility or triangulation number. We then utilize these point arrays to explain the immutable surface loops of bacteriophage MS2, the relative reactivity of surface lysines in CPMV and the non-quasiequivalent flexibility of the HBV dimers. We explain how using sister and double point arrays can function as predictive tools for site directed modifications in other systems. This success builds on our previous work showing that viruses place their protruding features along the great circles of the asymmetric unit, demonstrating that viruses indeed adhere to these geometric constraints.

Ticks transmit a wide variety of pathogens including bacteria, parasites and viruses. Over the last decade, numerous novel viruses have been described in arthropods, including ticks, and their characterization has provided new insights into RNA virus diversity and evolution. However, little is known about their ability to infect vertebrates. As very few studies have described the diversity of viruses present in ticks from the Caribbean, we implemented an RNA-sequencing approach on Amblyomma variegatum and Rhipicephalus microplus ticks collected from cattle in Guadeloupe and Martinique. Among the viral communities infecting Caribbean ticks, we selected four viruses belonging to the Chuviridae, Phenuiviridae and Flaviviridae families for further characterization and designing antibody screening tests. While viral prevalence in individual tick samples revealed high infection rates, suggesting a high level of exposure of Caribbean cattle to these viruses, no seropositive animals were detected. These results suggest that the Chuviridae- and Phenuiviridae-related viruses identified in the present study are more likely tick endosymbionts, raising the question of the epidemiological significance of their occurrence in ticks, especially regarding their possible impact on tick biology and vector capacity. The characterization of these viruses might open the door to new ways of preventing and controlling tick-borne diseases.

The replication of species A rotaviruses (RVAs) and other RNA viruses involves recruitment of and inter-action with the cellular organelles lipid droplets (LDs), both physically and functionally. Inhibition of en-zymes involved in the cellular fatty acid biosynthesis pathway or of cellular lipases that degrade LDs re-duces the functions of ‘viral factories’ (viroplasms for rotaviruses or replication compartments of other RNA viruses) and decreases the production of infectious progeny virus. Similarly, disturbance of the cellular lipid homeostasis in various ways blocks the replication of flaviviruses (hepatitis C viruses, Zikavirus, others), HIV-1, SARS-CoV-2, picornaviruses, noroviruses, influenza viruses and other negative-strand RNA viruses.

Marnaviridae viruses are abundant algal viruses that regulate the dynamics of algal blooms in aquatic environments. They employ a narrow host range because they merely lyse their own algal host species. This host-specific lysis is thought to correspond to the unique transmission mechanism of the Marnaviridae viruses. Here, we present the atomic structures of the full and empty capsids of Chaetoceros socialis forma radians RNA virus 1 built in 3.0 Å and 3.1 Å cryo-electron microscopy maps. The empty capsid structure and the structural variability provide insights on its assembly and uncoating intermediates. In conjunction with the previously reported atomic model of the Chaetoceros tenuissimus RNA virus type II capsid, we have identified the common and diverse structural features of the VP1 surface between the Marnaviridae viruses. We have also tested the potential usage of AlphaFold2 for structural prediction of the VP1s and a subsequent structural phylogeny for classifying Marnaviridae viruses by their hosts. These findings will be crucial for inferring the host-specific transmission mechanism in Marnaviridae viruses, which is decisive for monitoring and possibly regulating the occurrence and disappearance of algae blooms.

The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: 1. Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. 2. Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.

Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by “priming” and/or “activation” steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusiogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.

Sea cucumbers (Holothuroidea; Echinodermata) are ecologically significant constituents of benthic marine habitats. We surveilled RNA viruses inhabiting 8 species (representing 4 families) of holothurian collected from four geographically distinct locations by viral metagenomics, including a single specimen of Apostichopus californicus affected by a hitherto undocumented wasting disease. The RNA virome comprised genome fragments of both single-stranded positive sense and double stranded RNA viruses, including those assigned to the Picornavirales, Ghabrivirales, and Amarillovirales. We discovered an unconventional flavivirus genome fragment which was most similar to a shark virus. Ghabivirales-like genome fragments were most similar to fungal totiviruses in both genome architecture and homology, and likely infected mycobiome constituents. Picornavirales, which are commonly retrieved in host-associated viral metagenomes, were similar to invertebrate transcriptome-derived picorna-like viruses. Sequence reads recruited from the grossly normal A. californicus metavirome to nearly all viral genome fragments recovered from the wasting-affected A. californicus. The greatest number of viral genome fragments was recovered from wasting A. californicus compared to any other surveyed holothurian, including the grossly normal A. californicus, which reflects a pattern observed in viral metagenomics study of sea star wasting. These results expand the known host range of flaviviruses, and suggest that fungi and their viruses may play roles in holothurian ecology.

Amid the covid-19 pandemic, other diseases, including viruses, are still acting to the detriment of their seasonality and risk factors for contagion. For this reason, it is interesting to know the degree of impact of other viruses, mainly respiratory, in which they have similar symptoms, in diagnoses for contamination by the new coronavirus based on epidemiological surveys, via epidemiological weeks, in Brazil. To what extent there may be a hypothesis of confusion of contaminated data, harming the health system, with regard to the need for intensive care units and control of viruses, and negatively or positively implying in the control or uncontrolling of viruses in general.

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle-articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology after acute injury and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, Nuclear Magnetic Resonance Imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced-inflammation promote fast loss of muscle mass and reduction of fibers cross sectional area, by inducing muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin expression, that trigger protein breakdown, resulting in acute SkM fibers atrophy. After temporal reduction of inflammation and clearance of infectious viral particles, SkM atrophy persists until the late phase of infection. The genomic CHIKV and MAYV RNAs were still detected in SkM at the late phase, along with increased levels of chemokines and anti-inflammatory cytokines expression. In agreement with the involvement of inflammatory mediators on induced atrophy, neutralization of TNF and the reduction of oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogens expression and atrophic fibers, while increasing weight gain in treated mice. Taken together, these data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphaviruses pathogenesis and with description of potentially new targets of therapeutic intervention.

Enteric viruses are the major cause of gastroenteritis and enteric hepatitis worldwide, but in some areas like Saudi Arabia little data is known about their presence in water sources. The available information from clinical samples is not enough to figure their actual prevalence. The aim of this study was to gather information for the first time in Saudi Arabia on the presence of Norovirus (NoV) genogroup GI and GII, hepatitis A virus (HAV) and hepatitis E virus (HEV) in water. For this purpose, thirteen monthly samples were collected in lake Wadi Hanifa and surrounding wells from December 2014 to November 2015. Viruses were detected and quantified by Real-time RT-qPCR. Despite HEV findings were anecdotic, our results highlight interesting behaviors of the other viruses. There was a higher prevalence of noroviruses in Wadi Hanifa samples than in well water samples (46.43% vs12.5% of NoV GI; 66.67% vs8.33% of NoV GII). On the contrary, similar levels of HAV positivity were observed (40.48% in surface water vs 43.06% in well water). Also, a strong influence of flooding events on HAV and NoV GI occurrence was observed in both surface and well water samples, being NoV GII apparently not affected.

With the progress of immunotherapy in cancer, oncolytic viruses (OVs) are getting more and more attention during the past decade. Due to their cancer-selective and immunogenic property, OVs are considered ideal candidates to be combined with immunotherapy to increase both specificity and efficacy in cancer treatment. OVs preferentially replicate in and lyse cancer cells, generating pathogen-associated molecular patterns (PAMPs) and danger (damage)-associated molecular patterns (DAMPs). These signals trigger innate immune response to modulate the solid tumor microenvironment, resulting in in situ autovaccination leading to adaptive anti-virus and anti-tumor immunity. Here, we summarize the conceptual updates of oncolytic virotherapy, immunotherapy, and the strategies to enhance the virus-mediated anti-tumor immune response, including: 1. Arm OVs with cytokines to modulated innate and adaptive immunity; 2. Combine OVs with immune checkpoint inhibitors to release T cell inhibition; 3. Combine OVs with immune co-stimulators to enhance T cell activation.

Background: The Ibizan Hound is a canine breed native to the Mediterranean region, where leishmaniasis is an endemic zoonosis. Several studies indicate a low prevalence of this disease in dogs, whereas other canine breeds present a high prevalence. However, the molecular underlaying mechanisms yet remains unknown. Methods: In this study, we analyze the haplotypes of genes encode cytokines related to immune response of Leishmania infantum infection in twenty-four Boxer and twenty-four Ibizan hound apparently healthy using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. Results: The results show that several haplotypes of genes encoding Interleukin 6 (IL6) and Interferon gamma (IFNG) are related to Interferon gamma (IFN-γ), and Interleukins (IL) 2 and 18 serum levels. Our results indicate that the regulation of immune response is different in the two canine breeds analyzed and are related to the haplotype compositions of the genes encoding these cytokines. Conclusions: Future studies are needed to elucidate whether these differences and haplotypes are related to different phenotypes in immune response and expression gene regulation to L. infantum infection in dogs.

We introduce a magnetic bead-based sample preparation scheme for enabling a Raman spectroscopic differentiation of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) positive and negative samples. The beads were functionalized with the angiotensin-converting enzyme 2 (ACE2) receptor protein, which is used as recognition element to selectively enrich SARS-CoV-2 on the surface of the magnetic beads. Subsequent Raman measurements directly enable discriminating SARS-CoV-2 positive and negative samples. The proposed approach is applicable for other virus species, too, when the specific recognition element is exchanged. Series of Raman spectra were measured on three types of samples, namely SARS-CoV-2, Influenza A H1N1 virus and a negative control. For each sample type, eight independent replicates were considered. All spectra are dominated by the magnetic bead substrate and no obvious differences between sample types are apparent. In order to address the subtle differences in the spectra, we calculated different correlation coefficients, namely the Pearson coefficient and the Normalized Cross Correlation coefficient. By comparing the correlation with the negative control differentiating between SARS-CoV-2 and Influenza A virus is possible. This study provides a first step towards the detection and potential classification of different viruses with the use of conventional Raman spectroscopy.

Leishmaniasis is a disease caused by parasites of Leishmania genus and transmitted by sand fly vectors. Tegumentary leishmaniasis is the most prevalent clinical outcome in Latin America afflicting people from 18 countries. In Panama, leishmaniasis has annual incidence rates as high as 3000 cases, representing therefore one of the major public health problems. In endemic regions, L. panamensis is responsible for almost eighty percent of the human cases that present different clinical outcomes. This differences in disease outcomes could be result of the local interplay between L. panamensis variants and human hosts with different genetic backgrounds. The genetic diversity of L. panamensis in Panama has only been partially explored, and the variability reported for this species is based on few studies restricted to small populations and/or with poor resolutive markers at low taxonomic levels. Accordingly, in this study we explored the genetic diversity of sixty-nine L. panamensis isolates from different endemic regions of Panama using a MLST approach based on four housekeeping genes (Aconitase, ALAT, GPI and HSP70). Two to seven haplotypes per locus were identified, and regional differences in L. panamensis genetic diversity were observed. Genotype analysis evidenced the circulation of thirteen L. panamensis genotypes, a fact that might have important implications for the local control of the disease.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human rotavirus-specific immunity, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

Viruses are the most numerically abundant biological entities on Earth. As ubiquitous replicators of information molecules and agents of community change, viruses have potent effects on life on Earth and may play a critical role in human spaceflight missions, life detection missions to other planetary bodies, and in planetary protection. However, major knowledge gaps constrain our understanding of the Earth’s virosphere: 1) the role viruses play in biogeochemical cycles, 2) the origin(s) of viruses, and 3) the involvement of viruses in the evolution, distribution, and persistence of life. As viruses are the only replicators that span all known types of nucleic acids, an expanded experimental and theoretical toolbox built for Earth’s viruses will be pivotal for detecting and understanding life on Earth and beyond. Only by filling in these knowledge and technical gaps will we obtain an inclusive assessment of how to distinguish and detect life on other planetary surfaces. Meanwhile, space exploration requires life-support systems for the needs of humans, plants, and their microbial inhabitants. Viral effects on microbes and plants are essential for Earth’s biosphere and human health, but virus-host interactions in spaceflight are poorly understood. Viral relationships with their hosts respond to environmental changes in complex ways which are difficult to predict by extrapolating from Earth-based proxies. These relationships must therefore be studied in space to fully understand how spaceflight will modulate viral impacts on human health and life-support systems, including microbiomes. This review addresses key questions that must be examined to incorporate viruses into Earth system models, life-support systems, and life detection. Further, the results of tackling these questions will help in our efforts to develop planetary protection protocols and further our understanding of viruses in astrobiology.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing the avian pox disease, which is characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosys-tem. Several diagnostic methods are available and vaccination of birds can be an effective pre-ventive measure. To update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023, ten APV-positive samples were analyzed. A P4b gene fragment was amplified by PCR and the nucleotide sequence of the amplicons was deter-mined by Sanger sequencing. The sequences obtained were aligned using ClustalW, and a max-imum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on avipoxviruses in Portugal.

Herein, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV) sensing system created by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, derived from 6’-silalyllactose used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range from 1 x 100 TCID50/mL to 1 x 107 TCID50/mL, with a detection limit of 4 x 101 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while displaying weak fluorescence signals when exposed to NA-deficient viruses such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Taken together, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC capable of detecting the ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.

Coinfection of B virus (HBV) and hepatitis Delta virus (HDV) lead to severe forms of progressive liver diseases. However, commercially available antiviral drugs have shown considerable adverse effects and low efficacy in managing these patients. Thus, there is a pressing need to develop new, innovative, and evidence-based therapies for these pathological entities. In line with this, some investigators have attempted to develop anti-viral drugs targeting HBV/HDV coinfection. In another development, investigators have also attempted to establish immune therapy targeting for HBV infection as recovery or effective control of HBV infection also lead to efficient control of HDV infection. This review has discussed an account of the evolving antiviral and immune therapeutic drugs for HBV/HDV infection. Also, based on current scientific knowledge how we have described the pros and cons of these drugs as no suitable drug has been developed during the last forty years for treating HBV infection except the availability of two” repurposed drugs”; interferons and its derivatives and nucleoside analogs, for managing HBV or HBV/HDV coinfection. It seems that an evolving and innovative therapeutic regimen that combines both antiviral drugs and immune modulators may be the best and realistic option for management of HBV/HDV coinfection.

This review seeks to explain four features of viral respiratory illnesses that have perplexed many generations of microbiologists: (1) the seasonal occurrence of viral respiratory illness; (2) the occurrence of respiratory illness year-round in the Tropics; (3) the rapid response of illness to temperature drops in temperate regions; (4) the explosive arrival and rapid termination of epidemics caused by influenza and other respiratory viruses. I discuss the inadequacy of the popular explanations of seasonality, and propose a simple hypothesis, called Temperature Dependent Viral Tropism (TD-VT), that is compatible with the above and other features of respiratory illness. TD-VT notes that viruses can often transmit themselves more effectively if they moderate their pathogenicity (thereby maintaining the mobility of their hosts) and suggests that most endemic respiratory viruses accomplish this by developing thermal sensitivity, in the sense that they normally replicate rapidly only at temperatures below normal body temperature. This allows them to confine themselves to the upper respiratory tract and to avoid infecting the lungs, heart, gut etc. I review biochemical and tissue-culture studies that found that “wild” respiratory viruses often show natural thermal sensitivity within a range that supports organ-specific tropism within the human body, and I discuss the evident tendency for viral strains to adapt their thermal sensitivity to their local climate and season. I also explore the possible misinterpretation of early experiments where volunteers were inoculated nasally with viral samples and then chilled. Next, I discuss the practical implications of the TD-VT hypothesis for preventing and treating respiratory illness. Finally, I note that the hypothesis is very testable and make suggestions for the most important experiments to increase our understanding of the seasonality and pathogenicity of viral respiratory illness.

Adeno-associated viruses (AAVs, genus dependoparvovirus) are promising gene therapy vectors. In strains AAV1-12, the capsid gene VP1 encodes a recently discovered protein, MAAP, in an overlapping frame. MAAP binds the cell membrane by an unknown mechanism. We discovered that MAAP is also encoded in bovine AAV and in porcine AAVs (which have shown promise for gene transfer into muscle tissues), in which it is probably translated from a non-canonical start codon. MAAP is predicted to be mostly disordered except for a predicted C-terminal, membrane-binding amphipathic α-helix. MAAP has a highly unusual composition. In particular, it lacks internal methionines, and is devoid of tyrosines in most strains. Unexpectedly, we discovered that the N-terminus of VP1 also lacks several amino acids. In all AAVs that encode MAAP, the first 200 aas of VP1 are devoid of internal methionines, probably owing to a selection against ATG codons that could prevent translation of MAAP and of capsid isoforms (VP2, VP3). The N-terminus of VP1 also lacks cysteines, likely to avoid the formation of disulfide bridges when it becomes exposed outside of the capsid during post-endocytic trafficking. Finally, the region common to VP1 and VP2 lacks tyrosine in the vast majority of AAVs that encode MAAP. Avoiding these "forbidden" aas in MAAP and VP1 when creating recombinant AAV capsids might increase the efficiency of capsid design. Conversely, the presence of "forbidden" aas in some rare strains probably indicates that they have unusual properties that could help us understand the viral cycle.

Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and Chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus (Accession number: GSE118858, https://www.ncbi.nlm.nih.gov/gds/?term=GSE118858). RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins AaeLRIM1, AaeAPL1 and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses.

Visceral leishmaniasis (VL)is a fatal disease caused by the protozoa Leishmania infantum for which dogs are the main reservoirs. A vaccine against canine visceral leishmaniasis (CVL) could be an important tool in the control of human and CVL by reducing the infection pressure of L. infantum. Despite the CVL vaccine available on the market, the Brazilian Ministry of Health did not implement the use of it in their control programs. In this sense, there is an urgent need to develop more efficient vaccines. In this study, the association between two polymeric nanoformulations, [poly (D, L-lactic) acid (PLA) polymer] loading Leishmania amazonensis antigens, was evaluated as a potential immunobiological agent against VL using golden hamsters as an experimental model. The results indicated that no significant adverse reactions were observed in animals vaccinated with LAPSmP. LAPSmP presented similar levels of total anti-Leishmania IgG as compared to LAPSmG. The LAPSmP and LAPSmG groups showed an intense reduction in liver and spleen parasitic load by qPCR. The LAPSmP and LAPSmG vaccines showed exceptional results, indicating that they may be promising candidates as a VL vaccine.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1,077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in the 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, just 4 animals (2 roe deer and 2 wild boars) tested positive only in the spleen, and 5 animals (3 roe deer and 2 red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 ani-mals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Isothermal Nucleic Acid Amplification based-Lateral Flow Testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative to address existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

We investigated by Real-time PCR and end point PCR the presence of 10 potential zoonotic viruses in 91 small mammals sold as pets in pet shops of Southern Italy. The animals were analysed for: aichivirus, sapovirus, astrovirus, hepatitis A virus, noroviruses (GI and GII), rotavirus, circovirus, coronaviruses. Among the 9 species of small mammals analysed (golden hamsters, Java squirrels, mongolian gerbils, peruvian guinea pigs, pet mice, pet rats, dwarf rabbits, african hedgehogs and sugar gliders) 12 pet rats, 11 pet mice and 1 golden hamster tested positive to a PCR amplifying a conserved gene (RdRp) of coronavirus. However further characterizaton by sequencing gave not conclusive results. A pet rat resulted instead positive to aichivirus and its sequence showed similarity with a murine kobuvirus-1 strain identified in China. To our knowledge, this is the first study reporting the detection of aichivirus in rodents in Italy, the virus probably introduced through the importation of animals from Asia. Since living in close contact to humans, expecially with children, the continuous monitoring of the small mammals is very important to avoid the spread of new pathologies among animals and humans and further investigation is desirable in order to safeguard public health.