Sequence comparison of wtVN1203 and rgVN1203
To investigate the difference in pathogenicity between wtVN1203 and rgVN1203, we sequenced the entire genomes of both viruses. Although 10 of the 11 protein sequences were identically encoded by the two viruses, we found 11 SMs in the PB1 gene, 2 in the NP gene, and 1 in the NA gene of rgVN1203 (referred to hereafter as rgVN1203/SM-3) (). Because most of the SMs were in PB1, a key component of the viral RNA-dependent RNA polymerase (RdRp), we first compared the codon triplet frequency at the affected nt positions in the PB1 genes of wtVN1203 and rgVN1203/SM-3. The VN1203 HA binds preferentially to α-(2,3)–linked sialic acid receptors, and therefore the codon triplets were evaluated by using PB1 of strictly avian influenza viruses (n = 1,088). The bioinformatics analysis showed significant differences in codon triplet frequency between the wtVN1203 and rgVN1203/SM-3 PB1 genes, with ratios as high as 1:112.1 (). A change at the third position of the codon triplets in PB1 of rgVN1203/SM-3 resulted in both rare and frequent codon usage. It is noteworthy that 3 of 11 SMs in PB1, at nt positions 246, 261, and 354 of the ORF, resulted in aa changes in the PB1-F2 protein: T51→M, V56→A, and E87→G, respectively ().
Figure. 1 Sequence comparison of wtVN1203 and rgVN1203. (A) The positions of synonymous mutations (SMs) in the PB1, NP, and NA genes of rgVN1203/SM-3 in comparison with the wtVN1203 nucleotide sequences. (B) Alignment and location of mutations in the PB1-F2 protein. (more ...)
Comparison of codon usage bias at the positions of synonymous mutations in the PB1 gene of rgVN1203/SM-3 and wtVN1203 a.
Repair of synonymous mutations in rgVN1203/SM-3 virus enhance its lethality in mallard ducks
SMs do not necessarily cause changes in the aa sequence of the resulting protein, but their presence can affect gene transcription and translation processes [3
]. To assess the effect of the changes in the PB1, NP, and NA genes on pathogenicity, we generated recombinant viruses with repairs in all 3 genes (rgVN1203/R-3), repairs in the PB1 gene only (rgVN1203/R-PB1), or no repairs (rgVN1203/SM-3) and compared their lethality in mallard ducks. The parental wtVN1203 virus was also included in this experiment. We first determined the pathogenicity index (PI) of each virus on the basis of the frequency of death and disease signs within 10 days after inoculation (a.i.) via natural routes with 106
50% egg infectious doses (EID50
) of stock virus (). The wtVN1203 virus exhibited the highest PI (1.24), followed by rgVN1203/R-PB1 (1.01) and rgVN1203/R-3 (0.99). The PI of rgVN1203/SM-3 was the lowest (0.58) among the viruses. Severe disease signs (cloudy eyes, ataxia, and neurologic disorders) were more frequent in ducks inoculated with wtVN1203, rgVN1203/R-3, or rgVN1203/R-PB1 than in those inoculated with rgVN1203/SM-3 ().
Pathogenicity of wtVN1203, rgVN1203/R-3, rgVN1203/R-PB1, and rgVN1203/SM-3 in mallard ducks a.
Inoculation with either virus caused a body temperature increase (as much as 1.5 °C greater than that of PBS-inoculated controls) within 3 days (). However, after correction for the starting temperature in each group, we found no significant difference in body temperature within the first 4 days after inoculation (a.i.) (virus*time interaction, P=0.34). All ducks maintained their average body temperature from day 5 a.i. until the end of the experiment.
Body weight in the control group increased steadily during the 12 days of observation (). Ducks inoculated with wtVN1203, rgVN1203/R-3, or rgVN1203/R-PB1 lost body weight during the first 5 days, whereas ducks inoculated with rgVN1203/SM-3 experienced only a brief plateau in weight gain (virus*time effect, P<0.0001). By day 4, ducks inoculated with rgVN1203/SM-3 were significantly heavier than those inoculated with rgVN1203/R-PB1 (simple effects ANOVA, P=0.03). Although survival in the inoculated groups did not differ significantly (Kaplan-Meier, P=0.08), higher mortality was observed after inoculation with wtVN1203, rgVN1203/R-3, or rgVN1203/R-PB1 than after inoculation with rgVN1203/SM-3 (). The wtVN1203 virus caused the highest mortality (55%), followed by rgVN1203/R-3 and rgVN1203/R-PB1 (both 40%). The rgVN1203/SM-3 virus killed only 4 of 20 ducks (20%).
To investigate whether reduced virus replication explained the lower pathogenicity of rgVN1203/SM-3, we titrated virus shed from the trachea and cloaca (). Ducks inoculated with wtVN1203, rgVN1203/R-3, or rgVN1203/R-PB1 viruses shed virus for 5 days; those inoculated with rgVN1203/SM-3 shed for only 3 days. The wtVN1203, rgVN1203/R-3, and rgVN1203/R-PB1 viruses were shed at higher levels from both the trachea and cloaca (ANOVA: trachea, P=0.39; cloaca, P=0.6). The highest titers were detected in the tracheas of ducks inoculated with rgVN1203/R-PB1 or wtVN1203 on days 3 and 5, respectively. Of the day-3 cloacal titers, that of rgVN1203/SM-3 was the lowest; on day 5, only wtVN1203 was detectable. These findings suggest that unrepaired PB1 reduced the lethality of rgVN1203/SM-3 in mallard ducks and that repair of NP and NA did not have a distinguishable effect on virus pathogenicity.
Amino acid changes encoded within the PB1-F2 reading frame of rgVN1203/SM-3 decrease lethality in mallard ducks
Because sequence analysis revealed 3 aa mutations encoded by SMs within the PB1-F2 reading frame of rgVN1203/SM-3, we investigated the role of these molecular changes in decreased lethality in mallard ducks. We generated a recombinant virus in which only these 3 SMs (at PB1 nt 246, 261, and 354) were repaired (rgVN1203/R-PB1-F2) and compared its pathogenicity with that of rgVN1203/R-PB1 and rgVN1203/SM-3. Although the PI values of all recombinant viruses were higher than those in the first experiment (), the PI of rgVN1203/SM-3 (0.78) was again the lowest. The rgVN1203/R-PB1 and rgVN1203/R-PB1-F2 viruses had PI values of 1.60 and 1.48, respectively. Inoculation with rgVN1203/R-PB1 or rgVN1203/R-PB1-F2 caused a markedly greater frequency of severe clinical signs than inoculation with rgVN1203/SM-3 ().
Pathogenicity of rgVN1203/R-PB1, rgVN1203/R-PB1-F2, and rgVN1203/SM-3 in mallard ducks a.
All virus-inoculated ducks developed fever within 4 days a.i. (), but no significant difference in body temperature was observed between the three virus groups (virus*time interaction, P=0.57). The body temperature of some ducks decreased dramatically (~3°C below the mean) on day 5, and they died on the subsequent day. Ducks in all virus groups lost body weight within 5 days a.i. (), but the rate of weight loss did not differ (virus*time interaction, P=0.13). It is noteworthy that ducks inoculated with rgVN1203/SM-3 began to recover almost a full day earlier than the other ducks. The mortality rate was high in ducks inoculated with rgVN1203/R-PB1 (8/10) and rgVN1203/R-PB1-F2 (7/10) but considerably lower in ducks inoculated with rgVN1203/SM-3 (3/10) (; Kaplan-Meier, P=0.02).
Tracheal swab titers on day 3 a.i. were higher in the rgVN1203/R-PB1 and rgVN1203/R-PB1-F2 groups than in the rgVN1203/SM-3 group (ANOVA, P<0.016; ), suggesting that the lower lethality of rgVN1203/SM-3 reflected reduced virus replication. The pattern of cloacal swab titers on day 3 was similar, although no significant difference was observed (ANOVA, P=0.24), and only rgVN1203/R-PB1 was detectable on day 5. Overall, these results suggest that the 3 aa changes in PB1-F2 contributed to the reduced lethality of rgVN1203/SM-3 in mallard ducks.
Mutations in PB1-F2 decrease viral polymerase activity
To characterize the effect of the 3 aa changes in PB1-F2 on transcription and translation of the virus genome, we compared levels of viral polymerase activity in the presence of repaired vs. unrepaired PB1-F2 in a minigenome system. IAV-like ribonucleoprotein (RNP) complexes were reconstituted in DF-1 chicken fibroblast cells or 293T human embryonic kidney cells by transfecting the cells with combinations of plasmids expressing vRNA-like RNA containing a firefly luciferase reporter gene under the control of an avian polymerase I (Pol I) promoter (for DF-1) or a human Pol I promoter (for 293T) and with other plasmids for expression of the RNP-associated viral proteins PB2, PB1, PA, and NP. Transfection efficiency was monitored in each sample by expression of the Renilla luciferase protein (dual-luciferase assay). The viral polymerase complex with unrepaired PB1-F2 had significantly less activity in DF-1 cells (20%-40% less; ANOVA, P
=0.05; ) and 293T cells (35–50% less; ANOVA, P
<0.0001; ) 24 h post-transfection (p.t.). The similar patterns of activity in the two cell lines showed that the activity was not cell type-dependent. These results indicate that the 3 aa changes in PB1-F2 directly or indirectly interfere with the function of the viral polymerase. These findings are similar to those observed in HEK293 cells by another group [17
Figure. 4 Viral polymerase activity in cultured cells. (A) DF-1 cells and (B) 293T cells were transfected with a plasmid containing an antisense firefly luciferase reporter gene flanked by a Pol I promoter and terminator sites, and with the Pol I/II-driven constructs (more ...)
Mutations in PB1-F2 lower replication and transcription rates of viral RNAs
We next investigated whether the higher activity of RNP complexes containing corrected PB1 or corrected PB1-F2 would affect the accumulation of different species of viral RNA in virus-infected cells. DF-1 cells were infected at an MOI of 1 with rgVN1203/R-3, rgVN1203/R-PB1, rgVN1203/R-PB1-F2, or rgVN1203/SM-3. Cells were then lysed and total RNAs were isolated 4, 6, 8, and 10 h p.i. The presence of viral vRNA, cRNA, and mRNA was confirmed by primer extension assay using radioactively labeled PB1 gene-specific primers for detection of viral negative-strand and positive-strand RNAs derived from the PB1 gene segment. Small cellular 5s rRNA served as a loading control for total RNA. The 3 viral RNA species of all 4 viruses appeared to increase through 6 h p.i. (). The viral vRNA levels in cells infected with rgVN1203/SM-3 were lower than in those infected with the other 3 viruses (Tukey’s HSD, P<0.0001). Levels of cRNA and mRNA were lower in cells infected with rgVN1203/SM-3 than in cells infected with either rgVN1203/R-PB1 (cRNA: P<0.018; mRNA: P<0.0001) or rgVN1203/R-PB1-F2 (cRNA: P<0.044; mRNA: P<0.0001) at most time points. Viral mRNA dominated the viral RNA population and reached the highest levels 6 h after infection with rgVN1203/R-PB1 and rgVN1203/R-PB1-F2 (Tukey’s HSD: P<0.0001). Overall, viral cRNA was less abundant than vRNA and mRNA.
Figure. 5 Viral RNA accumulation. DF-1 cells were infected with the indicated recombinant viruses at MOI = 1. Total RNA was isolated at the indicated time points and analyzed by Primer extension assays using PB1 gene-specific primers. Total RNA loading was monitored (more ...)