Hydrophobic Residues at the Intracellular Domain of the M2 Protein Play an Important Role in Budding and Membrane Integrity of Influenza Virus.

M2 protein of influenza virus plays an important role in virus budding, including membrane scission and vRNP packaging. Three hydrophobic amino acids (91F, 92V, and 94I) at the intracellular domain of the M2 protein constitute a hydrophobic motif, also known as the LC3-interacting region (LIR), whereas the role of this motif remains largely unclear. To explore the role of the 91-94 hydrophobic motif for influenza virus, all three hydrophobic amino acids were mutated to either hydrophilic S or hydrophobic A, resulting in two mutant viruses (WSN-M2/SSS and WSN-M2/AAA) in the background of WSN/H1N1. The results showed that the budding ability of the M2/SSS protein was inhibited and the bilayer membrane integrity of the WSN-M2/SSS virion was impaired based on transmission electron microscopy (TEM), which in turn abolished the resistance to trypsin treatment. Moreover, the mutant WSN-M2/SSS was dramatically attenuated in mice. In contrast, the AAA mutations did not have a significant effect on the budding of the M2 proteins or the bilayer membrane integrity of the viruses, and the mutant WSN-M2/AAA was still lethal to mice. In addition, although the 91-94 motif is an LIR, knocking out of the LC3 protein of A549 cells did not significantly affect the membrane integrity of the influenza viruses propagated on the LC3KO cells, which suggested that the 91-94 hydrophobic motif affected the viral membrane integrity and budding is independent of the LC3 protein. Overall, the hydrophobicity of the 91-94 motif is crucial for the budding of M2, bilayer membrane integrity, and pathogenicity of the influenza viruses. IMPORTANCE M2 plays a crucial role in the influenza virus life cycle. However, the function of the C-terminal intracellular domain of M2 protein remains largely unclear. In this study, we explored the function of the 91-94 hydrophobic motif of M2 protein. The results showed that the reduction of the hydrophobicity of the 91-94 motif significantly affected the budding ability of the M2 protein and impaired the bilayer membrane integrity of the mutant virus. The mouse study showed that the reduction of the hydrophobicity of the 91-94 motif significantly attenuated the mutant virus. All of the results indicated that the hydrophobicity of the 91-94 motif of the M2 protein plays an important role in budding, membrane integrity, and pathogenicity of influenza virus. Our study offers insights into the mechanism of influenza virus morphogenesis, particularly into the roles of the 91-94 hydrophobic motif of M2 in virion assembly and the pathogenicity of the influenza viruses.

Neurovirulence of avian influenza virus is dependent on the interaction of viral NP protein with host factor FMRP in the murine brain.

Avian influenza viruses (AIVs) are zoonotic viruses that exhibit a range infectivity and severity in the human host. Severe human cases of AIVs infection are often accompanied by neurological symptoms, however, the factors involved in the infection of the central nervous system (CNS) are not well known. In this study, we discovered that avian-like sialic acid (SA)-α2, 3 Gal receptor is highly presented in mammalian (human and mouse) brains. In the generation of a mouse-adapted neurotropic H9N2 AIV (SD16-MA virus) in BALB/c mice, we identified key adaptive mutations in its hemagglutinin (HA) and polymerase basic protein 2 (PB2) genes that conferred viral replication ability in mice brain. The SD16-MA virus showed binding affinity for avian-like SA-α2, 3 Gal receptor, enhanced viral RNP polymerase activity, increased viral protein production and transport that culminated in elevated progeny virus production and severe pathogenicity. We further established that host Fragile X Mental Retardation Protein (FMRP), a highly expressed protein in the brain that physically associated with viral nucleocapsid protein (NP) to facilitate RNP assembly and export, was an essential host factor for the neuronal replication of neurotropic AIVs (H9N2, H5N1 and H10N7 viruses). Our study identified a mechanistic process for AIVs to acquire neurovirulence in mice.IMPORTANCE Infection of the CNS is a serious complication of human cases of AIVs infection. The viral and host factors associated with neurovirulence of AIVs infection are not well understood. We identified and functionally characterized specific changes in the viral HA and PB2 genes of a mouse-adapted neurotropic avian H9N2 virus responsible for enhanced virus replication in neuronal cells and pathogenicity in mice. Importantly, we showed that host FMRP was a crucial host factor that was necessary for neurotropic AIVs (H9N2, H5N1 and H10N7 viruses) to replicate in neuronal cells. Our findings have provided insights into the pathogenesis of neurovirulence of AIV infection.

A study on the infection status and transmission of avian leukosis virus subgroup J in Hy-line brown roosters.

Avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increased pathogenicity and stronger horizontal and vertical transmission ability among different breeds. Although vertical transmission of ALV-J from infected hens through artificial insemination has been inferred from the detection of the p27 antigen in swabs and serum, there has been no further research on the transmission pattern of ALVs in roosters. In the present study, the positive rate of ALV increased significantly in an indigenous flock after detecting the p27 antigen via enzyme-linked immunosorbent assay (ELISA) and virus isolation in DF-1 cells. Viral sequence comparisons and an indirect fluorescent antibody assay showed that these isolates belonged to the ALV-J subgroup but formed a new branch in a phylogenetic tree when compared to domestic and foreign referential strains. The gp85 gene of the ALV-J isolated from hens and albumen was 94.1-99.7% identical to that in roosters, revealing that these isolates were quite likely transmitted to the hens and their offspring through the semen of ALV-infected roosters by artificial insemination from the Hy-line brown roosters. In addition, we defined four ALV-J infection states in plasma and semen of roosters (P+S+, P-S+, P+S-, and P-S-), which suggests that, in order to eradicate ALV in roosters, it is necessary to perform virus isolation using both semen and plasma. Additionally, ALV detection in semen by ELISA produced false-positive and false-negative results when compared to virus isolation in DF-1 cells. Collectively, our results suggested that an incomplete process of eradication of ALV from ALV-positive roosters led to the sporadic presence of ALV-J in laying hens.

An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts.

In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses.IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

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

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

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice.

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.

Acetylation at K108 of the NS1 protein is important for the replication and virulence of influenza virus.

Non-structural protein 1 (NS1) of influenza virus is a multifunctional protein that plays an important role in virus replication and virulence. In this study, an acetylation modification was identified at the K108 residue of the NS1 protein of H1N1 influenza virus. To further explore the function of the K108 acetylation modification of the NS1 protein, a deacetylation-mimic mutation (K108R) and a constant acetylation-mimic mutation (K108Q) were introduced into the NS1 protein in the background of A/WSN/1933 H1N1 (WSN), resulting in two mutant viruses (WSN-NS1-108R and WSN-NS1-108Q). In vitro and mouse studies showed that the deacetylation-mimic mutation K108R in the NS1 protein attenuated the replication and virulence of WSN-NS1-108R, while the constant acetylation-mimic mutant virus WSN-NS1-108Q showed similar replication and pathogenicity as the wild-type WSN virus (WSN-wt). The results indicated that acetylation at K108 of the NS1 protein has an important role in the replication and virulence of influenza virus. To further explore the potential mechanism, the type I interferon (IFN-I) antagonistic activity of the three NS1 proteins (NS1-108Q, NS1-108R, and NS1-wt) was compared in cells, which showed that the K108R mutation significantly attenuated the IFN-ß antagonistic activity of the NS1 protein compared with NS1-wt and NS1-108Q. Both NS1-wt and NS1-108Q inhibited the IFN-ß response activated by RIG-I CARD domain, MAVS, TBK1, and IRF3 more efficiently than the NS1-108R protein in cells. Taken together, the results indicated that acetylation at NS1 K108 is important for the IFN antagonistic activity of the NS1 protein and virulence of the influenza virus.

RNA-seq reveals the critical role of Lon protease in stress response and Brucella virulence.

The Brucella spp encounter stressful environment inside their host cells. The Lon protein is an important protease related to cellular protein degradation and resistance to stress in Brucella. However, the molecular mechanism between Lon protein and stress response was still unknown. In this study, it was found that the lon mutant exhibited obvious growth defect in TSB medium, compared with its parent strain. In addition, our results indicated that Lon protein was involved in resistance to various stress conditions and all the ß-lactam antibiotics tested. Although deletion of this protease did not affect Brucella virulence in macrophage, the mutant strain was significantly attenuated in mice infection model at 1 week post infection, and the expression level of several cytokine genes was significantly changed in vivo. To gain insight into the genetic basis for the distinctive phenotypic properties exhibited by the lon mutant strain, RNA-seq was performed, and the result showed that various genes involved in stress response, quorum sensing and transcriptional regulation were significantly altered in Δlon strain. Overall, these studies have preliminary uncovered the molecular mechanism between Lon protease, stress response and bacterial virulence.

Effects of Cr(VI)-induced calcium-sensing receptor activation on DF-1 cell pyroptosis.

This study aims to investigate the effects of Cr(VI)-induced calcium-sensing receptor (CaSR) activation on DF-1 cell pyroptosis. Previous studies show that Cr(VI) could accumulate in the body of chickens and change Ca levels. Hence, a Ca-related pathway may be an important mechanism participating in some pathological processes. Pyroptosis level, which is meditated by CaSR, increases under Cr(VI) accumulation. In the present study, pyroptosis was determined by flow cytometry to detect SYTOX blue and caspase-1 staining followed by morphological observation. Interleukin (IL)-1ß and IL-18 levels were detected by ELISA, while CaSR protein and [Ca2+]i contents were detected by Western blot and fluorescence microplate spectrophotometry, respectively. The results showed that Cr(VI) causes DF-1 cell pyroptosis in a time- and dose-dependent manner and that this effect is caspase-1 dependent. Further experiments indicated that pyroptosis could be induced by Cr(VI) and is accompanied by up-regulated [Ca2+]i content. CaSR inhibition led to decreases in pyroptosis level. Some mechanisms may be involved in Cr(VI)-triggered CaSR activation and enhance DF-1 cell pyroptosis. Taken together, the results of this study support future investigations on Cr(VI)-induced pyroptosis in DF-1 cells.

Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus.

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant's internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010-2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

Enhanced pathogenicity and neurotropism of mouse-adapted H10N7 influenza virus are mediated by novel PB2 and NA mutations.

The H10 subtype of avian influenza viruses (AIVs) circulates globally in wild birds and poultry, and this subtype has been shown to be increasingly prevalent in China. Among the various H10 viruses, H10N7 AIVs have caused repeated mammal and human infections. To investigate their genetic adaptation in mammals, we generated a mouse-adapted avian H10N7 variant (A/mallard/Beijing/27/2011-MA; BJ27-MA) which exhibited increased virulence in mice compared to wild-type virus and acquired neurotropism. Sequencing showed the absence of the widely recognized mammalian adaptation markers of E627K and D701N in PB2 in the mouse-adapted strain; instead, five amino acid mutations were identified: E158G and M631L in PB2; G218E in haemagglutinin (H3 numbering); and K110E and S453I in neuraminidase (NA). The neurovirulence of the BJ27-MA virus necessitated the combined presence of the PB2 and NA mutations. Mutations M631L and E158G of PB2 and K110E of NA were required to mediate increased virus replication and severity of infection in mice and mammalian cells. PB2-M631L was functionally the most dominant mutation in that it strongly upregulated viral polymerase activity and played a critical role in the enhancement of virus replication and disease severity in mice. K110E mutation in NA, on the other hand, significantly promoted NA enzymatic activity. These results indicate that the novel mutations in PB2 and NA genes are critical for the adaptation of H10N7 AIV in mice, and they could serve as molecular signatures of virus transmission to mammalian hosts, including humans.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

UNLABELLED: Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE: Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity.

Highly Pathogenic Avian Influenza H5N6 Viruses Exhibit Enhanced Affinity for Human Type Sialic Acid Receptor and In-Contact Transmission in Model Ferrets.

UNLABELLED: Since May 2014, highly pathogenic avian influenza H5N6 virus has been reported to cause six severe human infections three of which were fatal. The biological properties of this subtype, in particular its relative pathogenicity and transmissibility in mammals, are not known. We characterized the virus receptor-binding affinity, pathogenicity, and transmissibility in mice and ferrets of four H5N6 isolates derived from waterfowl in China from 2013-2014. All four H5N6 viruses have acquired a binding affinity for human-like SAα2,6Gal-linked receptor to be able to attach to human tracheal epithelial and alveolar cells. The emergent H5N6 viruses, which share high sequence similarity with the human isolate A/Guangzhou/39715/2014 (H5N6), were fully infective and highly transmissible by direct contact in ferrets but showed less-severe pathogenicity than the parental H5N1 virus. The present results highlight the threat of emergent H5N6 viruses to poultry and human health and the need to closely track their continual adaptation in humans. IMPORTANCE: Extended epizootics and panzootics of H5N1 viruses have led to the emergence of the novel 2.3.4.4 clade of H5 virus subtypes, including H5N2, H5N6, and H5N8 reassortants. Avian H5N6 viruses from this clade have caused three fatalities out of six severe human infections in China since the first case in 2014. However, the biological properties of this subtype, especially the pathogenicity and transmission in mammals, are not known. Here, we found that natural avian H5N6 viruses have acquired a high affinity for human-type virus receptor. Compared to the parental clade 2.3.4 H5N1 virus, emergent H5N6 isolates showed less severe pathogenicity in mice and ferrets but acquired efficient in-contact transmission in ferrets. These findings suggest that the threat of avian H5N6 viruses to humans should not be ignored.

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

H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro- or antivirulence role of PA-X in influenza viruses is virus strain-dependent.

Protective effect of Platycodon grandiflorus polysaccharide on apoptosis and mitochondrial damage induced by pseudorabies virus in PK-15 cells.

Previous studies have confirmed that Platycodon grandiflorus polysaccharide (PGPSt) has the effects of regulating immunity and anti-apoptosis, but its effect on mitochondrial damage and apoptosis caused by PRV infection is still unclear. In this research, the effects of PGPSt on the cell viability, mitochondria morphology, mitochondrial membrane potential and apoptosis caused by PRV based on PK-15 cells were respectively examined by CCK-F assay, Mito-Tracker Red CMXRos, JC-1 staining method and Western blot etc. CCK-F test results showed that PGPSt had a protective effect on the decrease of cell viability caused by PRV. The results of morphological observation found that PGPSt can improve mitochondrial morphology damage, mitochondrial swelling and thickening, and cristae fracture. Fluorescence staining test results showed that PGPSt alleviated the decrease of mitochondrial membrane potential and apoptosis in infected cells. The expression of apoptosis-related proteins showed that PGPSt down-regulated the expression of the pro-apoptotic protein Bax and up-regulated the expression of the anti-apoptotic protein Bcl-2 in infected cells. These results indicated that PGPSt protected against PRV-induced PK-15 cell apoptosis by inhibiting mitochondrial damage.

Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chickens, a pet cat, and humans at a backyard farm in Guangxi, China.

During an investigation in October 2018, two people with diarrhoea, mild abdominal pain, and mild arthralgia symptoms in Guangxi, China, were identified as infected by H9N2 avian influenza virus (AIV). Four H9N2 AIVs were isolated from one of two patients, a pet cat, and a dead chicken (two respective isolates from its lung and kidney tissues) bred by the patients at a backyard farm. Epidemiological investigation indicated that the newly bought chicken died first, and clinical syndromes appeared subsequently in the two owners and one cat. Furthermore, the two individuals possessed high H9N2-specific hemagglutination inhibition and microneutralization antibodies. Shared nucleotide sequence identity (99.9% - 100%) for all genes was detected in the four H9N2 isolates, and hemagglutinin (HA) T138A located on the receptor binding domain (RBD), resulted from nucleotide polymorphisms that were exclusively found in the isolate from the female patient. Moreover, HA K137N on the RBD was found in isolates from these three host species. Importantly, these four H9N2 isolates presented an exclusive binding preference for the human-type receptor (α2-6-SA), and could replicate and cause pathological changes in mice. Phylogenetic analyses showed that these four isolates clustered together and belonged to clade C1.2, lineage Y280. In addition, H9N2 viruses of human origin are genetically divergent and interspersed with the widespread poultry-origin H9N2 AIVs. All these results indicate a high risk of H9N2 AIVs in public health, and effective prevention and control measures against H9N2 AIVs should be considered and performed for both animal and human health.

The Mobility of Eurasian Avian-like M2 Is Determined by Residue E79 Which Is Essential for Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Identification of avian polyomavirus and its pathogenicity to SPF chickens.

The research aimed to study an Avian polyomavirus strain that was isolated in Shandong, China. To study the pathogenicity of APV in SPF chickens, and provide references for epidemiological research and disease prevention and control of APV. The genetic characterization of APV strain (termed APV-20) was analyzed and the pathogenicity of APV was investigated from two aspects: different age SPF chickens, and different infection doses. The results revealed that the APV-20 exhibits a nucleotide homology of 99% with the other three APV strains, and the evolution of APV In China was slow. In addition, the APV-20 infection in chickens caused depression, drowsiness, clustering, and fluffy feathers, but no deaths occurred in the infected chickens. The main manifestations of necropsy, and Hematoxylin and Eosin staining (HE) showed that one-day-old SPF chickens were the most susceptible, and there was a positive correlation between viral load and infection dose in the same tissue. This study showed that SPF chickens were susceptible to APV, and an experimental animal model was established. This study can provide a reference for the pathogenic mechanism of immune prevention and control of APV.

Characterization of four novel H5N6 avian influenza viruses with the internal genes from H5N1 and H9N2 viruses and experimental challenge of chickens vaccinated with current commercially available H5 vaccines.

Since 2014, highly pathogenic avian influenza H5N6 viruses have been responsible for outbreaks in poultry. In this study, four H5N6 virus strains were isolated from faecal samples of sick white ducks and dead chickens in Shandong in 2019. These H5N6 viruses were triple-reassortant viruses that have not been previously characterized. Their HA genes were derived from the H5 viruses and were closely related to the vaccine strain Re-11. Their NA genes all fell into the N6-like lineage and the internal gene were derived from H5N1 and H9N2 viruses. They all showed high pathogenicity in mice and caused lethal infection with high rates of transmission in chickens. Moreover, the SPF chickens inoculated with the currently used H5 (Re-11 and Re-12 strains)/H7 (H7-Re-2 strain) trivalent inactivated vaccines in China were completely protected from these four H5N6 viruses. Our study indicated the necessity of continued surveillance for H5 influenza A viruses and the importance of timely update of vaccine strains in poultry industry.

Eurasian Avian-like M1 Plays More Important Role than M2 in Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

Reassortant variant viruses generated between 2009 H1N1 pandemic influenza virus [A(H1N1)pdm09] and endemic swine influenza viruses posed a potential risk to humans. Surprisingly, genetic analysis showed that almost all of these variant viruses contained the M segment from A(H1N1)pdm09, which originated from Eurasian avian-like swine influenza viruses. Studies have shown that the A(H1N1)pdm09 M gene is critical for the transmissibility and pathogenicity of the variant viruses. However, the M gene encodes two proteins, M1 and M2, and which of those plays a more important role in virus pathogenicity remains unknown. In this study, the M1 and M2 genes of A(H1N1)pdm09 were replaced with those of endemic H3N2 swine influenza virus, respectively. The chimeric viruses were rescued and evaluated in vitro and in mice. Both M1 and M2 of H3N2 affected the virus replication in vitro. In mice, the introduction of H3N2 M1 attenuated the chimeric virus, where all the mice survived from the infection, compared with the wild type virus that caused 100 % mortality. However, the chimeric virus containing H3N2 M2 was still virulent to mice, and caused 16.6% mortality, as well as similar body weight loss to the wild type virus infected group. Compared with the wild type virus, the chimeric virus containing H3N2 M1 induced lower levels of inflammatory cytokines and higher levels of anti-inflammatory cytokines, whereas the chimeric virus containing H3N2 M2 induced substantial pro-inflammatory responses, but higher levels of anti-inflammatory cytokines. The study demonstrated that Eurasian avian-like M1 played a more important role than M2 in the pathogenicity of A(H1N1)pdm09 in mice.