Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.

Inhibitory effects of aprotinin on influenza A and B viruses in vitro and in vivo.

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza.

In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2.

Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation "storms" in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.

Antiviral activity of furanocoumarins isolated from Angelica dahurica against influenza a viruses H1N1 and H9N2.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Umbelliferae family) is an herbaceous, perennial plant native to northern and eastern Asia. The root of A. dahurica has traditionally been used under the name "Bai Zhi" as a medicinal plant for colds, dizziness, ulcers, and rheumatism. Moreover, it is also an important ingredient of various prescriptions, such as Gumiganghwal-Tang, for the common cold and influenza. AIM OF THE STUDY: Even though various biological activities of the root of A. dahurica have been reported along with its chemical components, the detailed mechanism of how it exerts anti-influenza activity at the compound level has not been studied. Therefore, we investigated the anti-influenza properties of furanocoumarins purified by bioactivity-guided isolation. MATERIALS AND METHODS: Bioactivity-guided isolation from a 70% EtOH extract of the root of A. dahurica was performed to produce four active furanocoumarins. The inhibition of cytopathic effects (CPEs) was evaluated to ascertain the antiviral activity of these compounds against influenza A (H1N1 and H9N2) viruses. The most potent compound was subjected to detailed mechanistic studies such as the inhibition of viral protein synthesis, CPE inhibition in different phases of the viral replication cycle, neuraminidase (NA) inhibition, antiapoptotic activity using flow cytometry, and immunofluorescence. RESULTS: The bioactivity-guided isolation produced four active furanocoumarins, isoimperatorin (1), oxypeucedanin (2), oxypeucedanin hydrate (3) and imperatorin (4) from the n-BuOH fraction. Among them, compound 2 (followed by compounds 1, 4 and 3) showed a significant CPE inhibition effect, which was stronger than that of the positive control ribavirin, against both H1N1 and H9N2 with an EC50 (µM) of 5.98 ± 0.71 and 4.52 ± 0.39, respectively. Compound 2 inhibited the synthesis of NA and nucleoprotein (NP) in a dose-dependent manner. In the time course assays, the cytopathic effects of influenza A-infected MDCK cells were reduced by 80-90% when treated with compound 2 for 1 and 2 h after infection and declined drastically 3 h after infection. The level of viral NA and NP production was markedly reduced to less than 20% for both proteins in compound 2 (20 µM)-treated cells compared to untreated cells at 2 h after infection. In the molecular docking analysis, compound 2 showed a stronger binding affinity for the C-terminus of polymerase acidic protein (PAC; -36.28 kcal/mol) than the other two polymerase subunits. Compound 2 also exerted an antiapoptotic effect on virus infected cells and significantly inhibited the mRNA expression of caspase-3 and Bax. CONCLUSION: Our results suggest that compound 2 might exert anti-influenza A activity via the inhibition of the early phase of the viral replication cycle, not direct neutralization of surface proteins, such as hemagglutinin and NA, and abnormal apoptosis induced by virus infection. Taken together, these findings suggest that furanocoumarins predominant in A. dahurica play a pivotal role in its antiviral activity. These findings can also explain the reasons for the ethnopharmacological uses of this plant as an important ingredient in many antiviral prescriptions in traditional Chinese medicine (TCM).

Viral interference between H9N2-low pathogenic avian influenza virus and avian infectious bronchitis virus vaccine strain H120 in vivo.

The interaction between a low pathogenic avian influenza virus (A/CK/TUN/145/2012), a H9N2 Tunisian isolate, and a vaccine strain (H120) of avian infectious bronchitis, administered simultaneously or sequentially three days apart to chicks during 20 days, was evaluated using ELISA antibody levels, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses and histopathology examination. First, the in vivo replication interference of avian influenza virus (AIV) and infectious bronchitis virus (IBV) was evaluated using qRT-PCR to detect accurately either AIV or IBV genomes or viral copy numbers during dual infections. Second, we have determined the amount of specific antibodies in sera of chick's infected with AIV alone, IBV alone, mixed AIV + IBV, IBV then AIV or AIV IBV 3 days later using an ELISA test. Finally, histopathological analyses of internal organs from inoculated chicks were realized. Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. According to our results, vaccine application was safe and do not interfere with AIV H9N2 infection, and does not enhance such infection. In conclusion, co-infection of chicks with AIV and IBV, simultaneously or sequentially, affected the clinical signs, the virus replication dynamics as well as the internal organ integrity. The results proposed that infection with heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated.

Viral interference between low pathogenic avian influenza H9N2 and avian infectious bronchitis viruses in vitro and in ovo.

BACKGROUND: Low pathogenic avian influenza (LPAI) H9N2 and infectious bronchitis virus (IBV) are important pathogens of poultry, causing important economic losses for the sector. Replication interference between these two viruses was described using cell cultures (CC) and embryonated chicken eggs (ECE). Chicken embryo lung (CEL) and ECE were simultaneously or sequentially infected with IBV vaccine strain (H120) and LPAIV-H9N2 (A/Ck/TUN/145/2012) to evaluate viral interactionsin vitro and in ovo, respectively. Real-time RT-PCR was developed to specifically quantify both AIV and IBV genomes as well as viral gene copy numbers during mixed infections. The amount of IL-1 beta, in supernatants of co-infected cell cultures, was determined using an ELISA assay. RESULTS: Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. CONCLUSION: It appears that either AIV or IBV has a negative impact on the other virus growth when they are inoculated simultaneously or sequentially. The ELISA results showed that higher level of secreted IL-1beta varies, depending on the viral interference conditions between both viruses, during mixed infections.

Intraspecies and interspecies transmission of mink H9N2 influenza virus.

H9N2 influenza A virus (IAV) causes low pathogenic respiratory disease and infects a wide range of hosts. In this study, six IAVs were isolated from mink and identified as H9N2 IAV. Sequence analysis revealed that the six isolates continued to evolve, and their PB2 genes shared high nucleotide sequence identity with H7N9 IAV. The six isolates contained an amino acid motif PSRSSR↓GL at the hemagglutinin cleavage site, which is a characteristic of low pathogenic influenza viruses. A serosurvey demonstrated that H9N2 IAV had spread widely in mink and was prevalent in foxes and raccoon dogs. Transmission experiments showed that close contact between H9N2-infected mink and naive mink, foxes and raccoon dogs resulted in spread of the virus to the contact animals. Furthermore, H9N2 challenge experiments in foxes and raccoon dogs showed that H9N2 IAV could infect these hosts. Virological and epidemiological surveillance of H9N2 IAV should be strengthened for the fur animal industry.

Avian influenza H9N2 virus isolated from air samples in LPMs in Jiangxi, China.

BACKGROUND: Recently, avian influenza virus has caused repeated worldwide outbreaks in humans. Live Poultry Markets (LPMs) play an important role in the circulation and reassortment of novel Avian Influenza Virus (AIVs). Aerosol transmission is one of the most important pathways for influenza virus to spread among poultry, from poultry to mammals, and among mammals. METHODS: In this study, air samples were collected from LPMs in Nanchang city between April 2014 and March 2015 to investigate possible aerosol transmission of AIVs. Air samples were detected for Flu A by Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR). If samples were positive for Flu A, they were inoculated into 9- to 10-day-old specific-pathogen-free embryonated eggs. If the result was positive, the whole genome of the virus was sequenced by MiSeq. Phylogenetic trees of all 8 segments were constructed using MEGA 6.05 software. RESULTS: To investigate the possible aerosol transmission of AIVs, 807 air samples were collected from LPMs in Nanchang city between April 2014 and March 2015. Based on RRT-PCR results, 275 samples (34.1%) were Flu A positive, and one virus was successfully isolated with embryonated eggs. The virus shared high nucleotide homology with H9N2 AIVs from South China. CONCLUSIONS: Our study provides further evidence that the air in LPMs can be contaminated by influenza viruses and their nucleic acids, and this should be considered when choosing and evaluating disinfection strategies in LPMs, such as regular air disinfection. Aerosolized viruses such as the H9N2 virus detected in this study can increase the risk of human infection when people are exposed in LPMs.

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent.

Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice.

BACKGROUND: H9N2 influenza viruses circulate globally and are considered to have pandemic potential. The hyper-inflammatory response elicited by these viruses is thought to contribute to disease severity. Calcitriol plays an important role in modulating the immune response to viral infections. However, its unknown whether calcitriol can attenuate the inflammatory response elicited by H9N2 influenza virus infection. METHODS: Human lung A549 epithelial cells were treated with calcitriol (100 nM) and then infected with an H9N2 influenza virus, or infected and then treated with calcitriol (30 nM). Culture supernatants were collected every 24 h post infection and the viral growth kinetics and inflammatory response were evaluated. Calcitriol (5 mg/kg) was administered daily by intraperitoneal injection to BABL/c mice for 15 days following H9N2 influenza virus infection. Mice were monitored for clinical signs of disease, lung pathology and inflammatory responses. RESULTS: Calcitriol treatment prior to and post infection with H9N2 influenza significantly decreased expression of the influenza M gene, IL-6, and IFN-ß in A549 cells, but did not affect virus replication. In vivo, we found that calcitriol treatment significantly downregulated pulmonary inflammation in mice 2 days post-infection, but increased the inflammatory response 4 to 6 days post-infection. In contrast, the antiviral cytokine IFN-ß was significantly higher in calcitriol-treated mice than in the untreated infected mice at 2 days post-infection, but lower than in untreated infected mice on days 4 and 8 post-infection. The elevated levels of pro-inflammatory cytokines and the decreased levels of antiviral cytokine are consistent with the period of maximum body weight loss and the lung damage in calcitriol-treated mice. CONCLUSIONS: These results suggest that calcitriol treatment might have a negative impact on the innate immune response elicited by H9N2 infection in mice, especially at the later stage of influenza virus infection. This study will provide some novel insights into the use of calcitriol to modulate the inflammatory response elicited by influenza virus infection in humans.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE: Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

Differential replication properties among H9N2 avian influenza viruses of Eurasian origin.

Avian influenza H9N2 viruses have become panzootic in Eurasia causing respiratory manifestations, great economic losses and occasionally being transmitted to humans. To evaluate the replication properties and compare the different virus quantification methods, four Eurasian H9N2 viruses from different geographical origins were propagated in embryonated chicken egg (ECE) and Madin-Darby canine kidney epithelial cell systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), Hemagglutination (HA) test and quantitative real time RT-PCR (qRT-PCR). The cellular morphology was analyzed using immunofluorescence (IF) and cellular ELISA was used to screen the sensitivity of the viruses to amantadine. The Eurasian wild type-H9N2 virus produced lower titers compared to the three G1-H9N2 viruses at respective time points. Detectable titers were observed earliest at 16 h post inoculation (hpi), significant morphological changes on cells were first observed at 32 hpi. Few nucleotide and amino acid substitutions were noticed in the HA, NA and NS gene sequences but none of them are related to the known conserved region that can alter pathogenesis or virulence following a single passage in cell culture. All studied H9N2 viruses were sensitive to amantadine. The G1-H9N2 viruses have higher replication capabilities compared to the European wild bird-H9N2 probably due to their specific genetic constitutions which is prerequisite for a successful vaccine candidate. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period.

Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adoption in mice.

The worldwide circulation of H9N2 avian influenza virus in poultry, the greater than 2.3 % positive rate for anti-H9 antibodies in poultry-exposed workers, and several reports of human infection indicate that H9N2 virus is a potential threat to human health. Here, we found three mutations that conferred high virulence to H9N2 virus in mice after four passages. The PB2-E627K substitution rapidly appeared at the second passage and played a decisive role in virulence. Polymerase complexes possessing PB2-E627K displayed 16.1-fold higher viral polymerase activity when compared to the wild-type virus, which may account for enhanced virulence of this virus. The other two substitutions (HA-N313D and HA-N496S) enhanced binding to both α2,3-linked and α2,6-linked sialic acid receptors; however, the HA-N313D and N496S substitutions alone decreased the virulence of mouse-adapted virus. Furthermore, this mouse-adapted virus was still not transmissible among guinea pigs by direct contact (0/3 pairs). Our findings show that adaption in mice enhanced the viral polymerase activity and receptor-binding ability, which resulted in a virulent phenotype in mice but not a transmissible phenotype in guinea pigs, indicating that host factors play an important role in adaptive evolution of influenza in new hosts.

A Vero-cell-adapted vaccine donor strain of influenza A virus generated by serial passages.

A cell culture-based vaccine production system is preferred for the large-scale production of influenza vaccines and has advantages for generating vaccines against highly pathogenic influenza A viruses. Vero cells have been widely used in human vaccine manufacturing, and the safety of these cells has been well demonstrated. However, the most commonly used influenza-vaccine donor virus, A/Puerto Rico/8/1934 (PR8) virus, does not grow efficiently in Vero cells. Therefore, we adapted the PR8 virus to Vero cells by continuous passaging, and a high-growth strain was obtained after 20 passages. Sequence analysis and virological assays of the adapted strain revealed that mutations in four viral internal genes (NP, PB1, PA and NS1) were sufficient for adaptation. The recombinant virus harboring these mutations (PR8-4mut) displayed accelerated viral transport into the nucleus and increased RNP activity. Importantly, the PR8-4mut could serve as a backbone donor virus to support the growth of the H7N1, H9N2 and H5N1 avian viruses and the H1N1 and H3N2 human viruses in Vero cells without changing its pathogenicity in either chicken embryos or mice. Thus, our work describes the generation of a Vero-adapted, high-yield PR8-4mut virus that may serve as a promising candidate for an influenza-vaccine donor virus.

Expression profile and histological distribution of IFITM1 and IFITM3 during H9N2 avian influenza virus infection in BALB/c mice.

The H9N2 avian influenza virus is a pandemic threat which has repeatedly caused infection in humans and shows enhanced replication and transmission in mice. Previous reports showed that host factors, the interferon-inducible transmembrane (IFITM) protein, can block the replication of pathogens and affect their pathogenesis. BALB/c mice are routine laboratory animals used in influenza virus research, but the effects of H9N2 influenza virus on tissue distribution and expression pattern of IFITM in these mice are unknown. Here, we investigated the expression patterns and tissue distribution of IFITM1 and IFITM3 in BALB/c mice by infection with H9N2 AIV strains with only a PB2 residue 627 difference. The results showed that the expression patterns of ITITM1 and IFITM3 differ in various tissues of BALB/c mice at different time points after infection. IFITM1 and IFITM3 showed cell- and tissue-specific distribution in the lung, heart, liver, spleen, kidney and brain. Notably, the epithelial and neuronal cells all expressed the proteins of IFITM1 and IFITM3. Our results provide the first look at differences in IFITM1 and IFITM3 expression patterns in BALB/c mice infected by H9N2 influenza viruses. This will enhance research on the interaction between AIV and host and further will elucidate the pathogenesis of influenza virus infection based on the interferon-inducible transmembrane (IFITM) protein.

Precision-cut intestinal slices as a culture system to analyze the infection of differentiated intestinal epithelial cells by avian influenza viruses.

Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.

Expression pattern of NLRP3 and its related cytokines in the lung and brain of avian influenza virus H9N2 infected BALB/c mice.

BACKGROUND: H9N2 avian influenza virus (AIV) becomes the focus for its ability of transmission to mammals and as a donor to provide internal genes to form the new epidemic lethal influenza viruses. Residue 627 in PB2 has been proven the virulence factor of H9N2 avian influenza virus in mice, but the detailed data for inflammation difference between H9N2 virus strains with site 627 mutation is still unclear. The inflammasome NLRP3 is recently reported as the cellular machinery responsible for activation of inflammatory processes and plays an important role during the development of inflammation caused by influenza virus infection. METHODS: In this study, we investigated the expression pattern of NLRP3 and its related cytokines of IL-1ß and TNF-α in BALB/c mice infected by H9N2 AIV strains with only a site 627 difference at both mRNA and protein levels at different time points. RESULTS: The results showed that the expression level of NLRP3, IL-1ß and TNF-α changed in the lung and brain of BALB/c mice after infection by VK627 and rVK627E. The immunohistological results showed that the positive cells of NLRP3, IL-1ß and TNF-α altered the positive levels of original cells in tissues and infiltrated inflammatory cells which caused by H9N2 infection. CONCLUSIONS: Our results provided the basic data at differences in expression pattern of NLRP3 and its related cytokines in BALB/c mice infected by H9N2 influenza viruses with only a site 627 difference. This implied that NLRP3 inflammasome plays a role in host response to influenza virus infection and determines the outcome of clinical manifestation and pathological injury. This will explain the variable of pathological presentation in tissues and enhance research on inflammation process of the AIV H9N2 infection.

Passaging impact of H9N2 avian influenza virus in hamsters on its pathogenicity and genetic variability.

INTRODUCTION: Avian influenza viruses of the H9N2 subtype have been reported to cause human infections. This study demonstrates the impact of nasal viral passaging of avian H9N2 in hamsters on its cross species-pathogenic adaptability and variability of amino acid sequences of the hemagglutinin (HA) and neuraminidase (NA) stalk. METHODOLOGY: Three intranasal passagings of avian H9N2 in hamsters P1, P2, and P3 were accomplished. Morbidity signs and lesions were observed three days post viral inoculation. The HA test was used for presumptive detection of H9N2 virus in the trachea and lungs of the hamsters challenged with the differently passaged viruses. Different primers were used for PCR amplification of the HA1 and NA stalk regions of the differently passaged H9N2 viruses, followed by sequence alignment. RESULTS: The morbidity signs indicated low pathogenicity of the differently passaged H9N2 viruses in hamsters. The frequency of gross and microscopic lesions in the tracheas and lungs were insignificantly different among hamsters challenged with the differently passaged H9N2 viruses (p > 0.05). There was 100% similarity in the amino acid sequence of the HA gene of most passaged viruses. The amino acid sequence of the neuraminidase in the third passaged H9N2 virus recovered from lungs showed a R46P mutation that might have a role in the pathogenic adaptability of P3 viruses in hamsters' lungs. CONCLUSIONS: The apparent adaptation of avian H9N2 virus to mammalian cells is in agreement with the World Health Organization's alertness for a possible public health threat by this adaptable virus.

H9N2 avian influenza infection altered expression pattern of sphiogosine-1-phosphate receptor 1 in BALB/c mice.

BACKGROUND: The pathological damage inflicted by virulent AIV strains is often caused by inducing a positive feedback loop of cytokines in immune cells that cause excessive inflammation. Previous research has shown that a G protein-coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1), plays a crucial role in the development of excessive inflammation in influenza virus infection (Cell 146:861-862, 2011; Cell 146:980-991, 2011). BALB/c mice are common laboratory animals used in research of influenza virus; however the effects of influenza infections on expression patterns of S1PR1 in mice are unknown. METHODS: We investigated the expression patterns of S1PR1 in normal BALB/c mice and those infected by two distinct H9N2 AIV strains, one (A/chicken/Guangdong/V/2008,V) highly pathogenic, and the other (A/chicken/Guangdong/Ts/2004,Ts), non-pathogenic in mice, using quantitative PCR and immunohistochemistry (IHC) to detect S1PR1 mRNA and protein, respectively. RESULTS: S1PR1 mRNA was ubiquitously expressed in all the tissues examined, and significant differences were seen in mRNA expression between infected Ts, V and control mice in detected tissues, heart, liver, spleen, kidney and brain. S1PR1 protein was expressed in the cytoplasm and also demonstrated quantitative changes in expression in the various tissues between mice infected with the two strains of AIV. CONCLUSIONS: Our results provided the first look at differences in S1PR1 expression patterns in BALB/c mice infected by non-pathogenic and highly pathogenic H9N2 influenza viruses. This information will not only be helpful in designing experiments to better understand the role of S1PR1 in virus-host interactions but also in developing novel anti-influenza agents to minimize the mortality and morbidity associated with highly virulent strains in avian and human populations.