Avian Influenza Virus In Mice Research Articles

Phosphorylation of PB2 at serine 181 restricts viral replication and virulence of the highly pathogenic H5N1 avian influenza virus in mice

Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.

Exploring the alternative virulence determinants PB2 S155N and PA S49Y/D347G that promote mammalian adaptation of the H9N2 avian influenza virus in mice

The occurrence of human infections caused by avian H9N2 influenza viruses has raised concerns regarding the potential for human epidemics and pandemics. The molecular basis of viral adaptation to a new host needs to be further studied. Here, the bases of nucleotides 627 and 701 of PB2 were changed according to the uncoverable purine-to-pyrimidine transversion to block the development of PB2 627K and 701N mutations during serial passaging in mice. The purpose of this experiment was to identify key adaptive mutations in polymerase and NP genes that were obscured by the widely known host range determinants PB2 627K and 701N. Mouse-adapted H9N2 variants were obtained via twelve serial lung-to-lung passages. Sequence analysis showed that the mouse-adapted viruses acquired several mutations within the seven gene segments (PB2, PB1, PA, NP, HA, NA, and NS). One variant isolate with the highest polymerase activity possessed three substitutions, PB2 S155N, PA S49Y and D347G, which contributed to the highly virulent and mouse-adaptative phenotype. Further studies demonstrated that these three mutations resulted in increased polymerase activity, viral transcription and replication in mammalian cells, severe interstitial pneumonia, excessive inflammatory cellular infiltration and increased growth rates in mice. Our results suggest that the substitution of these three amino acid mutations may be an alternative strategy for H9N2 avian influenza viruses to adapt to mammalian hosts. The continued surveillance of zoonotic H9N2 influenza viruses should also include these mammalian adaptation markers as part of our pandemic preparedness efforts.

An immune-enhanced multivalent DNA nanovaccine to prevent H7 and H9 avian influenza virus in mice

H7 avian influenza virus has caused multiple human infections and poses a severe public health threat. In response to the highly variable nature of AIVs, a novel, easily regenerated DNA vaccine has great potential in treating or preventing avian influenza pandemics. Nevertheless, DNA vaccines have many disadvantages, such as weak immunogenicity and poor in vivo delivery. To further characterize and solve these issues and develop a novel H7 AIV DNA vaccine with enhanced stability and immunogenicity, we constructed nine AIV DNA plasmids, and the immunogenicity screened showed that mice immunized with pβH7N2SH9 elicited stronger hemagglutination-inhibiting (HI) antibodies than other eight plasmid DNAs. Then, to address the susceptibility to degradation and low transfection rate of DNA vaccine in vivo, we developed pβH7N2SH9/DGL NPs by encapsulating the pβH7N2SH9 within the dendrigraft poly-l-lysines nanoparticles. As expected, these NPs exhibited excellent physical and chemical properties, were capable of promote lymphocyte proliferation, and induce stronger humoral and cellular responses than the naked pβH7N2SH9, including higher levels of HI antibodies than naked pβH7N2SH9, as well as the production of cytokines, namely, IL-2, IFN-α. Taken together, our results suggest that the construction of an immune-enhanced H7-AIV DNA nanovaccine may be a promising strategy against most influenza viruses.

Rapid emergence of a PB2 D701N substitution during adaptation of an H9N2 avian influenza virus in mice.

H9N2 avian influenza viruses (AIVs) have been isolated frequently from multiple avian species and, occasionally, from humans. To explore the potential molecular basis of cross-species transmission of H9N2 AIVs, an H9N2 AIV (A/chicken/Zhejiang/221/2016) was serially passaged in mouse lung. The results showed that the mouse-adapted H9N2 virus exhibited higher virulence and replicated more efficiently in mouse lung and liver. Whole-genome sequencing showed an amino acid substitution, D701N, in the PB2 protein, which is likely associated with the increased replicative ability of H9N2 virus in mice. The rapid emergence of adaptive substitutions indicates the necessity of continuous monitoring of H9N2 virus in poultry.

Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals.

Avian influenza virus (AIV) can evolve multiple strategies to combat host antiviral defenses and establish efficient infectivity in mammals, including humans. H9N2 AIV and its reassortants (such as H5N6 and H7N9 viruses) pose an increasing threat to human health; however, the mechanisms involved in their increased virulence remain poorly understood. We previously reported that the M1 mutation T37A has become predominant among chicken H9N2 isolates in China. Here, we report that, since 2010, this mutation has also been found in the majority of human isolates of H9N2 AIV and its emerging reassortants. The T37A mutation of M1 protein enhances the replication of H9N2 AIVs in mice and in human cells. Interestingly, having A37 instead of T37 increases the M1 protein stability and resistance to proteasomal degradation. Moreover, T37 of the H9N2 M1 protein is phosphorylated by protein kinase G (PKG), and this phosphorylation induces the rapid degradation of M1 and reduces viral replication. Similar effects are also observed in the novel H5N6 virus. Additionally, ubiquitination at K187 contributes to M1-37T degradation and decreased replication of the virus harboring T37 in the M1 protein. The prevailing AIVs thereby evolve a phospho-resistant mutation in the M1 protein to avoid viral protein degradation by host factors, which is advantageous in terms of replication in mammalian hosts.

Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions.

In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

The virulence modulator PA-X protein has minor effect on the pathogenicity of the highly pathogenic H7N9 avian influenza virus in mice

PA-X is a novel discovered accessory protein encoded by the PA mRNA of the influenza A virus. Accumulated studies have demonstrated the crucial role of this protein in regulating the virulence of various subtypes of influenza virus, including H1N1, H5N1, H9N2, H1N2, H3N8 and H3N2 virus. However, the role of PA-X protein in regulating the virulence of the highly pathogenic avian H7N9 virus was unknown. In this study, we firstly generated two recombinant H7N9 viruses which have lower PA-X expression level than the parental H7N9 virus. We then systematically compared their difference in virus replication, polymerase activity, virulence and virus-induced host immune responses in mice. The results showed that the PA-X deficient viruses significantly increased viral replication in madin darby canine kidney cells and slightly increased viral replication in mouse lung. In addition, loss of PA-X expression significantly increased viral polymerase activity and alleviated the host-shutoff activity mediated by the parental PA protein. However, in contrast with the usual function of PA-X in regulating the virulence in different subtype influenza virus, no obvious effect on viral virulence in mice was observed by H7N9 PA-X protein. Furthermore, among the 12 kinds of cytokines and 2 kinds of complement derived components that we tested, the PA-X deficiency viruses only induced significantly higher expression levels of MX1 than the parental virus. Altogether, these results showed that PA-X has little effect on viral virulence and viral induced innate immune response of the H7N9 subtype virus. Our study adds further information for the growing understanding of the complexity of PA-X in regulating viral virulence and host innate immune response of different influenza virus.

Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice.

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health. However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging. A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA. Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.

Amino acid substitutions involved in the adaptation of a novel H7N7 avian influenza virus in mice

The H7N7 avian influenza viruses can infect humans and poses a great threat to human health. To identify the amino acid substitutions that are associated with adaptation of avian-origin H7N7 virus to mammals, adaptation of the H7N7 virus was carried out by serial lung-to-lung passage in mice. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E525G, M645I, and D701N), NP (I475V), HA(D103N), and NA(K142E) proteins. The adapted H7N7 virus was more virulent in mice than the wild-type virus. Our results suggest that continued surveillance of poultry populations for these substitutions in the H7N7 virus is required.

Two mutations in viral protein enhance the adaptation of waterfowl-origin H3N2 virus in murine model

After serial passage of a waterfowl-origin H3N2 subtype avian influenza virus in BALB/c mice, we obtained H3N2 mouse-adapted variants and identified eight amino acid substitutions in five viral proteins in our previous study. Here, we analyze the key mutations determining viral pathogenicity in mammals. We found that both PB2-D701N mutation and M1-M192V mutation were implicated in the viral pathogenic phenotypic variation of H3N2 avian influenza virus in mice. Furthermore, we found that PB2-D701N could enhance viral replication in vitro and in vivo and expanded viral tissue tropism. Our data suggest that PB2-D701N and M1-M192V are the virulence markers of H3N2 avian influenza virus, and these markers can be used in the trans-species transmission surveillance for the H3N2 avian influenza virus.

PB2 and hemagglutinin mutations confer a virulent phenotype on an H1N2 avian influenza virus in mice.

We previously obtained mouse-adapted variants of H1N2 avian influenza virus that contained PB2-L134H, PB2-I647L, PB2-D701N, HA-G228S, and M1-D231N mutations. Here, we analyzed the effects of these mutations on viral pathogenicity in a mammalian model. By evaluating the virulence of mouse-adapted H1N2 variants at different generations, we found that the PB2-D701N and HA-G228S mutations both contribute to the virulence of this virus in mammals. Furthermore, we found that the PB2-D701N and HA-G228S mutations both enhance the ability of the virus to replicate in vivo and in vitro and that the PB2-D701N substitution results in an expansion of viral tissue tropism. These results suggest that the PB2-D701N mutation and the HA-G228S mutation are the major mammalian determinants of H1N2 virus. These results help us to understand more about the mechanisms by which influenza viruses adapt to mammals, and monitoring of these mutations can be used in continuous influenza surveillance to assess the pandemic potential of avian influenza virus variants.

Deep sequencing of the mouse lung transcriptome reveals distinct long non-coding RNAs expression associated with the high virulence of H5N1 avian influenza virus in mice

ABSTRACT Long non-coding RNAs (lncRNAs) play multiple key regulatory roles in various biological processes. However, their function in influenza A virus (IAV) pathogenicity remains largely unexplored. Here, using next generation sequencing, we systemically compared the whole-transcriptome response of the mouse lung infected with either the highly pathogenic (A/Chicken/Jiangsu/k0402/2010, CK10) or the nonpathogenic (A/Goose/Jiangsu/k0403/2010, GS10) H5N1 virus. A total of 126 significantly differentially expressed (SDE) lncRNAs from three replicates were identified to be associated with the high virulence of CK10, whereas 94 SDE lncRNAs were related with GS10. Functional category analysis suggested that the SDE lncRNAs-coexpressed mRNAs regulated by CK10 were highly related with aberrant and uncontrolled inflammatory responses. Further canonical pathway analysis also confirmed that these targets were highly enriched for inflammatory-related pathways. Moreover, 9 lncRNAs and 17 lncRNAs-coexpressed mRNAs associated with a large number of targeted genes were successfully verified by qRT-PCR. One targeted lncRNA (NONMMUT011061) that was markedly activated and correlated with a great number of mRNAs was selected for further in-depth analysis, including predication of transcription factors, potential interacting proteins, genomic location, coding ability and construction of the secondary structure. More importantly, NONMMUT011061 was also distinctively stimulated during the highly pathogenic H5N8 virus infection in mice, suggesting a potential universal role of NONMMUT011061 in the pathogenesis of different H5 IAV. Altogether, these results provide a subset of lncRNAs that might play important roles in the pathogenesis of influenza virus and add the lncRNAs to the vast repertoire of host factors utilized by IAV for infection and persistence.

Multiple adaptive amino acid substitutions increase the virulence of a wild waterfowl-origin reassortant H5N8 avian influenza virus in mice

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD50) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo, and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice.

PB2 and HA mutations increase the virulence of highly pathogenic H5N5 clade 2.3.4.4 avian influenza virus in mice.

H5 clade 2.3.4.4 influenza A viruses pose a potential threat to public health and are a cause of public concern. Here, we generated mouse-adapted viruses of a waterfowl-origin H5N5 virus (H5 clade 2.3.4.4) to identify adaptive changes that confer increased virulence in mammals. After two passages, we obtained a mouse-adapted H5N5 virus that contained single amino acid substitutions in the PB2 (E627K) and hemagglutinin (HA) (F430L) proteins. We then analyzed the impact of these individual amino acid substitutions on viral pathogenicity to mammals. The 50% mouselethal dose (MLD50) of the H5N5 virus containing the PB2-E627K substitution or the HA-F430L substitution was reduced 1000-fold or 3.16-fold, respectively. Furthermore, we found that PB2-E627K enhanced viral replication kinetics in vitro and in vivo. These results suggest that the PB2-E627K and HA-F430L substitutions are important for adaptation of H5N5 AIVs to mammals. These findings emphasize the importance of continued surveillance of poultry for H5N5 AIVs with these amino acid substitutions.

Rapid acquisition adaptive amino acid substitutions involved in the virulence enhancement of an H1N2 avian influenza virus in mice

Although H1N2 avian influenza virus (AIV) only infect birds, documented cases of swine infection with H1N2 influenza viruses suggest this subtype AIV may pose a potential threat to mammals. Here, we generated mouse-adapted variants of a H1N2 AIV to identify adaptive changes that increased virulence in mammals. MLD50 of the variants were reduced >1000-fold compared to the parental virus. Variants displayed enhanced replication in vitro and in vivo, and replicate in extrapulmonary organs. These data show that enhanced replication capacity and expanded tissue tropism may increase the virulence of H1N2 AIV in mice. Sequence analysis revealed multiple amino acid substitutions in the PB2 (L134H, I647L, and D701N), HA (G228S), and M1 (D231N) proteins. These results indicate that H1N2 AIV can rapidly acquire adaptive amino acid substitutions in mammalian hosts, and these amino acid substitutions collaboratively enhance the ability of H1N2 AIV to replicate and cause severe disease in mammals.

ITRAQ-based quantitative proteomics reveals important host factors involved in the high pathogenicity of the H5N1 avian influenza virus in mice.

We previously reported a pair of H5N1 avian influenza viruses which are genetically similar but differ greatly in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly lethal to mice, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is avirulent. In this study, to investigate the host factors that account for their virulence discrepancy, we compared the pathology and host proteome of the CK10- or GS10-infected mouse lung. Moderate lung injury was observed from CK10-infected animals as early as the first day of infection, and the pathology steadily progressed at later time point. However, only mild lesions were observed in GS10-infected mouse lung at the late infection stage. Using the quantitative iTRAQ coupled LC-MS/MS method, we first found that more significantly differentially expressed (DE) proteins were stimulated by GS10 compared with CK10. However, bio-function analysis of the DE proteins suggested that CK10 induced much stronger inflammatory response-related functions than GS10. Canonical pathway analysis also demonstrated that CK10 highly activated the "Acute Phase Response Signaling," which results in a wide range of biological activities in response to viral infection, including many inflammatory processes. Further in-depth analysis showed that CK10 exacerbated acute lung injury-associated responses, including inflammatory response, cell death, reactive oxygen species production and complement response. In addition, some of these identified proteins that associated with the lung injury were further confirmed to be regulated in vitro. Therefore, our findings suggest that the early increased lung injury-associated host response induced by CK10 may contribute to the lung pathology and the high virulence of this virus in mice.

Amino acid substitutions involved in the adaptation of a novel highly pathogenic H5N2 avian influenza virus in mice.

BackgroundH5N2 avian influenza viruses (AIVs) can infect individuals that are in frequent contact with infected birds. In 2013, we isolated a novel reassortant highly pathogenic H5N2 AIV strain [A/duck/Zhejiang/6DK19/2013(H5N2) (6DK19)] from a duck in Eastern China. This study was undertaken to understand the adaptive processes that led enhanced replication and increased virulence of 6DK19 in mammals. 6DK19 was adapted to mice using serial lung-to-lung passages (10 passages total). The virulence of the wild-type virus (WT-6DK19) and mouse-adapted virus (MA-6DK19) was determined in mice. The whole-genome sequences of MA-6DK19 and WT-6DK19 were compared to determine amino acid differences.FindingsAmino acid changes were identified in the MA-DK19 PB2 (E627K), PB1 (I181T), HA (A150S), NS1 (seven amino acid extension “WRNKVAD” at the C-terminal), and NS2 (E69G) proteins. Survival and histology analyses demonstrated that MA-6DK19 was more virulent in mice than WT-6DK19.ConclusionOur results suggest that these substitutions are involved in the enhanced replication efficiency and virulence of H5N2 AIVs in mammals. Continuing surveillance for H5N2 viruses in poultry that are carrying these mutations is required.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0612-5) contains supplementary material, which is available to authorized users.

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans.

PA-X-associated early alleviation of the acute lung injury contributes to the attenuation of a highly pathogenic H5N1 avian influenza virus in mice.

PA-X is a novel discovered accessory protein encoded by the PA mRNA. Our previous study demonstrated that PA-X decreases the virulence of a highly pathogenic H5N1 strain A/Chicken/Jiangsu/k0402/2010 in mice. However, the underlying mechanism of virulence attenuation associated with PA-X is still unknown. In this study, we compared two PA-X-deficient mutant viruses and the parental virus in terms of induction of pathology and manipulation of host response in the mouse lung, stimulation of cell death and PA nuclear accumulation. We first found that down-regulated PA-X expression markedly aggravated the acute lung injury of the infected mice early on day 1 post-infection (p.i.). We then determined that loss of PA-X expression induced higher levels of cytokines, chemokines and complement-derived peptides (C3a and C5a) in the lung, especially at early time point’s p.i. In addition, in vitro assays showed that the PA-X-deficient viruses enhanced cell death and increased expression of reactive oxygen species (ROS) in mammalian cells. Moreover, we also found that PA nuclear accumulation of the PA-X-null viruses accelerated in MDCK cells. These results demonstrate that PA-X decreases the level of complement components, ROS, cell death and inflammatory response, which may together contribute to the alleviated lung injury and the attenuation of the virulence of H5N1 virus in mice.

Glycine at Position 622 in PB1 Contributes to the Virulence of H5N1 Avian Influenza Virus in Mice.

ABSTRACTWe isolated two H5N1 viruses, A/duck/Hunan/S4020/2008 (DK/08) and A/chicken/Guangxi/S2039/2009 (CK/09), from live-bird markets during routine surveillance and found that these two viruses are genetically similar but differ in their replication and virulence in mice. The CK/09 virus is lethal for mice with a 50% mouse lethal dose (MLD50) of 1.6 log10 50% egg infectious doses (EID50), whereas the DK/08 virus is nonpathogenic for mice with an MLD50 value of 6.2 log10 EID50. We explored the genetic basis of the virulence difference of these two viruses by generating a series of reassortant viruses and mutants in the lethal virus CK/09 background and evaluating their virulence in mice. We found that the PB1 gene of the DK/08 virus dramatically attenuated the virulence of the CK/09 virus and that the amino acid at position 622 in PB1 made an important contribution. We further demonstrated that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impaired the binding of PB1 to viral RNA, thereby dramatically decreasing the polymerase activity and attenuating H5N1 virus virulence in mice. Our results identify a novel virulence-related marker of H5N1 influenza viruses and provide a new target for live attenuated vaccine development.IMPORTANCE H5N1 avian influenza viruses have caused the deaths of nearly 60% of the humans that they have infected since 1997 and clearly represent a threat to public health. A thorough understanding of the genetic basis of virulence determinants will provide important insights for antiviral drug and live attenuated vaccine development. Several virulence-related markers in the PB2, PA, M1, and NS1 proteins of H5N1 viruses have been identified. In this study, we isolated two H5N1 avian influenza viruses that are genetically similar but differ in their virulence in mice, and we identified a new virulence-related marker in the PB1 gene. We found that the mutation of glycine (G) to aspartic acid (D) at position 622 in PB1 partially impairs the binding of PB1 to viral RNA, thereby attenuating H5N1 virus virulence in mice. This newly identified virulence-related marker could be applied to the development of live attenuated vaccines against H5N1 influenza.