Avian Influenza Virus Subtype H5N1 Research Articles

Karaterisasi Virus Avian Influenza Subtipe H5N1 Isolat Lapang Asal Bali Untuk Kandidat Vaksin

A research on the isolation and characterization of the Avian Influenza H5N1 subtype field isolate has been carried out at the BSL-3 Laboratory of PT Sanbio Laboratories, Bogor. The aim of the study was to prepare a candidate for the H5N1 subtype Avian Influenza virus vaccine. Virus isolates were taken from field isolates from Bali. A total of seven field H5N1 AI subtypes from Bali were characterized in Bogor. The isolates were: isolate 3A, isolate 4A, isolate 9C, isolate 10 A, isolate 10 C, isolate P65, isolate P67. The passage of isolates was carried out on 9-day-old embryonic Specific Pathogenic (SPF) chicken eggs by injecting 0.1 mL of SPF isolates/eggs through the allantoic cavity. Each isolate was placed in five SPF eggs and then incubated in an incubator at 37 C and candled every day. Since day 2-4 post inoculation, embryo death has occurred. The eggs are harvested by their allantoic fluid and tested for haemagglutination test(HA/HI). The HI test results were confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using the front primer FPHA232_13 (ATTGGTTAYCATGCAAAYAACTCG) and the back primer BPHA232_597 (GGAAAYATAGGTRGTTGGRTTYTGATAG) The results were five of the seven isolates were positive AI subtype B5 585 - 581 The five isolates of AI subtype H5N1 were subsequently sequenced, the results were all positive for AI virus subtype H5N1 clade 2.3.2. Each field isolate was given the name A / Chicken / Bali3A / GAY / 2019; A / Chicken / Bali9C / GAY / 2019; A / Chicken / BaliA4 / GAY / 2019; A / Chicken / Bali10A / GAY / 2016 and A / Chicken / Bali10C / GAY / 2019. One A / Chicken / Bali 9C / GAY / 2016 isolate was subsequently repeated 7 times until a stable H5N1 subtype AI virus titer was obtained. The results of matching with bioinformatics turned out that A / Chicken / Bali 9C / GAY / 2016 isolates had a kinship of 98.62% with AI subtype H5N1 Banyuwangi, amounting to 98.45% with AI subtype H5N1 Lamongan, amounting to 98.10% with AI-H5N1 Lumajang, 97.58% with AI-H5N1 Kediri, 97.07% with AIH5N1 Blitar, 96.72% with AI-H5N1 Denpasar, 96.72% with AI-H5N1 Buleleng and 96.72% with AI-H5N1 Sukoharjo. The conclusion is one of isolate namely A / Chicken / Bali 9C / GAY / 2019 including AI subtype H5N1 clade 2.3.2, is’t stable at passage on SPF eggs, has a kinship of 96.72% with A / duck / Sukoharjo / BBVW-1428- 9/2012, the virus content is 106.9 ELD50 so it is potential for vaccine candidates.

Evolution of H9N2 avian influenza viruses in Iran, 2017-2019.

Since its first detection in 1998, avian influenza virus (AIV) subtype H9N2 has been enzootic in Iran. To better understand the evolutionary history of H9N2 viruses in Iran, we sequenced 15 currently circulating H9N2 viruses from domestic poultry during 2017-2019 and performed phylogenetic analysis of complete genome sequences. Phylogenetic analyses indicated that the Iranian H9N2 viruses formed multiple well-supported monophyletic groups within the G1-lineage of H9N2 virus. Our analysis of viral population dynamics revealed an increase in genetic diversity until 2007, corresponding to the multiple introductions and diversification of H9N2 viruses into multiple genetic groups (named Iran 1-4 subgroups), followed by a sudden decrease after 2008. Only the Iran 4 subgroup has survived, expanded, and currently circulates in Iran. The H9N2 viruses possessed many molecular markers associated with mammalian adaption in all gene segments, except neuraminidase gene. Considering the presence of mammalian host-specific markers, the public health threat of H9N2 viruses continues. Molecular analysis showed that Iranian H9N2 strains have continued to evolve and recent strains have multiple amino acid changes and addition of potential N-glycosylation on the antigenic sites of haemagglutinin. Continued antigenic and molecular surveillance of H9N2 viruses in poultry and mammals would be required to monitor further increments in viral evolution and their potential threat to public health.

Establishing a Multicolor Flow Cytometry to Characterize Cellular Immune Response in Chickens Following H7N9 Avian Influenza Virus Infection.

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.

Effect of Baicalin on Bacterial Secondary Infection and Inflammation Caused by H9N2 AIV Infection in Chickens.

H9N2 subtype avian influenza virus (H9N2 AIV) is a low pathogenic virus that is widely prevalent all over the world. H9N2 AIV causes immunosuppression in the host and often leads to high rates of mortality due to secondary infection with Escherichia. Due to the drug resistance of bacteria, many antibiotics are not effective in the treatment of secondary bacterial infection. Therefore, the purpose of this study is to find effective nonantibiotic drugs for the treatment of H9N2 AIV infection-induced secondary bacterial infection and inflammation. This study proves, for the first time, that baicalin, a Chinese herbal medicine, can regulate Lactobacillus to replace Escherichia induced by H9N2 AIV, so as to resolve the intestinal flora disorder. In addition, baicalin can effectively prevent intestinal bacterial translocation of SPF chickens' post-H9N2 AIV infection, thus inhibiting secondary bacterial infection. Furthermore, baicalin can effectively treat H9N2 AIV-induced inflammation by inhibiting intestinal structural damage, inhibiting damage to ileal mucus layer construction and tight junctions, improving antioxidant capacity, affecting blood biochemical indexes, and inhibiting the production of inflammatory cytokines. Taken together, these results provide a new theoretical basis for clinical prevention and control of H9N2 AIV infection-induced secondary bacterial infection and inflammation.

H5N1 avian influenza virus without 80-84 amino acid deletion at the NS1 protein hijacks the innate immune system of dendritic cells for an enhanced mammalian pathogenicity.

NS gene is generally considered to be related to the virulence of highly pathogenic avian influenza virus (AIV). In recent years, the strains with five amino acids added to the 80-84 positions of the NS1 protein have become prevalent in H5N1 subtype AIVs isolated from mammals. However, the pathogenicity and mechanism of this pattern in mammals remain unclear. In this study, H5N1 subtype AIVs without 80-84 amino acids of the NS1 protein (rNSΔ5aa ) and a mutant virus (rNS5aa-R ) with no deletion of 80-84 amino acids of the NS1 protein were used to determine the pathogenicity in mice. Our results showed that rNS5aa-R possessed an enhanced pathogenicity compared with rNSΔ5aa in vivo and in vitro, which was accompanied by high expression of IL-6, MX1 and CXCL10 in murine lungs. Furthermore, we found that rNS5aa-R increased the infection ability to dendritic cells (DCs). Besides, rNS5aa-R enhanced the expression of phenotypic markers (CD80, CD86, CD40 and MHCII), activation marker CD69, inflammatory cytokines (IL-6, TNF-α and IL-10) and antagonized interferon (IFN-α) of DCs, in comparison to rNSΔ5aa . Moreover, rNS5aa-R induced DCs to quickly migrate into nearby cervical lymph nodes by highly upregulating CCR7, and CD86 showed a high expression on the migrated DCs. We also found that rNS5aa-R -infected DCs significantly promoted the allogeneic CD4+ T-cell proliferation. These findings suggested that rNS5aa-R strongly induced the innate immune response compared with the rNSΔ5aa , which is conducive to activate a wide immune response, resulting in a strong cytokine storm and causing an enhanced pathogenicity of H5N1 subtype AIVs in mammals.

Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49–68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl.

Evaluation of the Viricidal Efficacy of Four Chemical Disinfectants against Emerged H7N9 Avian Influenza Virus in China

Background: Establishment of scientific disinfection measures in domestic poultry farms and live-poultry markets (LPMs) are critical to prevent the spread of H7N9 subtype avian influenza that outbreaks in poultry and infection in humans. The current study was aimed to evaluate the efficacy of four common disinfectants to inactivate H7N9 subtype avian influenza virus (AIV). Methods: According to the disinfection technical specification, the average inactivation logarithmic values of four disinfectants against the epidemic H7N9 subtype AIV, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder, and peroxyacetic acid solution, were systematically evaluated. Result: Our data showed that incubation of 0.005% (w/v) deciquam solution for 30 min contact time, or 0.05% (v/v) glutaral and benzalkonium bromide solution for 10 min, or 0.24 g/L sodium dichloroisocyanurate powder for 30 min, or 0.05% (v/v) peroxyacetic acid solution for 10 min, can completely inactivate H7N9 subtype AIV. These results suggested that glutaral and benzalkonium bromide solution as well as peroxyacetic acid solution at recommended concentrations can be effectively used to cut off the spread of H7N9 subtype AIV in poultry farms and LPMs, however, deciquam solution and sodium dichloroisocyanurate powder need to increase their working concentration.

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013-2017.

H9N2 subtype avian influenza virus (AIV) is an influenza A virus that is widely spread throughout Asia, where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens. To better understand the epidemicity and genetics of H9 subtype AIVs, we conducted active surveillance in live poultry markets (LPMs) in Hubei Province from 2013 to 2017.A total of 4798 samples were collected from apparent healthy poultry and environment. Real-time RT-PCR revealed that the positivity rate of influenza A was 26.6% (1275/4798), of which the H9 subtype accounted for 50.3% (641/1275) of the positive samples. Of the 132 H9N2 viral strains isolated, 48 representative strains were subjected to evolutionary analysis and genotyping. Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%-100% nucleotide homology, clustered with genotype 57, and had high homology with human H9N2 viruses isolated from 2013 to 2017 in China. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to change over time. Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein, and all isolates had I155T and Q226L mutations. Moreover, 44 strains had A558V mutations in the PB2 protein and four had E627V mutations, along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017. These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes, indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.

Taishan Pinus massoniana pollen polysaccharide inhibits H9N2 subtype influenza virus infection both in vitro and in vivo

The H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes that can be found throughout most countries. Currently, due to the neglect of low pathogenic avian influenza virus (LPAIV) and monotonous control technique, an expanding H9N2 virus epizootic have been arisen and causes great economic losses in the poultry industry. Therefore, novel anti-influenza drugs are necessary for the prevention and control of H9N2 AIV. Our previous studies have found that Taishan Pinus massoniana pollen polysaccharides (TPPPS) have antiviral effects, but whether they can inhibit the H9N2 AIV remains unclear. Here, we further investigated the effects of TPPPS on the H9N2 virus and its underlying mechanisms of action. We found that TPPPS significantly inhibited the replication of the H9N2 virus in a dose-dependent manner, especially during the period of virus adsorption in vitro. Transmission electron microscopy demonstrated that TPPPS reduce infection by interfering with virus entry into host cells rather than by interacting with the H9N2 virus particles. A fluorescence quantitative PCR (qPCR) assay and an animal experiment were performed to evaluate the anti-viral effect of TPPPS in vivo. As expected, the lungs of chickens treated with TPPPS had fewer lesions and lower virus contents compared with the PBS group. In addition, pre-treatment with TPPPS clearly enhanced host disease resistance and delayed infection by the H9N2 virus. Taken together, our results reveal that TPPPS suppress H9N2 virus replication both in vitro and in vivo and therefore shows promising as an anti-AIV agent.

Development of ssDNA Aptamers for Diagnosis and Inhibition of the Highly Pathogenic Avian Influenza Virus Subtype H5N1

Avian influenza (AI) has severely affected the poultry industry worldwide and has caused the deaths of millions of birds. Highly pathogenic avian influenza virus is characterized by high mortality and the ability to transmit from birds to humans. Early diagnosis is difficult because of the variation in pathogenicity and the genetic diversity between virus subtypes. Therefore, development of a sensitive and accurate diagnostic system is an urgent priority. We developed ssDNA aptamer probes to detect AI viruses. Through seven rounds of SELEX to search for a probe specific to the highly pathogenic AI virus subtype H5N1, we identified 16 binding aptamers and selected two with the highest binding frequency. These two aptamers had strong binding affinities and low detection limits. We found that they could bind more specifically to H5N1, as compared to other subtypes. Furthermore, these aptamers inhibited hemagglutination, which is caused by the virus surface protein hemagglutinin. Our results indicate that our screened aptamers are effective molecular probes for diagnosing H5N1 and can be used as therapeutic agents to inhibit viral surface proteins. Sensitive diagnosis and suppression of avian influenza will help maintain a stable and healthy livestock industry, as well as protect human health.

Development of a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for detection of H7N9 subtype avian influenza virus.

To establish a rapid detection method for H7N9 avian influenza virus (AIV), monoclonal antibodies (mAbs) against hemagglutinin (HA) of H7N9 were developed to establish an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). AC-ELISA achieved high specificity and sensitivity, with a detection limit of 3.9 ng/mL for H7N9 HA protein (A/Zhejiang/DTID-ZJU01/2013), and 2-2 HA unit/100 μL for live H7N9 AIV. The inter- and intra-assay coefficient of variation was less than 10%. Compared with conventional virus isolation detection, the sensitivity and specificity were 94.96% and 88.24%, respectively. AC-ELISA proved to be a rapid and practical technique for the detection of H7N9 AIV.

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.

Genetic incompatibilities and reduced transmission in chickens may limit the evolution of reassortants between H9N2 and panzootic H5N8 clade 2.3.4.4 avian influenza virus showing high virulence for mammals

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa, and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk, we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA, and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA, and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals.

Contributions of HA1 and HA2 Subunits of Highly Pathogenic Avian Influenza Virus in Induction of Neutralizing Antibodies and Protection in Chickens.

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes a devastating disease in poultry. Vaccination is an effective method of controlling avian influenza virus (AIV) infection in poultry. The hemagglutinin (HA) protein is the major determinant recognized by the immune system of the host. Cleavage of the HA precursor HA0 into HA1 and HA2 subunits is required for infectivity of the AIV. We evaluated the individual contributions of HA1 and HA2 subunits to the induction of HPAIV serum neutralizing antibodies and protective immunity in chickens. Using reverse genetics, recombinant Newcastle disease viruses (rNDVs) were generated, each expressing HA1, HA2, or HA protein of H5N1 HPAIV. Chickens were immunized with rNDVs expressing HA1, HA2, or HA. Immunization with HA induced high titers of serum neutralizing antibodies and prevented death following challenge. Immunization with HA1 or HA2 alone neither induced serum neutralizing antibodies nor prevented death following challenge. Our results suggest that interaction of HA1 and HA2 subunits is necessary for the display of epitopes on HA protein involved in the induction of neutralizing antibodies and protection. These epitopes are lost when the two subunits are separated. Therefore, vaccination with either a HA1 or HA2 subunit may not provide protection against HPAIV.

Association of wild bird densities around poultry farms with the risk of highly pathogenic avian influenza virus subtype H5N8 outbreaks in the Netherlands, 2016.

Highly pathogenic (HP) avian influenza viruses (AIV) can spread globally through migratory birds and cause massive outbreaks in commercial poultry. AIV outbreaks have been associated with proximity to waterbodies, presence of waterfowl or wild bird cases near poultry farms. In this study, we compared densities of selected HPAI high‐risk wild bird species around 7 locations (H farms) infected with HPAIV H5N8 in the Netherlands in 2016–2017 to densities around 21 non‐infected reference farms. Nine reference farms were in low‐lying water‐rich areas (R‐W) and 12 in higher non‐water‐rich areas (R‐NW). Average monthly numbers/km2 of Eurasian wigeons, tufted ducks, Anatidae (ducks, geese and swans) and Laridae (gulls) were calculated between September and April in rings of 0–1, 1–3, 3–6 and 6–10 km around the farms. Linear mixed model analyses showed generally higher bird densities for H and R‐W compared to R‐NW farms between October and March. This was most striking for Eurasian wigeons, with in peak month December 105 (95% CI:17–642) and 40 (7–214) times higher densities around H and R‐W farms, respectively, compared to R‐NW farms. Increased densities around H farms for Eurasian wigeons and Anatidae were more pronounced for distances up to 10 km compared to 0–1 km that mostly consists of the farm yard, which is an unattractive habitat for waterfowl. This distance effect was not observed in gulls, nor in tufted ducks that live on large open waterbodies which are unlikely to be within 0–1 km of farms. This study provides insights into spatio‐temporal density dynamics of HPAI high‐risk birds around farms and their associations with poultry outbreaks. The outcomes indicate that knowledge of environmental and ecological drivers for wild bird presence and abundance may facilitate identification of priority areas for surveillance and biosecurity measures and decisions on establishments of poultry farms to reduce risk of HPAI outbreaks.

Genetic variability of avian influenza virus subtype H5N8 in Egypt in 2017 and 2018.

Since the incursion of avian influenza virus subtype H5N8 in Egypt in late 2016, it has spread rapidly, causing severe losses in poultry production. Multiple introductions of different reassorted strains were observed in 2017. In this study, a genetic characterization of the HA gene was carried out with 31 isolates selected from different governorates and sectors. Fifteen isolates were selected for NA gene sequence analysis. The HA and NA genes were divided into two subgroups (I and II) with positive selection pressure identified at positions 174 and 29, respectively. The HA gene contained two novel mutations in the antigenic sites, A and E. The HA nucleotide sequence identity ranged from 77 to 90% with different vaccine seeds. Full-genome sequence analysis was carried out for eight viruses, representing different governorates and sectors, to identify the predominant reassorted strain in Egypt. All viruses were similar to a reassorted strain of clade 2.3.4.4b that has been identified in Germany, among other countries. Analysis of these viruses revealed mutations specific to Egyptian strains and not the original virus characterized in 2017 (A/duck/Egypt/F446/2017), with a novel antiviral resistance marker, V27A, indicating resistance to amantadine in the M2 protein of two strains. The results indicate increased variability of circulating H5N8 viruses compared to earlier viruses sequenced in 2016 and 2017. The predominant reassorted virus circulating in 2017 and 2018 originated from an early 2017 strain. It is important to continue this surveillance of avian influenza viruses to monitor the evolution of circulating viruses.

A Well-Defined H9N2 Avian Influenza Virus Genotype with High Adaption in Mammals was Prevalent in Chinese Poultry Between 2016 to 2019.

H9N2 subtype avian influenza virus (AIV) is widely prevalent in poultry, and the virus is becoming adaptive to mammals, which poses pandemic importance. Here, BALB/c mice were employed as a model to evaluate the adaption in mammals of 21 field H9N2 viruses isolated from avian species between 2016 to 2019 in China. The replication capacity of the viruses was evaluated in the lungs of mice. The pathogenicity of the viruses were compared by weight loss and lung lesions from infected mice. The whole genomic sequences of the viruses were further characterized to define the associated phenotypes of the H9N2 viruses in vitro and in vivo. The results showed that most viruses could replicate well and cause lesions in the mouse lungs. The propagation capacity in MDCK cells and damage to respiratory tissues of the infected mice corresponded to relative viral titers in the mouse lungs. Further genome analysis showed that all of the H9N2 viruses belonged to the same genotype, G57, and contained a couple of amino acid substitutions or deletions that have been demonstrated as avian-human markers. Additionally, nine amino acids residues in seven viral proteins were found to be correlated with the replication phenotypes of the H9N2 viruses in mammals. The study demonstrated that a well-defined H9N2 AIV genotype with high adaption in mammals was prevalent in China in recent years. Further investigations on the role of the identified residues and continuous surveillance of newly identified mutations associated with host adaption should be strengthened to prevent any devastating human influenza pandemics.

Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge.

Newcastle disease virus (NDV) and H9N2 subtype Avian influenza virus (AIV) are two notorious avian respiratory pathogens that cause great losses in the poultry industry. Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses, while an attenuated live vaccine bears the risk of mutation. Dendritic cell (DC) targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens. In this study, DC-binding peptide (DCpep)-decorated chimeric virus-like particles (cVLPs), containing NDV haemagglutinin-neuraminidase (HN) and AIV haemagglutinin (HA), were developed as a DC-targeting mucosal vaccine candidate. DCpep-decorated cVLPs activated DCs in vitro, and induced potent immune stimulation in chickens, with enhanced secretory immunoglobulin A (sIgA) secretion and splenic T cell differentiation. 40μg cVLPs can provide full protection against the challenge with homologous, heterologous NDV strains, and AIV H9N2. In addition, DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly, as indicated by robust sIgA secretion and a reduced virus shedding period. Taken together, this chimeric VLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

Characterization and Phylogenetic Analysis of the Hemagglutinin Gene in H9 Influenza Viruses from Chickens in Morocco from 2017 to 2019.

Since the early 2000s, the avian influenza virus (AIV) subtype H9N2 has been widely circulating in North African countries, including Libya, Tunisia, and Egypt. For unknown reasons, H9N2 was not detected in Moroccan farms until the end of 2016, and at present, it is endemic in poultry. This study was carried out to evaluate the evolution of H9 AIVs in Morocco from 2017 to 2019. In this study, 16 H9 viruses collected from 2017 to 2019 in Morocco were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Moroccan H9 viruses were closely related to viruses isolated from African and Middle Eastern countries, and the hemagglutinin gene resembled those of viruses of G1-like lineage, and multiple mammalian host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Our findings suggest that attention be given to the control of H9N2 influenza viruses in chickens and continuous monitoring of the prevalence and pathogenic potential of AIV H9N2 in the poultry population in Morocco.

Pengaruh polisakarida dalam panax ginseng untuk vaksinasi flu burung (H5N1)

Avian Influenza disease is caused by an avian influenza virus infection that became one of the biggest causes of death in the world. The subtype of the virus present in Indonesia is the H5N1 virus. According to the World Health Organization (WHO), in 2012, the outbreak of the H5N1 subtype avian influenza virus was first reported in 1996, Guangdong Province, South China, and spread to Indonesia since 2003. The number of death from avian influenza outbreak (H5N1) in Indonesia was recorded to be the highest in the world, with the number of death was 151 of 183 people who positively infected with the virus. The H5N1 virus can enter and infect humans when the body’s immune system of the person is low. Panax ginseng belongs to the Araliaceae family, which contains several components, such as ginsenosides, polyacetylenes, polyphenols, and polysaccharides. The Polysaccharides in Panax ginseng have an immunomodulatory function that can increase cytokine production and activate macrophages as an effort to improve and increase the body’s immune system. Therefore, polysaccharides in Panax ginseng affect avian influenza vaccination because it enhances the body’s immune response.