Avian leukosis virus usurps the cellular SERBP1 protein to enhance its transcription and promote productive infections in avian cells.

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which cluster separately from the well-characterized subgroups A, B, C, D, E, and J in sequence analysis, and exhibits a specific host range and a unique pattern of superinfection interference. Avian leukosis virus subgroup K replicate more slowly in avian cells than other ALV strains, leading to escaped detection during ALV eradication, but the underlying mechanism are largely unknown. In our previous study, we have reported that JS11C1 and most of other suspected ALV-K strains possessed unique mutations in the U3 region. Here, we selected 5 mutations in some important transcriptional regulation elements to explore the possible factor contributing for the lower activity of LTR, including CA-TG mutation in the CAAT box, 21 nt deletion in the CAAT box, A-G and A-T mutations in the CArG boxes, 11 nt insertion in the PRE boxes, and C-T mutation in the TATA box. On the basis of infectious clone of JS11C1, we demonstrated that the 11 nt fragment in the PRE boxes was associated with the transcription activity of LTR, the enhancer ability of U3, and the replication capacity of the virus. Notably, we determined the differential U3-protein interaction profile of ALVs and found that the 11 nt fragment specifically binds to cellular SERPINE1 mRNA binding protein 1 (SERBP1) to increase the LTR activity and enhance virus replication. Collectively, these findings reveal that a 11 nt fragment in the U3 gene contributed to its binding ability to the cellular SERBP1 to enhance its transcription and the infectious virus productions in avian cells. This study highlighted the vital role of host factor in retrovirus replication and thus provides a new perspective to elucidate the interaction between retrovirus and its host and a molecular basis to develop efficient strategies against retroviruses.

Virome characterization of diarrheic red-crowned crane (G. japonensis).

BACKGROUND: The red-crowned crane is one of the vulnerable bird species. Although the captive population has markedly increased over the last decade, infectious diseases can lead to the death of young red-crowned cranes while few virological studies have been conducted. METHODS: Using a viral metagenomics approach, we analyzed the virome of tissues of the dead captive red-crowned crane with diarrhea symptoms in Dongying Biosphere Reserve, Shandong Province, China and feces of individual birds breeding at the corresponding captive breeding center, which were pooled separately. RESULTS: There is much more DNA and RNA viruses in the feces than that of the tissues. RNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Parvoviridae, associated with enteric diseases were detected in the tissues and feces. Genomes of the picornavirus, genomovirus, and parvovirus identified in the study were fully characterized, which further suggested that infectious viruses of these families were possibly presented in the diseased red-crowned crane. CONCLUSION: RNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Genomoviridae and Parvoviridae were possibly the causative agent for diarrhea of red-crowned crane. This study has expanded our understanding of the virome of red-crowned crane and provides a baseline for elucidating the etiology for diarrhea of the birds.

Illumina high-throughput sequencing for the genome of emerging fowl adenovirus D species and C species simultaneously.

In recent years, clinical cases of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) have been emerging and increasing in chicken flocks worldwide. Mixed infections with 2 or more fowl adenovirus (FAdV) serotypes were common in these cases. Herein, we collected a clinical sample that was positive for FAdV from 40-day-old broilers with IBH and HPS symptoms in Shandong province of China and determined the complete genome of FAdVs on the Illumina HiSeq4000 platform. The results showed that the sample contained 2 FAdV strains of D species and C species and named SD1763-1 and SD1763-2 respectively. The genome of SD1763-1 strain was 43,913 nt in length, with a G+C content of 53.51%, whereas SD1763-2 strain was 43,721 nt in length, with a G+C content of 54.87%. Sequence alignment and phylogenetic analysis revealed that strain SD1763-1 was clustered together with serotype 2/11 of FAdV-D, and SD1763-2 was clustered together with FAdV-4. There is no recombination between the genomes of the 2 viruses of FAdV-D and FAdV-C in the present study. This is the first report of obtaining 2 genomic sequences of FAdV strains simultaneously by direct use of deep sequencing in one clinical individual chicken sample, which provided direct evidence for mixed infections of adenovirus serotypes in the clinic and enriched the genome data to explore the geographic biomarkers and virulence signatures of the genus Aviadenovirus.

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.

Isolation and Identification of Subgroup J Avian Leukosis Virus Inducing Multiple Systemic Tumors in Parental Meat-Type Chickens.

Avian leukosis virus (ALV) continues evolving to obtain new genomic characters to enhance its pathogenicity. In the present study, an ALV-J strain LH20180301 was isolated from broiler breeder chickens that reached the speak of paralyzation before 20-week-old. The necropsy chickens showed subcutaneous and muscular hemorrhage, and developed tumors in multiple organs including bone, liver, spleen, and kidney. The complete provirus was then cloned and sequenced to investigate the molecular characteristics and oncogenicity etiology of this virus associated with the outbreak of disease. The genomic structure of the reported ALV-J strain LH20180301 was highly conservative with other ALVs. Recombination events between the virus with endogenous virus were identified in the viral genome. Compared with the ALV-J original HPRS-103 strain, the major recombination sites of the viral genome with ev-1 were located in 5' UTR-gag and 3' UTR regions. Phylogenetic analysis of group specific antigen gp85 encoding protein showed that the LH20180301 branched with ALV-J prevalent in "yellow chickens" of local breeds in South China. Nine amino acids (N58, D60, K70, A71, K108, N112, N113, N121, R272) in the gp85 were highly conserved among ALV-J isolates before 2012, but various mutations were found in the late isolates including LH20180301. In addition, the LH20180301 strain also had the same deletion pattern of 3' UTR with them. Therefore, LH20180301 might derive from the same ancestor with those viruses and may be the trend of ALV-J evolution in China. The defined new genomic characters in the gp85 and 3' UTR region of ALV-J might provide the molecular basis for its enhanced oncogenicity.

Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8b isolate from China.

In this study, we determined and analyzed the complete nucleotide sequence of the genome of a fowl adenovirus isolate SD1356 in China and examined its pathogenicity in specific pathogen-free chick embryos and newly hatched chicks. The full genome of SD1356 was 44,454 nucleotides in length with 58.1% G + C content. Sequence alignment and phylogenetic analysis revealed that strain SD1356 was clustered together belonging to serotype 8b of fowl adenoviruses E species (FAdV-8b). No regions homologous to early regions E1, E3, and E4 of mastadenoviruses were recognized, and being very similar to the typical organization of FAdV-E genomes. All infected embryos died 4-6 d post-inoculation with visible lesions, such as hyperemic, stunting, and clubbed down, etc. Additionally, adenovirus was found in tissues or cloacal swabs of all infected birds and most of the contact uninfected controls, despite lack of clinical signs and pathological changes. Together, our study describes the genomic characteristics of an FAdV-8b strain isolated in China. The reported FAdV-8b strain SD1356 is fetal to chick embryos and possesses horizontal transmission capacity in chickens.

Newcastle Disease Virus Infection Interferes With the Formation of Intestinal Microflora in Newly Hatched Specific-Pathogen-Free Chicks.

Newcastle disease virus (NDV) infection leads to disproportion of intestinal tract microbiol population in chickens. Whether vertical infection of NDV affects the formation of a healthy and diverse intestinal community in newly hatched chicks, which might further perturb the establishment of a normal intestinal mucosal immunity, is unclear. This study examined the effects of NDV infection of chick embryos on the formation of the intestinal microbiome of chicks at hatch using 16S rRNA genes pyrosequencing. Eleven-day-old specific-pathogen-free chicken eggs were inoculated via intra-allantoic way with Class I NDV strain. At hatch, chicks were randomly selected and their duodenal and cecal contents were extracted and examined for the composition of gut microflora by Illumina sequencing of the V3+V4 region of the 16S rRNA genes. The results showed that the duodenal flora possesses a greater sample richness and higher microbial diversity as compared with the ceca flora in newly hatched chicks. In addition, there is a clear association with loss of important bacterial population in concert with an enrichment of potentially pathogenic population and NDV infections, both in the duodenum and ceca. It is also increasingly observed that the NDV infection may be associated with the dysbiosis of gut flora. This study presented a profile of the early intestinal microbiota in specific-pathogen-free chicks at hatch and strongly indicates that NDV infection interferes with the formation of intestinal microbiome in newly hatched chicks.

16S rRNA genes Illumina sequencing revealed differential cecal microbiome in specific pathogen free chickens infected with different subgroup of avian leukosis viruses.

Intestinal flora play important roles in the pathogenisis of many pathogens. This study examined the cecal microbiome of chickens infected with avian leukosis virus (ALV) using 16S rRNA genes Illumina sequencing. One-day-old specific pathogen free chicks were inoculated in the abdomen with subgroup J or K of ALV. At 21-day-old, chickens positive for ALV viremia were selected and their cecal contents were extracted and examined for the composition of gut microflora by illumina sequencing of the V3+V4 region of the 16S rRNA genes. The results showed that there is a clear association with loss of important bacterial populations in concert with an enrichment of potentially pathogenic populations and ALV infections, despite of the virus subgroups. In addition, ALV-K infected chickens revealed a preference for opportunistic pathogens in Firmicutes such as Staphylococcus and Weissella and some genus from Bacillales. Whereas, ALV-J infected chickens were characterized by a larger number of notable pathogens like Escherichia-Shigella from Proteobacteria, and other condition pathogens including Enterococcus and members of Erysipelotrichaceae from Firmicutes, and members of Helicobacteraceae from Bacteroidetes. Collectively, our results suggest that relative abundance data from the cecal microbiome differentiates healthy chickens from those infected with ALVs. Most importantly, there was a significant difference in the gut microbiome of chickens infected with ALV-K compared to those with ALV-J infected ones. This strongly suggests that ALV infection may be associated with the microbiome and there may be multiple underlying mechanisms by which the microbiome is involved in the pathogenisis of different subgroup of ALV infections.

Exploration of the BF2*15 major histocompatibility complex class I binding motif and identification of cytotoxic T lymphocyte epitopes from the H5N1 influenza virus nucleoprotein in chickens.

The binding motif of BF2*15 major histocompatibility complex (MHC) class I was explored by analyzing the interaction between an infectious bronchitis virus octapeptide and BF2*15, and the cytotoxic T lymphocyte (CTL) epitope from the nucleoprotein (NP) of H5N1 virus was identified using experimental methods. Computational methods, including homology modeling, molecular dynamics simulation, and molecular docking analysis, were used. The recombinant plasmid pCAGGS-NP was constructed, and NP expression was confirmed by indirect immunofluorescence and Western blot in transfected 293T cells. Antibodies against NP in pCAGGS-NP-inoculated specific-pathogen-free chickens were detected by enzyme-linked immunosorbent assay (ELISA). Interferon γ (IFN-γ) mRNA was quantified, and IFN-γ production was evaluated using quantitative reverse transcription PCR and capture ELISA, respectively. CD8(+) T-lymphocyte proliferation was detected using flow cytometric analysis. The BF2*15 MHC class I binding motif "x-Arg/Lys-x-x-x-Arg/Lys" was explored. Quantification of chicken IFN-γ mRNA, evaluation of IFN-γ production, and measurement of CD8(+) T-lymphocyte proliferation confirmed that the peptide NP67-74 of H5N1 was the BF2*15 MHC-class-I-restricted CTL epitope.

[Lentivirus Delivery of the Short Hairpin RNA Targeting NDV P Gene Inhibits Production of the Newcastle Disease Virus in Chicken Embryo Fibroblasts and Chicken Embryos].

Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.

Interactive mechanism between avian infectious bronchitis S1 protein T cell peptide and avian MHC I molecule.

This study aims to construct a 3D structure of the avian major histocompatibility complex (MHC)-ß2M complex through homology modelling technology, perform molecular docking of the predicted infectious bronchitis virus (IBV) S1 protein potential epitope peptide Sp6 (NQFYIKLT) and the avian MHC-ß2M complex, and demonstrate the interactive mechanism between Sp6 and MHC using molecular dynamical simulations. The peptide Sp6 and the non-related peptide NP89-97 (PKKTGGPIY) were used to stimulate in vitro recombinant plasmid (pCAGGS-S1) avian splenic lymphocytes. Flow cytometric results show that CD8(+) T lymphocytes reproduce stimulated by the Sp6 and the nonrelated peptide proliferate by 34.8% and 2.6%, respectively. Meanwhile, fluorescent quantitative PCR results show that the secretion of IFN-γ in avian splenic lymphocytes increases after Sp6 stimulation. These data suggest that Sp6 can induce the activated avian lymphocytes in vitro to produce CTL, which is the CTL epitope in IBV S1.

Attenuation mechanism of virulent infectious bronchitis virus strain with QX genotype by continuous passage in chicken embryos.

The virulent isolate SDZB0808 of QX-type infectious bronchitis virus (IBV) was continuously passaged in chicken embryos for 110 generations. The safety and immune efficacy of the 110th generation of IBVs (P110) were evaluated. Damage was not found in the appearance of the 3-day-old specific-pathogen-free (SPF) chicks immunized with 10(4.5) EID50 (median embryo infective dose) of P110 by intranasal and ocular administration. At 14 d after the vaccination with 10(4.5) EID50 of P110, all the 3-day-old SPF chicks were immune from the attack of the homologous virulent strain SDZB0808 and the heterologous virulent strain SDIB821/2012. The whole genome sequencing of SDZB0808 of different generations (P1-P110) indicated that the replicase 1a sequences of P60-P110 all lost a length of 30bp in the same region. Specific primers were designed according to the differences in the genomes of P1-P110. SYBR Green I real-time quantitative PCR was adopted to analyze the proportion of the viruses with 30bp deletion in P60, P100, and P110. Results showed that with the passage in chicken embryos, the proportion of the viruses with 30bp deletion gradually increased. Almost 100% of the viruses in the P110 had 30bp deletion in the replicase 1a sequence. Therefore, the attenuation of IBV's virulence may be the outcome of directional screening in the chicken embryos. This work confirmed the high safety and immune efficacy of P110 in SPF chickens. Thus, P110 can serve as an attenuated IBV vaccine candidate.

Genetic, pathogenic and antigenic diversity of Newcastle disease viruses in Shandong Province, China.

Thirty-one Newcastle disease viruses (NDVs) isolated from domestic and wild birds in Shandong Province, China (2006-2014) were characterized genetically, pathogenically and antigenically. Phylogenetic analysis classified the viruses into a single genotype under Class I, and four genotypes under Class II. The nineteen viruses classified in genotype VII of Class II were velogenic, while the other viruses were either mesogenic or lentogenic to chickens. Some NDV isolates (17/23) showed no neutralizing reactivity with a monoclonal antibody developed against the HN protein of the LaSota strain, reflecting the mutation at the related antigenic epitope. When challenged with two genotype VII NDV isolates, LaSota-vaccinated SPF chickens were prevented from disease development, but virus shedding was detected for at least 5 days post challenge. Circulation of the same NDV isolate for up to 13 years suggested the role of an environmental reservoir in NDV perpetuation, and reinforced the importance of strict biosecurity measures in addition to vaccination for disease control.

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals.

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.

[Complete genomic analysis of a novel infectious bronchitis virus isolate].

The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005, while insertions and deletions were located within the S1 gene. These results indicated that CK/CH/SD09/005 is a novel variant that may be derived from the QX-like strains that are prevalent in China. Multiple genetic mechanisms, including recombinations, mutations, insertions and deletions, are likely to have contributed to the emergence of this IBV strain.

Analysis of the phylogeny of Chinese H9N2 avian influenza viruses and their pathogenicity in mice.

We isolated nineteen strains of H9N2 influenza virus from farms across five northern Chinese provinces between 2001 and 2012. Sequence analysis of the genes for the two surface glycoproteins revealed that residue 226 of the hemagglutinin (HA) of eight isolates was a leucine. A T300I mutation in three strains resulted in the loss of a potential glycosylation site. The P315S mutation in seven strains added a potential glycosylation site in HA. The isolates CK/HN/323/08 and CK/HN/321/08 had a full-length neuraminidase (NA) that differed from those seen in other isolates. Phylogenetic and molecular analysis revealed that the nineteen strains shared common ancestry with strains BJ/94 and G1. We examined eight gene sequences in the present study and concluded that the HA and NS genes appeared to be derived directly from BJ/94. The remaining six genes evolved from different reference strains. Specifically, the NA and PA genes of CK/HN/321/08 and CK/HN/323/08 clustered with the G9 and Y439 branch, respectively, and the PB2 genes of CK/SD/513/11 and CK/GS/419/12 were in an unknown lineage. We found evidence that seven new genotypes had undergone intra-subtype reassortment. A mouse infection experiment with six selected isolates showed that five of these isolates were able to replicate in mouse lungs without adaptation. Viral replication in infected mice resulted in minimal weight loss, suggesting that these H9N2 avian influenza viruses had low virulence in mammals. Our findings highlight the genetic and biological diversity of H9N2 avian influenza viruses circulating in China and emphasize the importance in continuing surveillance of these viruses so as to better understand the potential risks they pose to humans.

Characterization of an avian influenza virus of subtype H4N6 isolated from ducks in the northern China.

In 2010, an H4N6 avian influenza virus (AIV) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China. This virus was named A/duck/Shandong/1/2010 (H4N6) (DK/SD/1/2010 hereafter). The gene sequence of the virus was determined, and genetic and evolutionary analyses were conducted by combining related sequences in GenBank. Results indicated that seven genes of DK/SD/1/2010 (PB2, PB1, PA, HA, NP, M, and NS) originate from the Eurasian lineage. Another gene, the NA gene, originated from both the Eurasian and the North American lineages. The amino acid sequence near the cleavage site of DK/SD/1/2010 HA (PKKASR↓GLF) corresponded to the characteristics of AIV of low pathogenicity. Animal inoculation tests showed that the virus cannot replicate in chickens and mice. Therefore, DK/SD/1/2010 may be a recombinant virus formed by influenza virus genes from different sources through complicated restructuring and evolution in ducks that is avirulent to chickens and mice.

Two amino acid residues in ion channel protein M2 and polymerase protein PA contribute to replication difference of H5N1 influenza viruses in mice.

A/swine/Fujian/1/2001 (FJ1) and A/Duck/Zhejiang/52/2000 (DK52) are H5N1 influenza viruses that are lethal in chickens. However, in mice, FJ1 is highly pathogenic, whereas DK52 cannot replicate at all. In this study, we used reverse genetics to demonstrate that amino acid residues at position 54 of polymerase acidic protein (PA) and position 26 of ion channel protein M2 of FJ1 and DK52 are important determinants for replication in mice. In addition, we prove that M2 and PA proteins contribute to the replication of H5N1 viruses in mice.

[Biological characteristics of three Newcastle disease virus isolates and entire genome sequences analysis].

Three Newcastle disease virus (NDV) strains recovered from ND outbreaks in chickens and duck flocks in north china during 2009 to 2011 were completely sequenced and biologically characterized. All the strains were velogenic and had the velogenic motif 112R-R-Q-K-R-F117 which was consistent with the results of biological tests. Analysis of the variable region (nucleotide 47 to 420) of the F gene indicated that the three isolates belonged to genotype VII d. Cross hemagglutination inhibition test indicated that the antigen homology between three isolates and LaSota were 82.5%-89.4%, the homology between the two isolates from chicken was 90%. A cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by SDLY01 isolate showed that LaSota vaccine could provide complete protection against SDLY01, however virus discharge could be detected on fifth day. Challenge experiment in which Cherry Valley duck of 30 day old challenged with SD03 strain indicated that cherry valley duck had no disease in experiment period, but virus discharge could be detected from Larynx and cloaca until fifth day. Genome length of three NDV isolates was 15192bp and belonged to genotype VII d. Sequence analysis clarified that the whole genomic sequence of these three isolates shared high homology with NDV virus strains isolated from goose and duck over the same period, which elucidated that NDV isolated from goose, duck or chicken had close genetics and epidemiological relationship.

Avian influenza virus and Newcastle disease virus (NDV) surveillance in commercial breeding farm in China and the characterization of Class I NDV isolates.

In order to determine the actual prevalence of avian influenza virus (AIV) and Newcastle disease virus (NDV) in ducks in Shandong province of China, extensive surveillance studies were carried out in the breeding ducks of an intensive farm from July 2007 to September 2008. Each month cloacal and tracheal swabs were taken from 30 randomly selected birds that appeared healthy. All of the swabs were negative for influenza A virus recovery, whereas 87.5% of tracheal swabs and 100% cloacal swabs collected in September 2007, were positive for Newcastle disease virus isolation. Several NDV isolates were recovered from tracheal and cloacal swabs of apparently healthy ducks. All of the isolates were apathogenic as determined by the MDT and ICPI. The HN gene and the variable region of F gene (nt 47-420) of four isolates selected at random were sequenced. A 374 bp region of F gene and the full length of HN gene were used for phylogenetic analysis. Four isolates were identified as the same isolate based on nucleotide sequences identities of 99.2-100%, displaying a closer phylogenetic relationship to lentogenic Class I viruses. There were 1.9-9.9% nucleotide differences between the isolates and other Class I virus in the variable region of F gene (nt 47-420), whereas there were 38.5-41.2% nucleotide difference between the isolates and Class II viruses. The amino acid sequences of the F protein cleavage sites in these isolates were 112-ERQERL-117. The full length of HN gene of these isolates was 1851 bp, coding 585 amino acids. The homology analysis of the nucleotide sequence of HN gene indicated that there were 2.0-4.2% nucleotide differences between the isolates and other Class I viruses, whereas there were 29.5-40.9% differences between the isolates and Class II viruses. The results shows that these isolates are not phylogenetically related to the vaccine strain (LaSota). This study adds to the understanding of the ecology of influenza viruses and Newcastle disease viruses in ducks and emphasizes the need for constant surveillance in times of an ongoing and expanding epidemic of AIV and NDV.