Fowl adenovirus serotype 4 (FAdV-4) strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province, China. The isolate was cultured in primary chicken embryo kidney cells. A study of pathogenicity indicated that SD1511 readily infected 7-35-d-old chickens by intramuscular injection and intranasal and oral routes, causing 50%-100% mortality. The 35-d-old chickens suffered more severe infection than 7- and 21-d-old chickens with mortality highest in the intramuscular injection group. The serum from surviving chickens showed potent viral neutralizing capability. The complete genome of SD1511 was sequenced and analyzed. The strain was found to belong to the FAdV-4 cluster with more than 99% identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations, including deletions of open reading frame 27 (ORF27), ORF48, and part of ORF19. Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.
Animals ; Antibodies, Neutralizing ; Aviadenovirus/pathogenicity* ; Cell Line ; Chick Embryo/virology* ; Chickens/virology* ; China ; Gene Deletion ; Genetic Variation ; Genome ; Genome, Viral ; Genomics ; Kidney/virology* ; Liver/virology* ; Open Reading Frames ; Poultry Diseases/virology* ; Serogroup ; Viral Load ; Virulence ; Virus Diseases/virology*

In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.
Amino Acid Sequence ; Animals ; Chick Embryo ; Coronavirus Infections ; veterinary ; virology ; Ducks ; virology ; Female ; Infectious bronchitis virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment

A quail-origin subtype of the influenza virus was isolated from a human-infecting H7N9 subtype of the avian influenza virus found in a live poultry market and was given the name A/Quail/Hangzhou/1/ 2013 (H9N2). We analyzed the whole genome of this virus and its biologic characteristics. Sequence analyses suggested that the: HA and NS genes belonged to a CK/BJ/1/94-like lineage; NA, NP, PA and PB1 genes belonged to a SH/F/98-like lineage; M and PB2 genes belonged to a G1-like lineage. Analyses of key amino acids showed that the cleavage site in HA protein was PSRSSR ↓ GL, and that the HA protein had a human receptor-binding site with Leu226. Deletion of amino acids 69 - 73 was detected in the stalk of NA protein, the M2 protein had an Asn31 mutation, and the NS1 protein had two mutations at Ser42, Ala149. The intravenous pathogenicity of this virus was 0.36. A study in chickens suggested that all inoculated birds shed the virus from the trachea and cloaca on the third day post-infection (p. i. ) until 11 days. All chickens that had direct contact shed the virus on the second day p. i. until 8 days. Results of virus reisolation suggested that lung and tracheal tissues could shed the virus in 5 days, whereas the other organs could shed the virus in 3 days. These results suggest that this virus strain is H9N2 subtype LPAIV, whose lineage is prevalent in mainland China. This research provides evidence on how to monitor and prevent the H9N2 subtype of the avian influenza virus.
Animals ; Chick Embryo ; Chickens ; China ; Genotype ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Phylogeny ; Quail ; virology

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.
Animals ; Chick Embryo ; Chickens ; Down-Regulation ; Fibroblasts ; virology ; Gene Targeting ; Lentivirus ; genetics ; metabolism ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; physiology ; Phosphoproteins ; genetics ; metabolism ; Poultry Diseases ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

To analyze the antigenic and genetic characteristics of the influenza A (H3N2) virus in mainland China during the surveillance year of 2013-2014, the antigenic characteristics of H3N2 virus were analyzed using reference ferret anti-sera. The nucleotide sequences of the viruses were determined by Sanger dideoxy sequencing, phylogenetic trees were constructed with the neighbor-joining method, and the genetic characteristics of the viruses were determined in comparison to current vaccine strains. The results showed that most of the H3N2 viruses were antigenically closely related to the A/Victoria/361/2011 vaccine strain cell-propagated prototype virus (99.6%). Using the A/Texas/50/2012 egg isolate as the reference antigen, 15.1% of the viruses were found to be closely antigenically related to it, while 11.9% of strains were closely antigenically related to the egg-propagated epidemic strain, A/Shanghai-Changning/1507/2012. Phylogenetic analysis of HA genes indicated that the A(H3N2) viruses in this surveillance year were in the same clade, but no drug resistant mutation was identified in the NA genes. During the 2013-2014 influenza surveillance year, no significant genetic change was detected in either the HA or NA genes of the A(H3N2) viruses, while significant mutations were found in egg isolates resulting from their adaptation during propagation in eggs. The antigenic and genetic changes should be investigated in a timely manner to enable the selection of an appropriate vaccine strain in China.
Animals ; Antigenic Variation ; Base Sequence ; Chick Embryo ; China ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Mutation ; Phylogeny

To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Coronavirus Infections ; veterinary ; virology ; Infectious bronchitis virus ; chemistry ; genetics ; growth & development ; metabolism ; Poultry Diseases ; virology ; Protein Structure, Tertiary ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; metabolism ; Transfection ; Vero Cells

OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology

In order to determine the challenge dose of pigeon paramyxovirus type 1 (PPMV-1) inactivated vaccine (S-1 strain). The virus titer of PPMV-1 E5 allantoic fluid (Chuansha strain) was determined using SPF chicken embryos in this research. After inoculating 30-day-old and 120-day-old pigeons with low-HI antibody against PPMV-1 (HI antibody < or =2) with different doses of PPMV-1 (Chuansha strain), the clinical symptoms and histopathological lesions of the challenged pigeons were examined. The results showed that the minimal lethal dose (MLD) of PPMV-1 (Chuansha strain) was 102.5 ELD50, so we determined that 10(5.5) ELD50, which was 1000 times the MLD, could be taken as the challenge dose in the vaccine efficacy test for PPMV-1 inactivated vaccine (S-1 strain).
Animals ; Antibodies, Viral ; immunology ; Bird Diseases ; immunology ; mortality ; virology ; Chick Embryo ; Columbidae ; immunology ; virology ; Newcastle Disease ; immunology ; mortality ; virology ; Newcastle disease virus ; immunology ; pathogenicity ; Phylogeny ; Viral Vaccines ; immunology ; Virulence

In order to investigate the prevalence and track genetic and antigenic evolutions of infectious bronchitis virus (IBV) and their prevalence in Guangxi, China since 1985, gene amplification and sequencing and virus neutralization (VN) test on chicken embryo tracheal organ cultures were used in genotyping and serotyping of 28 IBV isolates during 2009-2011 in Guangxi. The results of N gene sequencing and comparison showed that the 28 isolates and reference strains were classified into three groups, and most isolates belonged to group Ill, while the isolates in 1985-2008 belonged to groups IV and II. The data of VN test indicated that the 28 isolates belonged to 6 serotypes; among them, 71. 4% belonged to serotypes 1, 2, and 3, and 11 (39.3%) shared the same serotype with the current vaccine strains. Given the data of our previous study, it is found that prevalent serotypes and their proportions varied in different areas of Guangxi and during different periods. These data lay a good foundation for developing an oil-emulsified inactivated polyvalent vaccine containing local dominant serotypes for the effective prevention and control of infectious bronchitis.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Chickens ; China ; epidemiology ; Coronavirus Infections ; epidemiology ; immunology ; veterinary ; virology ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; epidemiology ; immunology ; virology

The distribution of glycosylation sites in HA proteins was various among H5 subtype avian influenza viruses (AIVs), however, the role of glycosylation sites to the virus is still unclear. In this study, avian influenza H5N1 viruses with deletion of the glycosylation sites in HA were constructed and rescued by site direct mutation and reverse genetic method, and their biological characteristics and virulence were determined. The result showed that the mutants were confirmed to be corrected by HA gene sequencing and Western blot analysis. The EID50 and TCID50 tested in SPF chick embryo and MDCK cells of a mutant rSdelta158 with deletion of glycosylation site at position 158 were slight lower than that of wild type rescued virus rS, and the plaque diameter of rSdelta158 was significant smaller than that of rS. The EID50 and TCID50 of mutants rSdelta169 and rSdelta290 with deletion of glycosylation sites at position 169 and 290, respectively, were slight higher than that of wild type rescued virus rS, the plaque diameters of rSdelta169 and rSdelta290 were similar as that of rS, but the plaque numbers of rSdelta169 and rSdelta290 were 10-fold higher than that to rS. On the other hand, the rSdelta158, rSdelta169 and rSdelta290 showed similar growth rate in chicken embryo fibroblast as rS. All viruses remained high pathogenicity to SPF chickens. Therefore, the growth of AIV can be affected by changes of glycosylation sites in HA protein, by which the effect is variable in different cells.
Amino Acid Motifs ; Animals ; Cell Line ; Chick Embryo ; Chickens ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; chemistry ; genetics ; growth & development ; metabolism ; Influenza in Birds ; virology ; Poultry Diseases ; virology