Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Live Bird Markets in Nigeria: A Potential Reservoir for H9N2 Avian Influenza Viruses.

Since 2006, multiple outbreaks of avian influenza (AI) have been reported in Nigeria involving different subtypes. Surveillance and molecular epidemiology have revealed the vital role of live bird markets (LBMs) in the dissemination of AI virus to commercial poultry farms. To better understand the ecology and epidemiology of AI in Nigeria, we performed whole-genome sequencing of nineteen H9N2 viruses recovered, from apparently healthy poultry species, during active surveillance conducted in nine LBMs across Nigeria in 2019. Analyses of the HA gene segment of these viruses showed that the H9N2 strains belong to the G1 lineage, which has zoonotic potential, and are clustered with contemporary H9N2 identified in Africa between 2016 and 2020. We observed two distinct clusters of H9N2 viruses in Nigeria, suggesting different introductions into the country. In view of the zoonotic potential of H9N2 and the co-circulation of multiple subtypes of AI virus in Nigeria, continuous monitoring of the LBMs across the country and molecular characterization of AIVs identified is advocated to mitigate economic losses and public health threats.

Antigenic and molecular characterization of low pathogenic avian influenza A(H9N2) viruses in sub-Saharan Africa from 2017 through 2019.

Sub-Saharan Africa was historically considered an animal influenza cold spot, with only sporadic highly pathogenic H5 outbreaks detected over the last 20 years. However, in 2017, low pathogenic avian influenza A(H9N2) viruses were detected in poultry in Sub-Saharan Africa. Molecular, phylogenetic, and antigenic characterization of isolates from Benin, Togo, and Uganda showed that they belonged to the G1 lineage. Isolates from Benin and Togo clustered with viruses previously described in Western Africa, whereas viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Viruses from Benin exhibited decreased cross-reactivity with those from Togo and Uganda, suggesting antigenic drift associated with reduced replication in Calu-3 cells. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2). Therefore, viral genetic and antigenic surveillance in Africa is of paramount importance to detect further evolution or emergence of new zoonotic strains.

Mink is a highly susceptible host species to circulating human and avian influenza viruses.

Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive "mixing vessels" for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

PA-X is an avian virulence factor in H9N2 avian influenza virus.

Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focusing on the natural avian host, chickens. We found that the G1 lineage A/chicken/Pakistan/UDL-01/2008 (H9N2) PA-X induced robust host shutoff in both mammalian and avian cells and increased virus replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.

Autophagy is involved in the replication of H9N2 influenza virus via the regulation of oxidative stress in alveolar epithelial cells.

BACKGROUND: Oxidative stress is an important pathogenic factor in influenza A virus infection. It has been found that reactive oxygen species induced by the H9N2 influenza virus is associated with viral replication. However, the mechanisms involved remain to be elucidated. METHODS: In this study, the role of autophagy was investigated in H9N2 influenza virus-induced oxidative stress and viral replication in A549 cells. Autophagy induced by H9N2 was inhibited by an autophagy inhibitor or RNA interference, the autophagy level, viral replication and the presence of oxidative stress were detected by western blot, TCID50 assay, and Real-time PCR. Then autophagy and oxidative stress were regulated, and viral replication was determined. At last, the Akt/TSC2/mTOR signaling pathways was detected by western blot. RESULTS: Autophagy was induced by the H9N2 influenza virus and the inhibition of autophagy reduced the viral titer and the expression of nucleoprotein and matrix protein. The blockage of autophagy suppressed the H9N2 virus-induced increase in the presence of oxidative stress, as evidenced by decreased reactive oxygen species production and malonaldehyde generation, and increased superoxide dismutase 1 levels. The changes in the viral titer and NP mRNA level caused by the antioxidant, N-acetyl-cysteine (NAC), and the oxidizing agent, H2O2, confirmed the involvement of oxidative stress in the control of viral replication. NAC plus transfection with Atg5 siRNA significantly reduced the viral titer and oxidative stress compared with NAC treatment alone, which confirmed that autophagy was involved in the replication of H9N2 influenza virus by regulating oxidative stress. Our data also revealed that autophagy was induced by the H9N2 influenza virus through the Akt/TSC2/mTOR pathway. The activation of Akt or the inhibition of TSC2 suppressed the H9N2 virus-induced increase in the level of LC3-II, restored the decrease in the expression of phospho-pAkt, phospho-mTOR and phospho-pS6 caused by H9N2 infection, suppressed the H9N2-induced increase in the presence of oxidative stress, and resulted in a decrease in the viral titer. CONCLUSION: Autophagy is involved in H9N2 virus replication by regulating oxidative stress via the Akt/TSC2/mTOR signaling pathway. Thus, autophagy maybe a target which may be used to improve antiviral therapeutics.

A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus.

Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.

Influenza Virus Neuraminidase Engages CD83 and Promotes Pulmonary Injury.

Influenza A viruses cause severe respiratory illnesses in humans and animals. Overreaction of the innate immune response to influenza virus infection results in hypercytokinemia, which is responsible for mortality and morbidity. However, the mechanism by which influenza induces hypercytokinemia is not fully understood. In this study, we established a mouse-adapted H9N2 virus, MA01, to evaluate the innate immune response to influenza in the lung. MA01 infection caused high levels of cytokine release, enhanced pulmonary injury in mice, and upregulated CD83 protein in dendritic cells and macrophages in the lung. Influenza virus neuraminidase (NA) unmasked CD83 protein and contributed to high cytokine levels. Furthermore, we provide evidence that CD83 is a sialylated glycoprotein. Neuraminidase treatment enhanced lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW264.7 cells. Anti-CD83 treatment alleviated influenza virus-induced lung injury in mice. Our study indicates that influenza virus neuraminidase modulates CD83 status and contributes to the "cytokine storm," which may suggest a new approach to curb this immune injury.IMPORTANCE The massive release of circulating mediators of inflammation is responsible for lung injury during influenza A virus infection. This phenomenon is referred to as the "cytokine storm." However, the mechanism by which influenza induces the cytokine storm is not fully understood. In this study, we have shown that neuraminidase unmasked CD83 protein in the lung and contributed to high cytokine levels. Anti-CD83 treatment could diminish immune damage to lung tissue. The NA-CD83 axis may represent a target for an interruption of influenza-induced lung damage.

Non-basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys.

H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.

PA from a Recent H9N2 (G1-Like) Avian Influenza a Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems.

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1EGY and H9N2EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2EGY, expressed individually and in combination in the genetic background of H5N1EGY, were genetically compatible with the other H5N1EGY segments. At 37 °C the replication efficiencies of H5N1EGY reassortants expressing the H9N2EGY polymerase subunits PB2 and PA (H5N1PB2-H9N2EGY, H5N1PA-H9N2EGY) were higher than the wild-type H5N1EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1PB2-H9N2EGY, but reduced for H5N1PA-H9N2EGY. At 33 °C and 39 °C, H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY replicated to higher levels than the wild-type H5N1EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1EGY. Genetic analysis of the polymerase-encoding segments revealed that the PAH9N2EGY and PB2H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1EGY enabled the mutant virus H5N1PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses.

H9N2 avian influenza viruses (AIVs) circulate in poultry throughout much of Asia, the Middle East, and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right and in the generation of novel zoonotic viruses, for example, H7N9. In recent years, it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titers in various systems, and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates that these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as the rapidly expanding geographical range seen in these viruses.IMPORTANCE Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia, an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here, we take representative viruses and investigate the genetic basis for this "fitness." A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes to viral replication as well as to virus accessory functions-however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.

Molecular characterization and pathogenesis of H9N2 avian influenza virus isolated from a racing pigeon.

H9N2 avian influenza viruses (AIVs) can cross species barriers and expand from birds tomammals and humans. It usually leads to economic loss for breeding farms and poses a serious threat to human health.This study investigated the molecular characteristics of H9N2 AIV isolated from a racing pigeon and its pathogenesis in BALB/c mice and pigeons. Phylogenetic analysis indicated that the H9N2 virus belonged to the Ck/BJ/94-like lineage, and acquired multiple specific amino acid substitutions that might contribute to viral transmission from birds to mammals and humans. A pathogenesis study showed that both mice and pigeons infected with H9N2 virus showed clinical signs and mortality. The H9N2 viruses efficiently replicated in mice and pigeons. In our study, high levels of viral shedding were detected in pigeons, but the infection was not transmitted to co-housed pigeons. Histopathological examination revealed the presence of inflammatory responses in the infected mice and pigeons. Immunohistochemical analysis showed the presence of H9N2 virus in multiple organs of the infected mice and pigeons. Moreover, the infected mice and pigeons demonstrated significant cytokine/chemokine production. Our results showed that the H9N2 virus can infect mice and pigeons, and can not be transmitted between pigeons through direct contact.

Supplemental dietary selenium enhances immune responses conferred by a vaccine against low pathogenicity avian influenza virus.

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens.

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity.

Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2.

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7-8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.

Adaptation of H9N2 Influenza Viruses to Mammalian Hosts: A Review of Molecular Markers.

As the number of human infections with avian and swine influenza viruses continues to rise, the pandemic risk posed by zoonotic influenza viruses cannot be underestimated. Implementation of global pandemic preparedness efforts has largely focused on H5 and H7 avian influenza viruses; however, the pandemic threat posed by other subtypes of avian influenza viruses, especially the H9 subtype, should not be overlooked. In this review, we summarize the literature pertaining to the emergence, prevalence and risk assessment of H9N2 viruses, and add new molecular analyses of key mammalian adaptation markers in the hemagglutinin and polymerase proteins. Available evidence has demonstrated that H9N2 viruses within the Eurasian lineage continue to evolve, leading to the emergence of viruses with an enhanced receptor binding preference for human-like receptors and heightened polymerase activity in mammalian cells. Furthermore, the increased prevalence of certain mammalian adaptation markers and the enhanced transmissibility of selected viruses in mammalian animal models add to the pandemic risk posed by this virus subtype. Continued surveillance of zoonotic H9N2 influenza viruses, inclusive of close genetic monitoring and phenotypic characterization in animal models, should be included in our pandemic preparedness efforts.

Dendrigraft poly-L-lysines delivery of DNA vaccine effectively enhances the immunogenic responses against H9N2 avian influenza virus infection in chickens.

Biodegradable nanomaterials can protect antigens from degradation, promote cellular absorption, and enhance immune responses. We constructed a eukaryotic plasmid [pCAGGS-opti441-hemagglutinin (HA)] by inserting the optimized HA gene fragment of H9N2 AIV into the pCAGGS vector. The pCAGGS-opti441-HA/DGL was developed through packaging the pCAGGS-opti441-HA with dendrigraft poly-l-lysines (DGLs). DGL not only protected the pCAGGS-opti441-HA from degradation, but also exhibited high transfection efficiency. Strong cellular immune responses were induced in chickens immunized with the pCAGGS-opti441-HA/DGL. The levels of IFN-γ and IL-2, and lymphocyte transformation rate of the vaccinated chickens increased at the third week post the immunization. For the vaccinated chickens, T lymphocytes were activated and proliferated, the numbers of CD3+CD4+ and CD4+/CD8+ increased, and the chickens were protected completely against H9N2 AIV challenge. This study provides a method for the development of novel AIV vaccines, and a theoretical basis for the development of safe and efficient gene delivery carriers.

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites.

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

Two genetically similar H9N2 influenza viruses isolated from different species show similar virulence in minks but different virulence in mice.

The H9N2 influenza virus has been frequently endemic in poultry, infected mammals and humans and has threatened public health. It is therefore imperative to understand the molecular mechanism enabling this virus to jump from avian to mammalian species. In this study, two H9N2 influenza viruses were isolated from the same region in eastern China but from different hosts; one was isolated from mink and named A/Mink/Shandong/WM01/2014(H9N2)(WM01), while the other was isolated from chicken and named A/Chicken/Shandong/LX830/2014(H9N2)(LX830). Sequencing and phylogenetic analysis showed that both H9N2 influenza viruses had similar genetic backgrounds. The results of infection in minks suggested that both viruses caused significant weight loss and pathological changes in the lungs. Mouse infection showed that LX830 was nonpathogenic in mice, but WM01 resulted in 25% mortality and pathological changes in the lungs, such as severe edema and diffused inflammation of the interalveolar septa. Comparison of the full genomes of both H9N2 influenza viruses showed 52-nucleotide-synonym mutations in 8 gene segments and 7-nucleotide-antonym mutations, resulting in 7 amino acid (AA) substitutions distributed in the PB1, PA, NA and M gene segments. None of these mutations did affect splicing of the M and NS gene segments at the nucleotide level or minor open reading frames (ORFs), such as PB1-F2 and PA-X. Phylogenetic analysis showed that both H9N2 influenza viruses belong to the prevalent epidemic genotype in Asia. Keywords: H9N2 influenza virus; chicken; minks; pathogenicity; phylogenetic.