Characterizing the Core Internal Gene Pool of H9N2 Responsible for Continuous Reassortment With Other Influenza A Viruses

Reassortment among avian influenza viruses is the main source of novel avian influenza virus subtypes. Studies have shown that the H9N2 virus often donates internal segments to generate novel reassortant avian influenza viruses, acting as a reassortment template. However, the characteristics of the internal pattern of reassortment remain unclear. In this article, we first defined the core gene pool of the internal segments of the H9N2 virus that provide templates for reassortment. We used genetic distance and sequence similarity to define typical clusters in the core gene pool. Then, we analyzed the phylogenetic relationships, feature vector distances, geographic distributions and mutation sites of strains related to the core gene pool. Strains in the same typical clusters have close phylogenetic relationships and feature vector distances. We also found that these typical clusters can be divided into three categories according to their main geographic distribution area. Furthermore, typical clusters in the same geographic area contain some common mutation patterns. Our results suggest that typical clusters in the core gene pool affect the reassortment events of the H9N2 virus in many respects, such as geographic distribution and amino acid mutation sites.

H9N2 virus-derived M1 protein promotes H5N6 virus release in mammalian cells: Mechanism of avian influenza virus inter-species infection in humans

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection.

Arctiin suppresses H9N2 avian influenza virus-mediated inflammation via activation of Nrf2/HO-1 signaling

Background H9N2 avian influenza viruses (AIVs) infect avian and mammalian hosts and provide internal genes for new emerging highly pathogenic avian viruses that cause severe pneumonia with high mortality, for which few medications are available. Arctiin, a bioactive lignan glycoside, has been reported to possess multiple pharmacological properties. However, the effect of arctiin on H9N2 virus infection is unclear. In the current study, we analyzed the effect of arctiin on H9N2 virus infection and the underlying molecular mechanism in vitro. Methods The antiviral effect against H9N2 virus was determined by plaque reduction assay (PRA) and progeny virus reduction assay. We employed MTT assay, qRT-PCR, ELISA, immunofluorescence and Western blotting to better understand the anti-inflammatory effect and corresponding mechanism of arctiin on H9N2 virus-infected cells. Results The results showed that arctiin had antiviral activity against H9N2 virus. Arctiin treatment reduced H9N2 virus-triggered proinflammatory cytokines, such as IL-6, and TNF-α. Moreover, arctiin significantly suppressed H9N2 virus-mediated expression of COX-2 and PGE2. Furthermore, we found that arctiin inhibited H9N2 virus-mediated activation of RIG-I/JNK MAPK signaling. Interestingly, arctiin treatment obviously reversed H9N2 virus-induced reduction of Nrf2, increased the nuclear translocation of Nrf2, and upregulated Nrf2 signaling target genes (HO-1 and SOD2). Zinc protoporphyrin (Znpp)—an HO-1 inhibitor—weakened the inhibitory effect of arctiin on H9N2 virus-induced RIG-I/JNK MAPK and proinflammatory mediators. Conclusion Taken together, these results suggested that the anti-inflammatory effects of arctiin on H9N2 virus infection may be due to the activation of Nrf2/HO-1 and blocked RIG-I/JNK MAPK signaling; thus, arctiin may be a promising agent for prevention and treatment of H9N2 virus infections.

Modeling and Proposal of a Black Phosphorus-based Nanostructure for Detection of Avian Influenza Virus in THz Region

In this paper, a multilayer/monolayer black phosphorus (BP)-based nanostructure is presented to detect the avian influenza virus. The nanostructur is a grating arrangement made of BP over SiO2 or Al2O3 substrate. To achieve the transmission spectrum, depend on the changes in the lateral length of BP, namely (L = 100, 150, 170 nm) as well as the complex refractive index of each of three viruses types (H1N1, H5N2, H9N2) in the THz range, the structure is numerically simulated by 3D Finite Difference Time Domain (3D-FDTD) method. The change in resonance frequency is greater for the H9N2 virus because the real part of its refractive index is relatively larger. Here, too, the rate of change is examined based on the different thicknesses of the H9N2 virus. Also, changes in the refractive index of the environment have been used to calculate important parameters in the sensors, such as sensitivity, FWHM, and figure of merit. Overall, this platform provides a promising platform for detecting influenza viruses.

Role of the amino acid mutations in the HA gene of H9N2 avian influenza virus under selective pressure in escape vaccine antibodies

It has been well-documented that some amino acid mutations in hemagglutinin (HA) of H9N2 avian influenza virus (H9N2 virus) alter the viral antigenicity, but little is reported about the role of antibody escape mutations in escape vaccine antibodies. In this study, we found that the evolution of F/98 strain in chicken embryos or chickens resulted in significant differences in immune escape, and identify the contribution of HA mutations to the antigenic variation and immune escape of H9N2 virus. Among amino acid mutations in the HA of the antigen variant viruses occurring in embryonated chicken eggs and/or chickens with or without the selection pressure of vaccine antibodies, the mutations, S145N, Q164L, A168T, A198V, M224K and Q234L, affect the antigen drift of H9N2 virus. Specially, the A198V mutation, located at the receptor-binding site on the head domain of HA, significantly contributed the antigenic variation of H9N2 virus. The mutation A198V or Q234L significantly improved the receptor binding activity, while S145N mutation decreased the receptor binding activity. Single S145N mutation could promote viral escape from polyclonal antibodies (pAbs) by preventing Ab binding physically, and single A198V mutation could promote viral escape from pAbs by enhancing the receptor binding activity. Additionally, either the mutation S145N or A198V did interfere with the immunogenicity of the inactivated vaccine, resulting in reduction of the protective efficiency of H9N2 inactivated vaccine, which contributed escape from the antibody-based immunity. Our findings provided an important reference for the accurate evaluation of the role of the amino acids mutation in HA affecting the antigenicity of H9N2 virus on immune escape, and delivered a new perspective for monitoring the adaptive evolution of H9N2 virus.ImportanceIn this study, the role of the HA mutations of H9N2 virus occurring with and without antibody selective pressure on escaping from the antibody-based immune response in host was analyzed. The results demonstrated that (i) the HA mutations S145N, Q164L, A168T, A198V, M224K, and Q234L occurring in the process of the adaptive evolution of H9N2 virus in embryonated chicken eggs and/or chickens could affect the antigenic variation of H9N2 virus. Among these mutations, the HA mutation A198V had the most significant effect on the antigenic variation; (ii) S145N mutation promoted viral escape from pAbs by preventing Abs binding physically; (iii) A198V mutation did promote viral escape from pAbs by enhancing the receptor binding activity; (iv) neither the HA mutation S145N or A198V interfered with the immunogenicity of the inactivated vaccine, resulting in reduction of the protective efficiency of H9N2 inactivated vaccine.

Novel reassortant of H9N2 avian influenza viruses isolated from chickens and quails in Egypt

Background and Aim: Poultry infections with H9N2 avian influenza viruses (AIVs) are endemic in Egypt. This study determined the genetic changes in the sequences of H9N2 AIVs isolated from chicken and quails in Egypt, including determining genetic reassortment and detecting the main genetic changes in hemagglutinin (HA) and neuraminidase (NA) genes. Materials and Methods: Swab samples were collected from chicken and quails, examined through reverse transcription-polymerase chain reaction, and AIVs from positive samples were isolated in embryonated chicken eggs. Complete genome sequencing and phylogenetic analyses were conducted for two H9N2 AIV isolates, and sequences of HA and NA gene segments were analyzed in another two isolates. Results: A novel reassortant virus was identified from a commercial chicken flock (A/chicken/Egypt/374V/2016) and quails from a live bird market (A/quail/Egypt/1253V/2016). The reassortant viruses acquired four genome segments from the classic Egyptian H9N2 viruses (HA, NA, NP, and M) and four segments from Eurasian AIVs (PB2, PB1, PA, and NS). Many genetic changes have been demonstrated in HA and NA genes. The isolated novel reassortant H9N2 virus from quails showed amino acid mutations in the antigenic sites on the globular head of the mature HA monomer matched with the parent Egyptian H9N2 virus. Conclusion: This work described the genetic characterization of a novel reassortment of the H9N2 virus in Egypt. The emergence of new reassorted AIV viruses and genome variability raises the concern of an influenza pandemic with zoonotic potentials.

Development of a Rapid Fluorescent Diagnostic System to Detect Subtype H9 Influenza A Virus in Chicken Feces

The circulation of the H9N2 virus results in significant economic losses in the poultry industry, and its zoonotic transmission highlights the need for a highly sensitive and rapid diagnostic and detection system for this virus. In this study, the performance of lateral flow test strips for a fluorescent immunochromatographic test (FICT) was optimized for the diagnosis of H9N2 virus-infected animal samples. The novel monoclonal antibodies (McAbs) against influenza A H9 viruses were developed, and two categories of McAbs with linear and conformational epitopes were compared for the performance of rapid diagnostic performance in the presence of feces sample at different time points (2, 4, and 6 days) post-infection (dpi). The limit of detection (LOD) of FICT and Kd values were comparable between linear and conformational epitope McAbs. However, superior performance of linear epitope McAbs pairs were confirmed by two animal studies, showing the better diagnostic performance showing 100% relative sensitivity in fecal samples at 6 dpi although it showed less than 80% sensitivity in early infection. Our results imply that the comparable performance of the linear epitope McAbs can potentially improve the diagnostic performance of FICT for H9N2 detection in feces samples. This highly sensitive rapid diagnostic method can be utilized in field studies of broiler poultry and wild birds.