A reverse genetics approach to study the pathogenesis of pneumonia virus of mice

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Abstract

Human and bovine respiratory syncytial viruses (HRSV and BRSV), along with
pneumonia virus of mice (PVM) are the members of the genus Pneumovirus in the
subfamily Pneumovirinae of the family Paramyxoviridae. Although both HRSV and
BRSV have been associated with important diseases in human and livestock, there is no
clearcut description of the molecular aspects of their pathogenesis. For HRSV, the lack
of a suitable study model is one of the main reasons hampering the study of aspects of
pathogenesis of the virus. HRSV infects a wide range of animal models, however most
of the common laboratory animal models are not sufficiently permissive to study the
infectivity of the virus.
PVM naturally infects mice and causes a disease indistinguishable from that of
HRSV in humans. Two strains of PVM have been described: strain 15 (Warwick) which
is not pathogenic and strain J3666 which is highly pathogenic. The main difference
between these two strains lies in the organisation of the gene encoding the attachment
(G) glycoprotein. The G gene in strain J3666 has two ORFs. The larger second ORF
codes for the G glycoprotein and is located downstream of the first ORF which has no
known function. The strain 15 G gene also contains two ORFs but in this case both the
first and main ORFs overlap each other.
The aim of the project was to investigate the molecular basis for pathogenesis of
PVM as a model for pneumoviruses. As a first step, the pathogenesis of PVM strain
J3666 was revaluated and the effect of consecutive tissue culture passages on the
pathogenicity of the virus was examined. It was shown that consecutive passages of
PVM strain J3666 caused attenuation of the virus. To investigate the possible mutations
causing the attenuation the majority of the virus genome was sequenced from three
passage stocks where the transition from pathogenic to non-pathogenic occurred. No
differences in the genome sequences for the three passage stocks were found. However,
sequence analysis of individual clones of the SH and G genes of the viruses showed
evidence that the stocks contained a mixed population of sequences.
A robust reverse genetics system was established to rescue recombinant PVM
from cDNA using co-transfection of plasmids coding for the ribonucleoprotein complex
of the virus (N, L, M2-1 and P proteins) and a cDNA copy of the virus genome cloned
under the control of the bacteriophage T7 RNA polymerase. Using this system, four
viruses differing in their G gene organisation were generated and used to infect mice to
study the effect of mutations on pathogenicity. It was shown that the viruses with the G
gene of strain 15 (Warwick) lacking the first ORF manifest a modest increase in their
pathogenicity compared to the non-pathogenic PVM strain 15(Warwick) parent. The
recombinant viruses containing the G gene organisation of strain J3666 showed the
highest level of pathogenicity.
The reverse genetics system was used to study the role of the first ORF in G
glycoprotein expression. Using a dicistronic minigenome construct, the effect of the
presence or absence of the first ORF in both the strain 15 and strain J3666 G gene
organisation was studied. It was shown that the presence of the first ORF of the G gene
in the strain 15 (Warwick) suppressed the expression of the G protein, while the first
ORF in the strain J3666 did not have any significant effect on G protein expression.

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