Adaptation Of Influenza Viruses Research Articles

Characterization of H5N1 avian influenza virus isolated from bird in Russia with the E627K mutation in the PB2 protein

Currently A(H5Nx) avian influenza viruses are globally widespread and continue to evolve. Since their emergence in 2020 novel highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b reassortant viruses have become predominant in the world and caused multiple infections in mammals. It was shown that some of A(H5N1) viruses mostly isolated from mammals contain an E627K mutation in the PB2 protein which can lead to adaptation of influenza viruses to mammalian cells. In 2023 in Russia we have isolated two highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses from birds one of which contained an E627K mutation in the PB2 protein. This virus had increased virulence in mice. Limited airborne transmission of the virus with the PB2-E627K mutation was observed between ferrets, in which infectious virus was detected in the nasal washings of the three of the twelve recipient ferrets, and clinical symptoms of the disease were observed in one case. Both viruses showed dominant binding to avian-type sialoside receptors, which was most likely the reason for the limited transmissibility. Thus, this study indicates a possible limited increase in the pandemic potential of A(H5N1) 2.3.4.4b viruses and highlights the importance of continuous avian influenza surveillance for pandemic preparedness and response.

Enhanced Diversifying Selection on Polymerase Genes in H5N1 Clade 2.3.4.4b: A Key Driver of Altered Species Tropism and Host Range Expansion.

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses have shown unprecedented host range and pathogenicity, including infections in cattle, previously not susceptible to H5N1. We investigated whether selection pressures on clade 2.3.4.4b viral genes could shed light on their unique epidemiological features. Our analysis revealed that while the gene products of clade 2.3.4.4b H5N1 primarily undergo purifying selection, there are notable instances of episodic diversifying selection. Specifically, the polymerase genes PB2, PB1, and PA exhibit significantly greater selection pressures in clade 2.3.4.4b than all earlier H5N1 virus clades. Polymerases play critical roles in influenza virus adaptation, including viral fitness, interspecies transmission, and virulence. Our findings provide evidence that significant selection pressures have shaped the evolution of the H5N1 clade 2.3.4.4b viruses, facilitating their expanded host tropism and the potential for further adaptation to mammalian hosts. We discuss how exogenous factors, such as altered bird migration patterns and increased host susceptibility, may have contributed to the expanded host range. As H5N1 viruses continue to infect new hosts, there is a greater risk of emergent novel variants with increased pathogenicity in humans and animals. Thus, comprehensive One Health surveillance is critical to monitor transmission among avian and mammalian hosts.

Identifying host-specific amino acid signatures for influenza A viruses using an adjusted entropy measure

BackgroundInfluenza A viruses (IAV) exhibit vast genetic mutability and have great zoonotic potential to infect avian and mammalian hosts and are known to be responsible for a number of pandemics. A key computational issue in influenza prevention and control is the identification of molecular signatures with cross-species transmission potential. We propose an adjusted entropy-based host-specific signature identification method that uses a similarity coefficient to incorporate the amino acid substitution information and improve the identification performance. Mutations in the polymerase genes (e.g., PB2) are known to play a major role in avian influenza virus adaptation to mammalian hosts. We thus focus on the analysis of PB2 protein sequences and identify host specific PB2 amino acid signatures.ResultsValidation with a set of H5N1 PB2 sequences from 1996 to 2006 results in adjusted entropy having a 40% false negative discovery rate compared to a 60% false negative rate using unadjusted entropy. Simulations across different levels of sequence divergence show a false negative rate of no higher than 10% while unadjusted entropy ranged from 9 to 100%. In addition, under all levels of divergence adjusted entropy never had a false positive rate higher than 9%. Adjusted entropy also identifies important mutations in H1N1pdm PB2 previously identified in the literature that explain changes in divergence between 2008 and 2009 which unadjusted entropy could not identify.ConclusionsBased on these results, adjusted entropy provides a reliable and widely applicable host signature identification approach useful for IAV monitoring and vaccine development.

Influenza A virus-specific memory T cell persistence and functional recall are retained during pregnancy

Abstract Influenza A virus (IAV)-specific memory T cells induced by infection or through vaccination with live attenuated cold adapted influenza virus (LAIV) vaccine strains are well known to be able to confer protection against secondary homotypic as well as heterotypic IAV challenge. Whether IAV-specific memory T cell responses induced by LAIV vaccination can persist and retain their capacity to protect the maternal-fetal unit when generated prior to the altered physiological state of pregnancy, during which systemic levels of several hormones with putative immunomodulatory functions increase, is largely unknown. We show here that LAIV A/Alaska/6/77 CR-29 (H3N2)-vaccination prior to pregnancy induces IAV-specific memory CD4 and CD8 T cells that do indeed persist and functionally respond during recall in a manner similar to memory cells present in non-pregnant mice. This recall response can fully protect pregnant dams as well as their offspring from the adverse outcomes of lethal heterotypic infection with A/Puerto Rico/8/1934 (PR8-H1N1) virus. Using an adoptive transfer model and congenic HNT TcR transgenic CD4 T cells specific for PR8, we verify the observations observed with endogenous memory T cells that the recovery, surface marker phenotype, transcription factor profile, and TNF, IFN-g, and IL-2 multi-cytokine producing capacity of memory CD4 T cells responding to infection are largely unaltered by the pregnancy environment. These observations thus support that LAIV-vaccination prior to pregnancy results in the generation of IAV-specific memory T cells that have the potential to universally protect the maternal-fetal unit from the adverse outcomes of seasonal as well as pandemic strains of IAV. Supported by grants from NIH (R21HD093948), the University of Central Florida Foundation and the Florida Education Fund McKnight Doctoral Dissertation Fellowship.

The Species-Specific 282 Residue in the PB2 Subunit of the Polymerase Regulates RNA Synthesis and Replication of Influenza A Viruses Infecting Bat and Nonbat Hosts.

Bat influenza viruses are genetically distant from classical influenza A viruses (IAVs) and show distinct functional differences in their surface antigens. Nevertheless, any comparative analyses between bat and classical IAV RNA polymerases or their specific subunits are yet to be performed. In this work, we have identified signature residues present in the bat influenza virus polymerase which are responsible for its altered fitness in comparison to the classical IAVs. Through comparative sequence and structural analysis, we have identified specific positions in the PB2 subunit of the polymerase, with differential amino acid preferences among bat and nonbat IAVs. Functional screening helped us to focus upon the previously uncharacterized PB2-282 residue, which is serine in bat virus but harbors highly conserved glutamic acid in classical IAVs. Introduction of E282S mutation in the human-adapted PB2 (influenza A/H1N1/WSN/1933) drastically reduces polymerase activity and replication efficiency of the virus in human, bat, and canine cells. Interestingly, this newly identified PB2-282 residue within an evolutionary conserved "S-E-S" motif, present across different genera of influenza viruses and serving as a key regulator of RNA synthesis activity of the polymerase. In contrast, bat influenza viruses harbor an atypical "S-S-T" motif at the same position of PB2, alteration of which with the human-like "S-E-T" motif significantly enhances its (H17N10/Guatemala/164/2009) polymerase activity in human cells. Together, our data indicate that the PB2-S282 residue may serve as an inherent restriction element of the bat virus polymerase, limiting its activity in other host species. IMPORTANCE Influenza A viruses are known for their ability to perform cross-species transmission, facilitated by amino acid alterations either in the surface antigen hemagglutinin (HA) or in the polymerase subunit PB2. Recent isolation of influenza A-like viruses from bats raised concern about their epizootic and zoonotic potential. Here, we identify a novel species-specific signature present within the influenza virus polymerase that may serve as a key factor in adaptation of influenza viruses from bat to nonbat host species. The PB2-282 residue, which harbors a highly conserved glutamic acid for influenza viruses across all genera (A, B, C, and D), encompasses an atypical serine in the case of bat influenza viruses. Our data show that the human-adapted polymerase, harboring a bat-specific signature (PB2-S282,) performs poorly, while bat PB2 protein, harboring a human-specific signature (PB2-E282), shows increased fitness in human cells.

Identification and Characterization of Swine Influenza Virus H1N1 Variants Generated in Vaccinated and Nonvaccinated, Challenged Pigs.

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

Label-Free, Multiplex Glycan Microarray Biosensor for Influenza Virus Detection.

Newly emerging influenza viruses adapted from animal species pose significant pandemic threats to public health. An understanding of hemagglutinin (HA) receptor-binding specificity to host receptors is key to studying the adaptation of influenza viruses in humans. This information may be particularly useful for predicting the emergence of a pandemic outbreak. Therefore, high-throughput sensing technologies able to profile HA receptor binding can facilitate studies of influenza virus evolution and adaptation in humans. As a step toward this goal, we have prepared glycan-based receptor analogue microarrays on the Arrayed Imaging Reflectometry (AIR) platform. These arrays demonstrate label-free, multiplex detection and discrimination between human and avian influenza viruses. Microarrays consisting of glycan probes with 2,6 and 2,3 linkages were prepared. After first confirming their ability to capture lectins (carbohydrate-binding proteins) with known specificities, we observed that the arrays were able to discriminate between and quantify human pandemic influenza A/California/07/2009 (H1N1pdm) and avian A/Netherlands/1/2000 (H13N8) influenza viruses, respectively. As the method may be expanded to large numbers of glycans (>100) and virus subtypes (H1-H18), we anticipate it can be applied to systematically evaluate influenza virus adaptation in humans. In turn, this will facilitate global influenza surveillance and serve as a new tool enabling health organizations, governments, research institutes, and laboratories to react quickly in the face of a pandemic outbreak.

“Genetic tuning” of avian influenza virus host adaptation from birds to humans

Abstract Avian influenza viruses can adapt to infect humans by accumulating mutations or reassortments, potentially leading to a pandemic. However, the adaptation dynamics of zoonotic avian influenza viruses in their transition from animal hosts to human hosts remains largely unknown. In a recent PNAS report, Liu et al. have presented deep sequencing data showing host adaptation by “genetic tuning” of a zoonotic H7N9 avian influenza virus to cross the species barrier to infect humans. This provides the first in-depth data on avian influenza virus adaptation at the bird-human interface. As a one-health approach, the deep sequencing approach as implemented by Liu et al. is applicable to the study of emerging zoonotic viruses and provides valuable data on “genetic tuning” in virus-host adaptation. This experimental design should be useful for studying other zoonotic viruses, including SARS-CoV-2 and the novel swine influenza virus, and helpful for mitigating future pandemic public health risks.

Modeling influenza virus infection in mature Wistar rats

Passage of influenza A virus (IAV) in embryonated chicken eggs, intranasal inoculation of rats with virus-containing material s, production of pulmonary homogenate, determination of IAV titer in embryonated chicken eggs, detection of histological changes in lung and pulmonary vessels. The article presents the results of the adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats. The infectious titer of the virus in the homogenates of infected rats lungs at the last stage of adaptation was 7.0 lg EID50/ml. Histological studies revealed pronounced changes in the respiratory tract (spasm of bronchioles, submucosal edema, desquamation of ciliated epithelium of bronchioles) and pulmonary vessels (spasm, desquamation and swelling of endotheliocytes, dissociation and swelling of the elastic membrane and media). In order to identify IAV in blood vessels and lung tissues, an immunohistochemical study was performed using monoclonal antibodies to NP antigen of IAV. The data obtained allow us to conclude that the strain of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) was adapted to mature Wistar rats maintaining virulent properties. The infectious titer of the virus at the last stage of adaptation was 7.0 lg EID50/ml. IAV identification is confirmed by immunohistochemical examination.

Truncation of PA-X Contributes to Virulence and Transmission of H3N8 and H3N2 Canine Influenza Viruses in Dogs.

Equine-origin H3N8 and avian-origin H3N2 canine influenza viruses (CIVs) prevalent in dogs are thought to pose a public health threat arising from intimate contact between dogs and humans. However, our understanding of CIV virulence is still limited. Influenza A virus PA-X is a fusion protein encoded in part by a +1 frameshifted open reading frame (X-ORF) in segment 3. The X-ORF can be translated in full-length (61-amino-acid) or truncated (41-amino-acid) form. Genetic analysis indicated that the X-ORFs of equine H3N8 and avian H3N2 influenza viruses encoded 61 amino acids but were truncated after introduction into dogs. To determine the effect of PA-X truncation on the biological characteristics of CIVs, we constructed four recombinant viruses on H3N8 and H3N2 CIV backgrounds bearing truncated or full-length PA-Xs. We observed that truncation of PA-X increased growth of both H3N8 and H3N2 CIVs in MDCK cells and suppressed expression from cotransfected plasmids in MDCK cells. Furthermore, truncation of PA-X enhanced viral pathogenicity in dogs, as shown by aggravated clinical symptoms and histopathological changes, increased viral replication in the respiratory system, and prolonged virus shedding. Additionally, CIVs with truncated PA-Xs were transmitted more efficiently in dogs. Global gene expression profiling of the lungs of infected dogs revealed that differentially expressed genes were mainly associated with inflammatory responses, which might contribute to the pathogenicity of PA-X-truncated CIVs. Our findings revealed that truncation of PA-X might be important for the adaptation of influenza viruses to dogs.IMPORTANCE Epidemics of equine-origin H3N8 and avian-origin H3N2 influenza viruses in canine populations are examples of successful cross-species transmission of influenza A viruses. Genetic analysis showed that the PA-X genes of equine H3N8 or avian H3N2 influenza viruses were full-length, with X-ORFs encoding 61 amino acids; however, those of equine-origin H3N8 or avian-origin H3N2 CIVs were truncated, suggesting that PA-X truncation occurred after transmission to dogs. In this study, we extended the PA-X genes of H3N8 and H3N2 CIVs and compared the biological characteristics of CIVs bearing different lengths of PA-X. We demonstrated that for both H3N8 and H3N2 viruses, truncation of PA-X increased virus yields in MDCK cells and enhanced viral replication, pathogenicity, and transmission in dogs. These results might reflect enhanced suppression of host gene expression and upregulation of genes related to inflammatory responses. Collectively, our data partially explain the conservation of truncated PA-X in CIVs.

SRSF10 inhibits the polymerase activity and replication of avian influenza virus by regulating the alternative splicing of chicken ANP32A

Compared with mammalian ANP32A, most avian-coded ANP32A contains a 33 amino acids insertion (ch-ANP32A-33) or a 29 amino acids insertion (ch-ANP32A-29), which can rescue the mammalian-restricted avian influenza virus polymerase activity, with ch-ANP32A-33 exhibiting a more potent phenotype. The alternative splicing of 3′ splice sites (SSs) of chicken ANP32A intron 4 generates full-length ch-ANP32A-33 and truncated ch-ANP32A-29. In this study, we found a splicing regulatory cis-element that affected the alternative splicing of 3′ SSs by block-scanning mutagenesis. RNA affinity purification and mass spectrometry showed that the SRSF10 bound to the splicing cis-element and the binding was further identified and confirmed by RIP experiment. Overexpression of SRSF10 changed the ratio of the two chicken ANP32A transcripts with the increased ch-ANP32A-29 and the decreased ch-ANP32A-33. The knockdown of both of the ch-ANP32A-33 and ch-ANP32A-29 was harmful to avian influenza virus polymerase activity in DF-1 cells, but the restoration and increasement of only ch-ANP32A-29 could not completely rescue the activity of avian influenza virus polymerase. Overexpression of SRSF10 negatively affected the polymerase activity and replication of avian influenza virus, and the expression of ch-ANP32A-33 could partially recover the decrease of polymerase activity of avian influenza virus. By contrast, SRSF10 had weak inhibition on the polymerase activity of mammalian adapted influenza virus and had no effect on the replication of mammalian adapted influenza virus. Taken together, we demonstrated that SRSF10 acts as a negative regulator in polymerase activity and replication of avian influenza virus by binding to the splicing cis-element to regulate the alternative splicing of chicken ANP32A intron 4 for the reduced ch-ANP32A-33 and increased ch-ANP32A-29.

Bearing the brunt: Mongolian khulan (Equus hemionus hemionus) are exposed to multiple influenza A strains

The majority of influenza A virus strains are hosted in nature by avian species in the orders of Anseriformes and Charadriformes. A minority of strains have been able to cross species boundaries and establish themselves in novel non-avian hosts. Influenza viruses of horses, donkeys, and mules represent such successful events of avian to mammal influenza virus adaptation. Mongolia has over 3 million domestic horses and is home to two wild equids, the Asiatic wild ass or khulan (Equus hemionus hemionus), and Przewalski’s horse (Equus ferus przewalskii). Domestic and wild equids are sympatric across most of their range in Mongolia. Epizootic influenza A virus outbreaks among Mongolian domestic horses have been frequently recorded. However, the exposure, circulation and relation to domestic horse influenza A virus outbreaks among wild equids is unknown. We evaluated serum samples of Asiatic wild asses in Mongolia for antibodies against influenza A viruses, using modified protein microarray technique. We detected antibodies against hemagglutinin (H) H1, H3, H5, H7, H8 and H10 influenza A viruses. Asiatic wild asses may represent a previously unidentified influenza A virus reservoir in an ecosystem shared with populations of domestic horses in which influenza strains circulate.

Effects of the PA-X and PB1-F2 Proteins on the Virulence of the 2009 Pandemic H1N1 Influenza A Virus in Mice.

There have been several previous reports showing that PA-X and PB1-F2 proteins can regulate innate immune responses and may play roles in the adaptation of influenza viruses to new hosts. In this research, we investigated, for the first time, the combined effects of PA-X and PB1-F2 proteins on viral virulence in mice. Based on the 2009 pH1N1 A/Guangdong/1057/2010 virus backbone, four viruses encoding different combinations of full-length or truncated PA-X and PB1-F2 proteins were rescued by a reverse genetic engineering system. We analyzed viral replication, host-shutoff activity, in vitro viral pathogenicity and in vivo host immune response. We found that simultaneously expressing the full-length PA-X and PB1-F2 proteins enhanced viral replication in vitro through increasing the accumulation of the RNP complex protein and enhanced viral pathogenicity in mice during the early stage of infection. Furthermore, PA-X and PB1-F2 simultaneously regulated the host innate response, and different forms of PB1-F2 proteins may have impacts on the host shutoff activity induced by the PA-X protein. Our results provide a better understanding of the mechanisms of PA-X and PB1-F2 proteins during viral replication, pathogenicity and host immune response.

Monoclonal antibody against N2 neuraminidase of cold adapted A/Leningrad/134/17/57 (H2N2) enables efficient generation of live attenuated influenza vaccines

Cold adapted influenza virus A/Leningrad/134/17/57 (H2N2) is a reliable master donor virus (Len/17-MDV) for preparing live attenuated influenza vaccines (LAIV). LAIVs are 6:2 reasortants that contain 6 segments of Len/17-MDV and the hemagglutinin (HA) and neuraminidase (NA) of contemporary circulating influenza A viruses. The problem with the classical reassortment procedure used to generate LAIVs is that there is limited selection pressure against NA of the Len/17-MDV resulting in 7:1 reassortants with desired HA only, which are not suitable LAIVs. The monoclonal antibodies (mAb) directed against the N2 of Len/17-MDV were generated. 10C4–8E7 mAb inhibits cell-to-cell spread of viruses containing the Len/17-MDV N2, but not viruses with the related N2 from contemporary H3N2 viruses. 10C4–8E7 antibody specifically inhibited the Len/17-MDV replication in vitro and in ovo but didn’t inhibit replication of H3N2 or H1N1pdm09 reassortants. Our data demonstrate that addition of 10C4–8E7 in the classical reassortment improves efficiency of LAIV production.

Diversity of Influenza A(H5N1) Viruses in Infected Humans, Northern Vietnam, 2004-2010.

Influenza viruses exist in each host as a collection of genetically diverse variants, which might enhance their adaptive potential. To assess the genetic and functional diversity of highly pathogenic avian influenza A(H5N1) viruses within infected humans, we used deep-sequencing methods to characterize samples obtained from infected patients in northern Vietnam during 2004–2010 on different days after infection, from different anatomic sites, or both. We detected changes in virus genes that affected receptor binding, polymerase activity, or interferon antagonism, suggesting that these factors could play roles in influenza virus adaptation to humans. However, the frequency of most of these mutations remained low in the samples tested, implying that they were not efficiently selected within these hosts. Our data suggest that adaptation of influenza A(H5N1) viruses is probably stepwise and depends on accumulating combinations of mutations that alter function while maintaining fitness.

Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells

We analyzed the adaptation of influenza viruses to growth in differentiated airway epithelial cells of a new host by passaging an avian H9N2 virus three times in porcine precision-cut lung slices (PCLS). Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins, and two in the HA protein. In this study, we characterized the PB2 mutation G685R by generating recombinant H9N2 viruses containing the PB2 single mutation alone or in combination with one of the HA mutations (A190V or T212I). When analyzed in porcine cells − a tracheal cell line (NPTr) or PCLS − the PB2-685 mutant did not provide a growth advantage and had no effect on the ciliary activity which is a virulence marker of swine influenza viruses. Pathogenicity for mice was also not increased by the single PB2 mutation. However, both double mutants (HA-190+PB2-685 and HA-212+PB2-685) showed significantly increased virulence in mice. Therefore, the mutations in the HA and PB2 proteins may confer early adaptation of an avian H9N2 virus to a mammalian host. In conclusion, we expect that a broader ensemble of mutations will be required to render an H9N2 virus virulent for pigs.

A single dose and long lasting vaccine against pandemic influenza through the controlled release of a heterospecies tandem M2 sequence embedded within detoxified bacterial outer membrane vesicles.

The influenza A virus undergoes genetic drift and shift, leaving the general population susceptible to emerging pandemic strains, despite seasonal flu vaccination. Here we describe a single dose influenza vaccine derived from recombinant outer membrane vesicles (rOMVs) that display an antigen-mapped heterospecies tandem sequence of the M2 protein from the influenza A virus, released over 30days from poly(lactic-co-glycolide) (PLGA) microparticles. Four weeks post vaccination, BALB/c mice developed high anti-M2e IgG titers that were equivalent to those generated at 8weeks in a typical prime/boost vaccine regimen. Challenge of mice with a lethal dose of mouse adapted influenza virus PR8 (H1N1) 10weeks post vaccination resulted in 100% survival for both rOMV single-dose microparticle and prime/boost vaccinated mice. Anti-M2e IgG1 and IgG2a antibody titers were weighted toward IgG1, but splenocytes isolated from rOMV single-dose microparticle vaccinated mice produced high levels of IFNγ relative to IL-4 in response to stimulation with M2e peptides, supporting a more Th1 biased immune response. The protective immune response was long lasting, eliciting sustained antibody titers and 100% survival of mice challenged with a lethal dose of PR8 six months post initial vaccination. Together, these data support the potential of controlled release rOMVs as an effective single dose, long lasting and rapidly effective vaccine to protect against influenza.

Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.

Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs.

The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a “triple-reassortment” H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs.

Selective Bottlenecks Shape Evolutionary Pathways Taken during Mammalian Adaptation of a 1918-like Avian Influenza Virus.

Avian influenza virus reassortants resembling the 1918 human pandemic virus can become transmissible among mammals by acquiring mutations in hemagglutinin (HA) and polymerase. Using the ferret model, we trace the evolutionary pathway by which an avian-like virus evolves the capacity for mammalian replication and airborne transmission. During initial infection, within-host HA diversity increased drastically. Then, airborne transmission fixed two polymerase mutations that do not confer a detectable replication advantage. In later transmissions, selection fixed advantageous HA1 variants. Transmission initially involved a “loose” bottleneck, which became strongly selective after additional HA mutations emerged. The stringency and evolutionary forces governing between-host bottlenecks may therefore change throughout host adaptation. Mutations occurred in multiple combinations in transmitted viruses, suggesting that mammalian transmissibility can evolve through multiple genetic pathways despite phenotypic constraints. Our data provide a glimpse into avian influenza virus adaptation in mammals, with broad implications for surveillance on potentially zoonotic viruses.