Emergence of avian H1N1 influenza viruses in pigs in China.

Avian influenza A viruses from Asia are recognized as the source of genes that reassorted with human viral genes to generate the Asian/57 (H2N2) and Hong Kong/68 (H3N2) pandemic strains earlier in this century. Here we report the genetic analysis of avian influenza A H1N1 viruses recently isolated from pigs in southern China, a host suspected to generate new pandemic strains through gene reassortment events. Each of the eight gene segments was of avian origin. Phylogenetic analysis indicates that these genes form an Asian sublineage of the Eurasian avian lineage, suggesting that these viruses are an independent introduction into pigs in Asia. The presence of avian influenza viruses in pigs in China places them in an optimal position for transmission to humans and may serve as an early warning of the emergence of the next human influenza virus pandemic.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

Proteotyping to establish gene origin within reassortant influenza viruses

The application of a rapid and direct proteotyping approach with which to identify the gene origin of viral antigens in a reassortant influenza strain is demonstrated. The reassortant strain, constructed for a vaccine against type A 2009 H1N1 pandemic influenza, contains genes derived from a wild-type pandemic strain (A/California/7/2009) and an egg adapted high-growth strain (denoted NYMC X-157) derived from an earlier A/Puerto Rico/8/34 strain. The proteotyping approach employs modern proteomics methods and high resolution mass spectrometry to correctly establish that the genes of the surface antigens, hemagglutinin and neuraminidase, are derived from the A/California/7/2009 strain while those for nucleoprotein and matrix protein M1 antigens are derived from the NYMC X-157 strain. This is achieved for both gel-separated antigens and those from a whole vaccine digest. Furthermore, signature peptides detected in the mass spectra of the digested antigens enable the engineered reassortant strain to be identified as a type A virus of the H1N1 subtype in accord with earlier studies. The results demonstrate that proteotyping approach provides a more direct and rapid approach over RT-PCR with which to characterize reassortant strains of the influenza virus at the molecular protein level. Given that these strains pose the greatest risk to human and animal health and have been responsible for all human pandemics of the 20th and 21st centuries, there is a vital need for the origins and evolutionary history of these strains to be rapidly established.     

Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses

The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian-human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs.     

Reassortment Patterns in Swine Influenza Viruses

Three human influenza pandemics occurred in the twentieth century, in 1918, 1957, and 1968. Influenza pandemic strains are the results of emerging viruses from non-human reservoirs to which humans have little or no immunity. At least two of these pandemic strains, in 1957 and in 1968, were the results of reassortments between human and avian viruses. Also, many cases of swine influenza viruses have reportedly infected humans, in particular, the recent H1N1 influenza virus of swine origin, isolated in Mexico and the United States. Pigs are documented to allow productive replication of human, avian, and swine influenza viruses. Thus it has been conjectured that pigs are the “mixing vessel” that create the avian-human reassortant strains, causing the human pandemics. Hence, studying the process and patterns of viral reassortment, especially in pigs, is a key to better understanding of human influenza pandemics. In the last few years, databases containing sequences of influenza A viruses, including swine viruses, collected since 1918 from diverse geographical locations, have been developed and made publicly available. In this paper, we study an ensemble of swine influenza viruses to analyze the reassortment phenomena through several statistical techniques. The reassortment patterns in swine viruses prove to be similar to the previous results found in human viruses, both in vitro and in vivo, that the surface glycoprotein coding segments reassort most often. Moreover, we find that one of the polymerase segments (PB1), reassorted in the strains responsible for the last two human pandemics, also reassorts frequently.     

The evolutionary emergence of pandemic influenza

Pandemic influenza remains a serious public health threat and the processes involved in the evolutionary emergence of pandemic influenza strains remain incompletely understood. Here, we develop a stochastic model for the evolutionary emergence of pandemic influenza, and use it to address three main questions. (i) What is the minimum annual number of avian influenza virus infections required in humans to explain the historical rate of pandemic emergence? (ii) Are such avian influenza infections in humans more likely to give rise to pandemic strains if they are driven by repeated cross-species introductions, or by low-level transmission of avian influenza viruses between humans? (iii) What are the most effective interventions for reducing the probability that an influenza strain with pandemic potential will evolve? Our results suggest that if evolutionary emergence of past pandemics has occurred primarily through viral reassortment in humans, then thousands of avian influenza virus infections in humans must have occurred each year for the past 250 years. Analyses also show that if there is epidemiologically significant variation among avian influenza virus genotypes, then avian virus outbreaks stemming from repeated cross-species transmission events result in a greater likelihood of a pandemic strain evolving than those caused by low-level transmission between humans. Finally, public health interventions aimed at reducing the duration of avian virus infections in humans give the greatest reduction in the probability that a pandemic strain will evolve.     

Full Factorial Analysis of Mammalian and Avian Influenza Polymerase Subunits Suggests a Role of an Efficient Polymerase for Virus Adaptation

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.     

Phylogenetic analysis of H1N1 sequences from pandemic infections during 2009 in India

Since April 2009, a serious pandemic infection has been rapidly spread across the world. These infections are caused due to the novel swine origin influenza A (H1N1) virus and hence these are commonly called as "Swine Flu". This new virus is the reassortment of avian, human and swine influenza viruses and thus it has a unique genome composition. There are 16 different types of hemagglutinin (HA) and 9 different types of neuraminidase (NA) that can be genetically and antigenetically differentiated. The first influenza A virus isolated from pigs was of the H1N1 subtype and these viruses have been reported to cause infection in pigs in many countries. The outbreak of this virus has been transmitted from pigs to humans. This new reassorted (exchange of genes) virus which is the cause of 2009 pandemic infections has the ability to spread from human to human. This spread of infection should be brought to an end. In this study, a phylogenetic analysis of the nucleotide sequences of the RNA segments of human H1N1 viruses was carried using MEGA version 4.0 to demonstrate the route map of infection to India. Phylogenetic analysis of the sequences from India, published in Influenza Virus Resource (a database that integrates information gathered from the Influenza Genome Sequencing Project of the National Institute of Allergy and Infectious diseases (NIAID) and the genbank of the (NCBI)) was retrieved and used for the analysis. The results showed that the various segments of the Indian isolates clustered well with the sequences from American, Asian and European countries and thus indicating the transmission of viruses from these places to India.     

Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses

Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak.     

A Novel Small Molecule Inhibitor of Influenza A Viruses that Targets Polymerase Function and Indirectly Induces Interferon

Influenza viruses continue to pose a major public health threat worldwide and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The antiviral cytokine, interferon (IFN) is an essential mediator of the innate immune response and influenza viruses, like many viruses, have evolved strategies to evade this response, resulting in increased replication and enhanced pathogenicity. A cell-based assay that monitors IFN production was developed and applied in a high-throughput compound screen to identify molecules that restore the IFN response to influenza virus infected cells. We report the identification of compound ASN2, which induces IFN only in the presence of influenza virus infection. ASN2 preferentially inhibits the growth of influenza A viruses, including the 1918 H1N1, 1968 H3N2 and 2009 H1N1 pandemic strains and avian H5N1 virus. In vivo, ASN2 partially protects mice challenged with a lethal dose of influenza A virus. Surprisingly, we found that the antiviral activity of ASN2 is not dependent on IFN production and signaling. Rather, its IFN-inducing property appears to be an indirect effect resulting from ASN2-mediated inhibition of viral polymerase function, and subsequent loss of the expression of the viral IFN antagonist, NS1. Moreover, we identified a single amino acid mutation at position 499 of the influenza virus PB1 protein that confers resistance to ASN2, suggesting that PB1 is the direct target. This two-pronged antiviral mechanism, consisting of direct inhibition of virus replication and simultaneous activation of the host innate immune response, is a unique property not previously described for any single antiviral molecule.     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Evolutionary complexities of swine flu H1N1 gene sequences of 2009

A recently emerged novel influenza A (H1N1) virus continues to spread globally. The pandemic caused by this new H1N1 swine influenza virus presents an opportunity to analyze the evolutionary significance of the origin of the new strain of swine flu. Our study clearly suggests that strong purifying selection is responsible for the evolution of the novel influenza A (H1N1) virus among human. We observed that the 2009 viral sequences are evolutionarily widely different from the past few years’ sequences. Rather, the 2009 sequences are evolutionarily more similar to the most ancient sequence reported in the NCBI Influenza Virus Resource Database collected in 1918. Analysis of evolutionary rates also supports the view that all the genes in the pandemic strain of 2009 except NA and M genes are derived from triple reassorted swine viruses. Our study demonstrates the importance of using complete-genome approach as more sequences will become available to investigate the evolutionary origin of the 1918 influenza A (H1N1) swine flu strain and the possibility of future reassortment events.     

The influenza pandemic of 2009: lessons and implications

Influenza is a moving target, which evolves in unexpected directions and is recurrent annually. The 2009 influenza A/H1N1 pandemic virus was unlike the 2009 seasonal virus strains and originated in pigs prior to infecting humans. Three strains of viruses gave rise to the pandemic virus by antigenic shift, reassortment, and recombination, which occurred in pigs as 'mixing vessels'. The three strains of viruses had originally been derived from birds, pigs, and humans. The influenza hemagglutinin (HA) and neuraminidase (NA) external proteins are used to categorize and group influenza viruses. The internal proteins (PB1, PB1-F2, PB2, PA, NP, M, and NS) are involved in the pathogenesis of influenza infection. A major difference between the 1918 and 2009 pandemic viruses is the lack of the pathogenic protein PB1-F2 in the 2009 pandemic strains, which was present in the more virulent 1918 pandemic strains. We provide an overview of influenza infection since 1847 and the advent of influenza vaccination since 1944. Vaccines and chemotherapy help reduce the spread of influenza, reduce morbidity and mortality, and are utilized by the global rapid-response organizations associated with the WHO. Immediate identification of impending epidemic and pandemic strains, as well as sustained vigilance and collaboration, demonstrate continued success in combating influenza.     

Emergence and dissemination of a swine H3N2 reassortant influenza virus with 2009 pandemic H1N1 genes in pigs in China

The 2009 pandemic influenza virus (pdm/09) has been frequently introduced to pigs and has reassorted with other swine viruses. Recently, H3N2 reassortants with pdm/09-like internal genes were isolated in Guangxi and Hong Kong, China. Genetic and epidemiological analyses suggest that these viruses have circulated in swine for some time. This is the first evidence that swine reassortant viruses with pdm/09-like genes may have become established in the field, altering the landscape of human and swine influenza.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Comparative analysis of hemagglutinin of 2009 H1N1 influenza A pandemic indicates its evolution to 1918 H1N1 pandemic

To gain insight into the possible origin of the hemagglutinin of 2009 outbreak, we performed its comparative analysis with hemagglutinin of influenza viral strains from 2005 to 2008 and the past pandemics of 1977, 1968, 1957 and 1918. This insilico analysis showed a maximum sequence similarity between 2009 and 1918 pandemics. Primary structure analysis, antigenic and glycosylation site analyses revealed that this protein has evolved from 1918 pandemic. Phylogenetic analysis of HA amino acid sequence of 2009 influenza A(H1N1) viruses indicated that this virus possesses a distinctive evolutionary trait with 1918 influenza A virus. Although the disordered sequences are different among all the isolates, the disordered positions and sequences between 2009 and 1918 isolates show a greater similarity. Thus these analyses contribute to the evidence of the evolution of 2009 pandemic from 1918 influenza pandemic. This is the first computational evolutionary analysis of HA protein of 2009 H1N1 pandemic.     

High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1

Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process has been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.     

Evolutionary and transmission dynamics of reassortant H5N1 influenza virus in Indonesia

H5N1 highly pathogenic avian influenza (HPAI) viruses have seriously affected the Asian poultry industry since their recurrence in 2003. The viruses pose a threat of emergence of a global pandemic influenza through point mutation or reassortment leading to a strain that can effectively transmit among humans. In this study, we present phylogenetic evidences for the interlineage reassortment among H5N1 HPAI viruses isolated from humans, cats, and birds in Indonesia, and identify the potential genetic parents of the reassorted genome segments. Parsimony analyses of viral phylogeography suggest that the reassortant viruses may have originated from greater Jakarta and surroundings, and subsequently spread to other regions in the West Java province. In addition, Bayesian methods were used to elucidate the genetic diversity dynamics of the reassortant strain and one of its genetic parents, which revealed a more rapid initial growth of genetic diversity in the reassortant viruses relative to their genetic parent. These results demonstrate that interlineage exchange of genetic information may play a pivotal role in determining viral genetic diversity in a focal population. Moreover, our study also revealed significantly stronger diversifying selection on the M1 and PB2 genes in the lineages preceding and subsequent to the emergence of the reassortant viruses, respectively. We discuss how the corresponding mutations might drive the adaptation and onward transmission of the newly formed reassortant viruses.