Effect Of PB1 T677M And PB2 I63T Mutations On Pathogenicity Of AIV H5N1

H5N1 avian influenza virus (AIV) can infect not only birds but also human and other mammalian animals, crossing the species barrier, posing severe threat to human health. H5N1 AIV, which had infected 525 human beings and caused 310 deaths from 1977 to February 2011 with a mortality rate of about 60%, has become one of the most severe infectious viral pathogens since it crossed the species barrier in 1997. But up to now, little is known about the pathogenesis of H5N1 virus on human.The plaque forming characteristic is one of the important phenotypes of H5N1 virus. Different H5N1 strains can form different plaques, and even the same human H5N1 strain can form the plaques with different size and morphous. If these different plaques formed from a same virus strain have different pathogenesis, it should be easier to identify the molecular determinants of pathogenesis, as there are only a few nucleic acid differences between these different plaques. The objective of the present study was to explore the molecular pathogenesis of AIV H5N1 virus based on the observation that A/Vientam/1194/2004 (H5N1) (VN1194) strains form the plaques with different sizes during its passage on mammalian cells or in mice. H5N1 variants with significant different pathogenicity were isolated and purified according to their different sizes of plaques by plaque forming assay. After identification of their different pathogenicity and genome sequences, their molecular pathogenesis was further explored by reverse genetics method. This study might provide new ideas for the research of H5N1 pathogenesis.. Isolation and sequence analysis of the variants with significant different pathogenicityThe plaque forming characteristic of VN1194 was assayed and homogeneous needlepoint-like small plaques were formed. However, after propagation only one passage in mice intranasally, mixed plaques with large and small sizes were observed in lungs whereas only homogeneous small-plaques existed in brains. The variants with small-plaques and large-plaques were isolated and purified separately from lungs and brains by plaque forming assay and their different pathogenicity to mice was detected. The results showed that the variants (MLL1 and MLL4) isolated from lungs with large-plaques were less pathogenic to mice, the variants (MB1, MB2) isolated from lungs or brains with small-plaques were highly pathogenic to mice.The sequence analyze results demonstrated that there were amino acid mutations in each one of the 4 variants. PB2 I63T and PB1 T677M were common amino acid mutations in attenuated variants (MLL1 and MLL4), however, no common amino acid mutations were found among highly pathogenic variants (MB1, MB2). In summary, the plaque forming assay for AIV H5N1 was constructed and the plaque forming property changes were observed when VN1194 virus propagated after one passage in mice. Homogeneous and needlepoint-like small plaques were still the same as in their human hosts. However, relatively larger plaques were isolated from mice lungs. Four variants with significant different pathogenicity were isolated and purified. PB2 I63T and PB1 T677M were common amino acid mutations in attenuated variants. The two common amino acid mutations might associate with the attenuated pathogenicity and these observations might be the bases to understand the molecular pathogeneses mechanism of AIV H5N1.. Functions of PB1 T677M and PB2 I63TReverse genetics is a very important method to study the molecular pathogeneses of influenza virus and its vaccine. The virus strains with desired amino acid mutation(s) were rescued by reverse genetics to study the functions of PB1 T677M and PB2 I63T of AIV H5N1. Firstly, eight-plasmid DNA transfection system for the rescue of infectious AIV H5N1 from cloned cDNA was constructed. Then, rVN1194 with no mutation, rVN1194:PB2-63T and rVN1194:PB1-677M with only one amino acid mutation (PB1 T677M or PB2 I63T), and rVN1194M with two amino acid mutations (PB1 T677M and PB2 I63T) were rescued respectively by eight-plasmid DNA transfection system. Finally, the rescued strains were assayed and identified by embryonated egg experiment, plaque forming assay, specific PCR and the whole genomic sequence analysis. The pathogenicity to mice of the different rescued strains was tested and compared.Four desired strains were successfully rescued by eight-plasmid DNA transfection system. The genome sequences of the rescued variants were exactly the same as their original sequences. The 4 rescued variants were all formed to homogeneous and needlepoint-like small plaques. The pathogenicity experiments showed that the rescued strain rVN1194M with both mutations (PB2 I63T and PB1 T677M) was less pathogenic than the other 3 strains (rVN1194, rVN1194:PB2-63T and rVN1194:PB1-677M). Furthermore, some of the mice inoculated with rVN1194 and rVN1194:PB2-63T suffered from neurologic symptoms and none of the mice presented with the same symptoms infected by rVN1194M and rVN1194:PB1-677M.Based on these results, PB1 T677M and PB2 I67T co-mutations might be a reason for the virulence attenuation of AIV H5N1. However, PB1 T677M mutation might associate with the virus neurotropism or the capability to enter into mice brains.. Research on mechanism of association between co-mutations and virulence attenuationWe investigated the relationship between pathogenicity and replicative efficiency of the rescued viruses in many ways to study the mechanism of virulence attenuation associated with co-mutations. Firstly, the replicative efficiency of 4 rescued strains was observed in different cell lines (A549, neuro-2a and RAW264.7). The results showed that rVN1194M (T677M and PB2 63T co-mutations) had lower replicative efficiency than the other 3 strains. This suggested that the lower replicative efficiency might be one of the mian reasons why rVN1194M was less pathogenic than the other 3 strains. Secondly, the relationship between replicative efficiency and polymerase activity was studied in molecular level. The polymerase activity of the rescued strains with PB2 T677M (rVN1194M and rVN1194:PB1- 677M) was significantly higher than the others (rVN1194 and rVN1194:PB2-63T) when PB1 and PB2 mRNA expression had no differences. This demonstrated that the lower replicative efficiency of rVN1194M had no direct relationship with polymerase activity. Finally, the viral loads in lungs of the mice infected by rVN1194M were lower than the other 3 strains tested on the third day and sixth day after intranasally inoculation. This indicated that the lower viral loads might be another important reason why the virus with co-mutations (rVN1194M) had attenuated virulence. Whereas, no viruses were detected in mice brains infected with strains with PB1 T677M mutation (rVN1194M and rVN1194:PB1- 677M). This was coinciding with the clinical manifest conducted by animal experiments. Virus with PB1 T677M mutation might result in the inability to enter into mice brains.Histopathology observation is a direct proof of viral virulence. The histopathology changes were compared in different organs of mice infected by rVN1194 and rVN1194M. The results showed mice infected with rVN1194 suffered more serious injuries in livers, spleens and kidneys than infected with rVN1194M. Furthermore viral antigens have been found in mice brains which were infected by rVN1194 but not rVN1194M. This indicated that PB1 T677M and PB2 I63T co-mutations mediated virulence attenuation resulted in fewer injuries to the livers, spleens, kidneys, and resulted in its inability to enter into mice brains.In summary, PB1 T677M and PB2 I63T co-mutations mediated virulence attenuation was discovered based on the sequence analysis of the variants with significant different pathogenicity which was isolated by plaque forming assay. The results indicated that the pathogenesis of H5N1 with PB1 T677M and PB2 I63T co-mutations was associated with its lower replication, fewer injuries to the livers, spleens, kidneys, and its inability to enter into mice brains. The discovery that virus with PB1 T677M mutation lose the ability to enter mice brains provides new proof to neurotropism of H5N1 virus. These results would provide a new strategy for vaccine development and the study of molecular mechanism. The present study also provides foundations for the new research areas of H5N1 pathogenisis.