Complete genome sequence of an isolate of duck enteritis virus from China.

Here, we present the complete genomic sequence of duck enteritis virus (DEV) strain SD, isolated in China in 2012. The virus was virulent in experimentally infected 2-month-old ducks. The DEV SD genome is 160,945 base pairs (bp) in length. The viral genome sequence, when compared to that of strain DEV CSC, which was isolated in 1962, showed three discontinuous deletions of 101 bp, 48 bp and 417 bp within the inverted repeats. A comparison of the amino acid (aa) sequences of all ORFs of the CSC and SD isolates demonstrated an11-aa deletion, two single-aa deletions, and one single-aa deletion in LORF3, UL47, UL4, respectively. Moreover, 38 single aa variations were also detected in 24 different ORFs. These results will further advance our understanding of the genetic variations involved in evolution.

Platycodon grandifloras polysaccharides inhibit mitophagy injury induced by Cr (VI) in DF-1 cells.

This study aimed to investigate the role of Platycodon grandiflorus polysaccharide (PGPS) in chromium (VI)-induced autophagy in a chicken embryo fibroblast cell lines (DF-1 cells). DF-1 cells were exposed to Cr (VI), PGPSt, and Cr (VI) + PGPSt, and their effects on cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and autophagy-related proteins were examined. The results showed that the cell viability was reduced after Cr (VI) treatment, and 3-MA, CsA or PGPSt suppressed this decrease. Cr (VI) treatment increased the ROS levels and decreased the MMP, thereby enhancing the expression of mitochondrial autophagy marker proteins (PINK1, Parkin, and LC3-II), inhibiting mitophagy autophagy protein TOMM20 expression, and promoting the degradation of autophagy-related marker p62. These changes led to exceeding mitochondrial autophagy and cell trauma and could be mitigated by PGPSt. Overall, our research showed that Cr (VI) can induce exceeding mitochondrial autophagy in DF-1 cells, whereas PGPSt can improve Cr (VI)-induced mitochondrial autophagy by inhibiting ROS and restoring MMP.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-ß promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1ß and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Complete genome sequence of an H7N3 avian influenza virus isolated from ducks in southern China.

We report here the complete genomic sequence of an H7N3 avian influenza virus (AIV) isolate, which was obtained from duck in 1996. This is the first report of this subtype of AIV being isolated from duck in Guangdong of Southern China. Genomic sequence and phylogenetic analyses showed that it was highly homologous with the wild bird virus A/ruddy turnstone/Delaware Bay/135/1996 (H7N3) and that all eight genes of this virus belonged to the North America gene pool. The availability of genome sequences is helpful to further investigations of epidemiology and evolution of AIV between waterfowl and wild birds.

Complete genomic sequence of an H5N1 influenza virus from a parrot in southern China.

An H5N1 avian influenza virus (AIV) designated A/Parrot/Guangdong/C99/2005 (H5N1) was first isolated from a sick parrot in Guangdong in southern China in 2005. The complete genome of this strain was analyzed. Genome sequence analysis showed that all 8 gene segments of the virus nucleotide had 99.0% homology to A/chicken/Henan/12/2004 (H5N1). Phylogenetic analysis demonstrated that all 8 gene segments of the virus were derived from the Eurasian lineage. The availability of genome sequences is useful to investigate the host range and genetic evolution of the H5N1 avian influenza virus in Southern China.

Full genome sequence of a recombinant H5N1 influenza virus from a condor in southern China.

In this study, we report the first genomic information on an H5N1 avian influenza virus (AIV) isolated from a condor in Guangdong Province in southern China in 2003. Full genome sequencing and phylogenetic analyses show that it is a recombinant virus containing genome segments derived from the Eurasia and North America gene pools. This will be useful for analyses of the evolution of H5N1 AIV in southern China.

Complete genome sequence of an H5N2 avian influenza virus isolated from a parrot in southern China.

We report the complete genome sequence of an H5N2 avian influenza virus (AIV) that was first isolated from a parrot in Guangdong in southern China in 2004. Genomic sequence and phylogenetic analyses showed that it was highly homologous with the North American H5N2 viruses and all eight genes of this virus belonged to the North American gene lineage. These data will help in the investigation of the epidemiology and host range of AIVs in southern China.

Complete genome sequence of an H10N8 avian influenza virus isolated from a live bird market in Southern China.

An H10N8 avian influenza virus (AIV), designated A/Duck/Guangdong/E1/2012 (H10N8), was isolated from a duck in January 2012. This is first report that this subtype of AIV was isolated from a live bird market (LBM) in Guangdong Province in southern China. Furthermore, the complete genome of this strain was analyzed. The availability of genome sequences is helpful to further investigations of epidemiology and molecular characteristics of AIV in southern China.

Complete genomic sequence of a novel natural recombinant H6N2 influenza virus from chickens in Guangdong, Southern China.

Here, we reported the complete genome sequence of a novel H6N2 avian influenza virus (AIV) isolated from chicken in Guangdong, Southern China, in 2011 which was a natural recombinant virus between the H6N2 and H5N1 subtypes. It will help to understand the epidemiology and molecular characteristics of H6N2 influenza virus in Southern China.

The Interaction between B87 Vaccine Strain and BC6/85 of Infectious Bursa Disease Virus in SPF Chickens.

This study was initiated to determine the interaction between two infectious bursal disease virus (IBDV) strains in the early stages of infection by detection and quantification of IBDV RNA in lymphoid and non-lymphoid tissues. SPF chickens were inoculated with single infection or dual infection by the mild strain B87 followed by the pathogenic strain BC6/85 at 0, 1, 2, and 3 days post-inoculation (dpi) with B87. Real-time RT-PCR assays were developed to examine the viral loads of the tissues collected at various time intervals. The results reveal that B87 could delay the time point of positive detection of the BC6/85 strain in the bursa of Fabricius from 1 dpi to 3 dpi, indicating that B87 interfered with the replication of BC6/85. The interference occurred when BC6/85 was inoculated at 2 dpi and 3 dpi with the B87 strain. Moreover, BC6/85 could affect the proliferation and duration of B87 in SPF chickens. The rates of positive detection for B87 decreased significantly during dual infection. The investigation of the interaction between the two strains is important for the implementation of appropriate control measures.

Identification, Genetic Analysis, and Pathogenicity of Classical Swine H1N1 and Human-Swine Reassortant H1N1 Influenza Viruses from Pigs in China.

Swine influenza virus causes a substantial disease burden to swine populations worldwide and poses an imminent threat to the swine industry and humans. Given its importance, we characterized two swine influenza viruses isolated from Shandong, China. The homology and phylogenetic analyses showed that all eight gene segments of A/swine/Shandong/AV1522/2011(H1N1) were closely related to A/Maryland/12/1991(H1N1) circulating in North America. The HA, NA, M, and NS genes of the isolate were also confirmed to have a high homology to A/swine/Hubei/02/2008(H1N1) which appeared in China in 2008, and the virus was clustered into the classical swine lineage. The gene segments of A/swine/Shandong/AV1523/2011(H1N1) were highly homologous to the early human H1N1 and H2N2 influenza viruses, except for the HA gene, and the virus was a reassortant H1N1 virus containing genes from the classical swine (HA) and human (NA, PB2, PB1, PA, NP, M, and NS) lineages. Both the viruses could cause lethal infection and replicate efficiently in the lungs, brains, spleens, and kidneys of mice. Histopathological examinations showed that AV1522 and AV1523 viruses caused a spectrum of marked pneumonia and meningoencephalitis according to the duration of infection, demonstrating a progression of respiratory disease and neurological disease over the course of infection that ultimately resulted in lethality for the infected mice. The changes in the pathogenicity of swine influenza viruses to mammals, accompanied with the continuous reassortment and evolution of the viruses, highlights the importance of ongoing epidemiological investigation.

Pathogenicity of different H5N6 highly pathogenic avian influenza virus strains and host immune responses in chickens.

The H5N6 highly pathogenic avian influenza virus (HPAIV) has been circulating in China since 2013. In this report, we describe our recent chicken experimental studies investigating the pathogenicity and transmission of four H5N6 HPAIV field strains of different origins (GS39, CK44, DK47 and CK74) and the host immune responses. Four-week-old specific-pathogen-free chickens were inoculated intranasally with one of the four H5N6 HPAIV strains (one strain per group). Among the contact chickens, the GS39 and CK74 strains caused 100 % mortality, the CK44 strain caused 80 % mortality, and the DK47 strain caused 40 % mortality. The viruses were effectively replicated in multiple tissues of the inoculated chickens, in which high viral titers were detected in virus-infected tissues, and significantly upregulated expression of immune-related genes was found in the infected chickens at 24 hpi. The chicken serum antibody levels increased from 5log2 at 7 dpe to 7.67-8log2 at 14 dpe. The major histocompatibility complex molecules were upregulated 21.22- to 32.98-fold in lungs and 5.10- to 18.47-fold in spleens. In summary, H5N6 viruses can replicate within chickens and be effectively transmitted between chickens. Our study contributes to further understanding the pathogenesis of clade 2.3.4.4 H5N6 avian influenza viruses in chickens.

Genetic Characteristics and Pathogenicity Analysis in Chickens and Mice of Three H9N2 Avian Influenza Viruses.

H9N2 avian influenza is a remarkable disease that has circulated in domestic poultry in large regions of China and posed a serious threat to the poultry industry. The H9N2 virus can not only infect mammals directly, but also provide gene segments to generate novel, but lethal human reassortants. Therefore, it is important to study the evolution, pathogenicity, and transmission of the H9N2 virus. In this study, three H9N2 viruses isolated from chickens in different layer farms were identified. Phylogenetic analysis revealed that these H9N2 viruses were all multiple genotype reassortants, with genes originating from Y280-like, F/98-like, and G1-like viruses. Animal studies indicated that the AV1535 and AV1548 viruses replicated efficiently in the lungs, tracheas, spleens, kidneys, and brains of chickens; the viruses shed for at least 11 days post-inoculation (DPI) and were transmitted efficiently among contact chickens. The AV1534 virus replicated poorly in chickens, shed for 7 DPI, and were not transmitted efficiently among contact chickens. The AV1534 virus replicated well in mice lungs and caused about 2% weight loss. The AV1535 and AV1548 viruses were not able to replicate in the lungs of mice. Our results indicate that we should pay attention to H9N2 avian influenza virus surveillance in poultry and changes in the pathogenicity of them to mammals.

Phylogeny, Pathogenicity, Transmission, and Host Immune Responses of Four H5N6 Avian Influenza Viruses in Chickens and Mice.

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood-brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.

H7N9 Avian Influenza Virus Is Efficiently Transmissible and Induces an Antibody Response in Chickens.

H7N9 viruses pose a threat to human health and they are no less harmful to the poultry industry than the H5N1 avian influenza viruses. However, the pathogenesis, transmissibility, and the host immune response of the H7N9 virus in chickens and mice remain unclear. In this study, we found that H7N9 viruses replicated in multiple organs of the chicken and viral shedding persisted up to 30 days postinoculation (DPI). The viruses were efficiently transmitted between chickens through direct contact. Notably, chickens infected with H7N9 had high antibody levels throughout the entire observation period and their antibody response lasted for 30 DPI. The expression levels of the pattern-recognition receptors and pro-inflammatory cytokines were found to be significantly upregulated in the brain using quantitative real-time PCR. The expression of TLR3, TLR7, MDA5, Mx, IL-1ß, IL-6, IFN-α, and IFN-γ were also significantly different in the lungs of infected chickens. We found that the viruses isolated from these birds had low pathogenicity in mice, produced little weight loss and could only replicate in the lungs. Our findings suggested that the H7N9 viruses could replicate in chickens and mice and be efficiently transmitted between chickens, which presented a significant threat to human and poultry health.

The genetic and phylogenetic analysis of a highly pathogenic influenza A H5N6 virus from a heron, southern China, 2013.

The H5N6 highly pathogenic avian influenza viruses (HPAIVs) have circulated within poultry in China since 2013. Infections of H5N6 in wild birds were reported since 2014. In order to investigate the infection history of H5N6 in wild birds, we conducted a retrospective analysis of H5 positive wild bird samples collected in 2013, the year H5N6 was discovered in poultry. We isolated a new HPAI H5N6 virus from a dead heron collected in 2013. The virus had high identity in all eight gene sequences to those collected from poultry in 2013 (for example, A/chicken/Shenzhen/1845/2013, 99.1%-99.7%). Our findings revealed that H5N6 HPAIVs infected wild birds in southern China since the emergence of H5N6 in poultry in 2013. The co-circulation of H5N6 between wild birds and poultry is very close, and should raise our attention more.

Pathogenicity, Transmission and Antigenic Variation of H5N1 Highly Pathogenic Avian Influenza Viruses.

H5N1 highly pathogenic avian influenza (HPAI) was one of the most important avian diseases in poultry production of China, especially in Guangdong province. In recent years, new H5N1 highly pathogenic avian influenza viruses (HPAIV) still emerged constantly, although all poultry in China were immunized with H5N1 vaccinations compulsorily. To better understand the pathogenicity and transmission of dominant clades of the H5N1 HPAIVs in chicken from Guangdong in 2012, we chose a clade 7.2 avian influenza virus named A/Chicken/China/G2/2012(H5N1) (G2) and a clade 2.3.2.1 avian influenza virus named A/Duck/China/G3/2012(H5N1) (G3) in our study. Our results showed that the chickens inoculated with 10(3) EID50 of G2 or G3 viruses all died, and the titers of virus replication detected in several visceral organs were high but different. In the naive contact groups, virus shedding was not detected in G2 group and all chickens survived, but virus shedding was detected in G3 group and all chickens died. These results showed that the two clades of H5N1 HPAIVs had high pathogenicity in chickens and the contact transmission of them was different in chickens. The results of cross reactive HI assay showed that antigens of G2 and G3 were very different from those of current commercial vaccines isolates (Re-4, Re-6, and D7). And to evaluate the protective efficacy of three vaccines against most isolates form Guangdong belonging to clade 2.3.2.1 in 2012, G3 was chosen to challenge the three vaccines such as Re-4, Re-6, and D7. First, chickens were immunized with 0.3 ml Re-4, Re-6, and D7 inactivated vaccines by intramuscular injection, respectively, and then challenged with 10(6) EID50 of G3 on day 28 post-vaccination. The D7 vaccine had 100% protection against G3 for chickens, the Re-6 vaccine had 88.9%, and the Re-4 vaccine only had 66.7%. Our results suggested that the D7 vaccine could prevent and control H5N1 virus outbreaks more effectively in Guangdong. From the above, it was necessary to conduct continuously epidemiological survey and study the pathogenicity and antigenic variation of avian influenza in Southern China.

New Reassortant H5N6 Highly Pathogenic Avian Influenza Viruses in Southern China, 2014.

New reassortant H5N6 highly pathogenic avian influenza viruses (AIVs) were isolated from apparently healthy domestic ducks in Southern China in 2014. Our results show that the viruses grew efficiently in eggs and replicated systemically in chickens. They were completely lethal in chicken (100% mortality), and the mean death time was 6 to 7 days post-inoculation. The viruses could transmit in chickens by naïve contact. BLAST analysis revealed that their HA gene was most closely related to A/wild duck/Shangdong/628/2011 (H5N1), and their NA genes were most closely related to A/swine/Guangdong/K6/2010 (H6N6). The other genes had the highest identity with A/wild duck/Fujian/1/2011(H5N1). The results of phylogenetic analysis showed that their HA genes clustered into clade 2.3.4.4 of the H5N1 viruses and all genes derived from H5 were Mix-like or H6-like viruses. Thus, the new H5N6 viruses were reassortmented of H5N1 and H6N6 virus. Therefore, the circulation of the new H5N6 AIVs may become a threat to poultry and human health.

A Novel H1N2 Influenza Virus Related to the Classical and Human Influenza Viruses from Pigs in Southern China.

Southern China has long been considered to be an epicenter of pandemic influenza viruses. The special environment, breeding mode, and lifestyle in southern China provides more chances for wild aquatic birds, domestic poultry, pigs, and humans to be in contact. This creates the opportunity for interspecies transmission and generation of new influenza viruses. In this study, we reported a novel reassortant H1N2 influenza virus from pigs in southern China. According to the phylogenetic trees and homology of the nucleotide sequence, the virus was confirmed to be a novel triple-reassortant H1N2 virus containing genes from classical swine (PB2, PB1, HA, NP, and NS genes), triple-reassortant swine (PA and M genes), and recent human (NA gene) lineages. It indicated that the novel reassortment virus among human and swine influenza viruses occurred in pigs in southern China. The isolation of the novel reassortant H1N2 influenza viruses provides further evidence that pigs are "mixing vessels," and swine influenza virus surveillance in southern China will provide important information about genetic evaluation and antigenic variation of swine influenza virus to formulate the prevention and control measures for the viruses.

PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice.

H5N1 influenza viruses with high lethality are a continuing threat to humans and poultry. Recently, H5N1 high-pathogenicity avian influenza virus (HPAIV) has been shown to transmit through aerosols between ferrets in lab experiments by acquiring some mutation. This is another deeply aggravated threat of H5N1 HPAIV to humans. To further explore the molecular determinant of H5N1 HPAIV virulence in a mammalian model, we compared the virulence of A/Duck/Guangdong/212/2004 (DK212) and A/Quail/Guangdong/90/2004 (QL90). Though they were genetically similar, they had different pathogenicity in mice, as well as their 16 reassortants. The results indicated that a swap of the PB2 gene could dramatically decrease the virulence of rgDK212 in mice (1896-fold) but increase the virulence of rgQL90 in mice (60-fold). Furthermore, the polymerase activity assays showed that swapping PB2 genes between these two viruses significantly changed the activity of polymerase complexes in 293T cells. The mutation Ser715Asn in PB2 sharply attenuated the virulence of rgDK212 in mice (2710-fold). PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice and 715 Ser in PB2 plays an important role in determining high virulence of DK212 in mice.