[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.

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.

[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.

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.

Diversified reassortant H9N2 avian influenza viruses in chicken flocks in northern and eastern China.

According to our previous study of the M genes of H9N2 avian influenza viruses (AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly emerged in northern and eastern China in addition to the existing A/chicken/Hong Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of seventeen H9N2 isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.

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.

[Molecular epidemiological analysis of class I Newcastle disease virus isolated from China in 2008].

Thirteen isolates of Class I Newcastle disease virus obtained from healthy poultry in China during 2008 were characterized genotypically in this study. All the isolates were proved to be lentogenic strains based on the deduced amino acid sequence of the Fusion protein gene. Molecular epidemiological analysis showed that 13 isolates could be subdivided into 2 distinct genotypes, 11 isolates belonged to genotype 2, and other 2 isolates belonged to genotype 3. Results indicated two genotypes of Class I Newcastle disease virus might widely exist in domestic poultry in China.

[Soluble expression of chicken interferon-gamma and antiviral activity of purified expression product].

OBJECTIVE: We cloned and expressed chicken interferon-gamma gene (chIFN-gamma), and detected the bioactivity of chIFN-gamma expressed in Escherichia coli (E. coli). METHODS: The chIFN-gamma mature protein gene was amplified by RT-PCR from spleen lymphocytes of chicken which were stimulated with concanavalin A (ConA) and then cloned into the prokaryotic expression vector pET-32a ( + ). Recombinant expression plasmid of pET-32a ( + )-chIFN-gamma was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression with IPTG induction. Purified soluble rchIFN-gamma proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The antiviral activity was detected by MDCK-VSV system. RESULTS: A 456 bp cDNA encoding chIFN-gamma mature protein gene was cloned and successfully expressed in E. coli with approximate molecular weight of 31.0 kDa, which could be recognized by anti-His mAb and rabbit polyclonal antibody against chlFN-gamma. The recombinant chIFN-gamma proteins were expressed to form inclusion bodies with a portion of soluble protein. The soluble rchIFN-gamma proteins were purified by Ni-NTA column under a native condition with the yield of 3.0 mg/L. The purified recombinant chIFN-gamma (rchIFN-gamma) proteins by 1:32 dilution could resist 100 TCID50 VSV virus attack. CONCLUSION: The purified rchIFN-gamma proteins by Ni-NTA column under a native condition had better antiviral activity, which will establish a basis for further developing new type antiviral interferon praeparatum.

Evolution analysis of the matrix (M) protein genes of 17 H9N2 chicken influenza viruses isolated in northern China during 1998-2008.

Matrix (M) protein genes of 17 H9N2 avian influenza viruses (AIVs) isolated from chickens in northern China during the last 10 years were completely sequenced and phylogenetically analyzed. Homology of nucleotide sequences in the M gene of 17 isolates was 92.7-99.9%. Phylogenetic analysis showed that 11 of the tested M genes belong to the A/chicken/HongKong/Y280/97 (Y280)-like lineage, while the other six belong to the A/Quail/HongKong/G1/97 (G1)-like lineage. This is also the first time that a G1-like M gene of a H9N2 virus was detected in chicken flocks in northern China. These newly appearing changes in M genes may be due to reassortment events of AIVs, or they may have come from the H9N2 strains of southern China which surged in northern China after translocation. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S31N in two isolates, which is the molecular characterization of amantadine resistance in AIVs. Results of this study suggest that long-term monitoring should be continued to track the transmission and evolution of H9N2 AIVs in chickens in China.