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

BACKGROUNDTo understand the characteristics of internal genes of two strains of influenza A(H9N2) virus isolated from men and on the basis of these to reveal the origin of these two strains of influenza A(H9N2)virus.

METHODSThe target gene was amplified by RT-PCR,the PCR product was ligated with P GEM-T Vector (Promega Company, USA) at 4 degrees, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction enzyme. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03)and Editseq (Version 3.69) softwares.

RESULTSInternal genes of the two strains of H9N2 virus were G9 lineage. There was a slight difference in nucleotide sequence in PA gene between the two strains, whereas another five gene segments were identical to each other.

CONCLUSIONThe genomes of the two strains of influenza A(H9N2)virus were G9 lineage.They were transmitted to men separately from different avian sources with different characteristics of gene of influenza A(H9N2) virus, respectively.

Animals ; Chick Embryo ; China ; Humans ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Phylogeny ; Viral Proteins ; genetics

BACKGROUNDTo understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus.

METHODSViruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.

RESULTSGenome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain.

CONCLUSIONSA/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.

Animals ; Base Sequence ; Chick Embryo ; Child ; Genome, Viral ; Humans ; Influenza A Virus, H9N2 Subtype ; Influenza A virus ; genetics ; Influenza, Human ; virology ; Phylogeny

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*

To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P < 0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; immunology ; pathogenicity ; Chick Embryo ; Fibroblasts ; virology ; Proliferating Cell Nuclear Antigen ; analysis ; immunology ; Viral Load ; immunology

Raw white rice has not been considered a good carrierfor oral vaccination, probably because of its antiviralactivity. Methods are required to overcome antiviralactivity in raw white rice. This study was carried out todetermine the effects of various treatments of raw whiterice on the survival of strain I-2 of Newcastle diseasevirus. These included cooking and baking the rice ormixing the rice with vegetable oil prior to coating withvaccine virus. The vaccine-coated rice was then stored for30min and 24h, followed by quantitative recovery of thevirus. Thirty min after mixing, uncooked, cooked, andbaked rice, and rice mixed with vegetable oil showed titersof 10(6.2), 10(7.2), 10(6.6), and 10(7.0) EID50/0.1ml, respectively.After storage for 24h at 22-25oC, the titers dropped to10(5.0), 10(6.5), 10(5.0), and 10(6.0) EID50/0.1ml for uncooked,cooked, baked, and oiled rice, respectively.
Animals ; Chick Embryo ; Chickens ; Cookery ; Newcastle Disease/*virology ; Newcastle disease virus/growth & development/*physiology ; Oryza sativa/chemistry/*virology ; Viral Vaccines/chemistry

According to 45 hemagglutinin (HA) gene sequences of H7 subtype of avian influenza virus (AIV), a pair of specific oligonucleotide primers was designed. We developed one step RT-PCR for detecting AIV subtype H7. Sensitivity to detection of allantoic fluid by one step RT-PCR reached 10(5.5) EID50/mL and detection of swab samples reached 10(3) EID50/mL. We simultaneity detected the tissue and swab samples infected with H7 subtypes AIV by one step RT-PCR and virus isolation method. The results showed that the sensitivity of the assay gave an excellent correlation with the conventional virus isolation method. H1-H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR. The results showed the assays were high specific, without cross-reaction with other subtypes or other avian diseases. Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of AIV subtype H7.
Animals ; Birds ; Chick Embryo ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H7N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza in Birds ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Based on the complete genome sequence of pigeon-origin Newcastle disease virus strain JS/07/04/ Pi(genotype VIb), nine overlapped fragments covering its full-length genome were amplified by RT-PCR. The fragments were connected sequentially and then inserted into the transcription vector TVT7/R resulting in the TVT/071204 which contained the full genome of strain JS/07/04/Pi. The TVT/071204 was co-transfected with three helper plasmids pCI-NP, pCI-P and pCI-L into the BSR cells, and the transfected cells and culture supernatant were inoculated into 9-day-old SPF embryonated eggs 60 h post-transfection. The HA and HI tests were conducted following the death of embryonated eggs. The results showed that the allantoic fluids obtained were HA positive and the HA could be inhibited by anti-NDV serum which indicated that the strain JS/07/04/Pi was rescued successfully. The rescued virus rNDV/071204 showed similar growth kinetics to its parental virus in CEF. The successful recovery of this strain would contribute to the understanding of the host-specificity of pigeon-origin NDV and to the development of the novel vaccines against the NDV infection in pigeons.
Animals ; Base Sequence ; CHO Cells ; Chick Embryo ; Columbidae ; virology ; Cricetinae ; Cricetulus ; DNA, Complementary ; genetics ; Fluorescent Antibody Technique, Indirect ; Molecular Sequence Data ; Newcastle disease virus ; genetics ; growth & development

Based on the genomic sequence of NDV08-004 strain (GenBank accession number FJ794269), seven pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pGEM-Teasy vector. The fragments (named A to G) were sub-cloned into transcription vector pOLTV5 according to the universal RE site and the plasmid named NDV08-004-pO which contained the full length cDNA of NDV08-004 strain was constructed. Three helper plasmids (pCI-NP, pCI-P and pCI-L) together with NDV08-004-pO were co-transfected into BSR T7/5 cells, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus. The virus was rescued successfully and identified by HA and RT-PCR and sequencing. The rescue system constructed in this study provided a good foundation for the further related research.
Animals ; Base Sequence ; Chick Embryo ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; Plasmids ; Reverse Genetics ; methods

OBJECTIVETo observe the effect of HSV-1 infection on the viability of the cultured chicken embryo telencephalon neurons in vitro.

METHODSPrimary culture model of chicken embryo of telencephalon neurons was established. Herpes simplex virus type 1 (HSV-1) was propagated in Vero cells and viral titer was measured by plaque forming method. Inverted microscope, transmission electron microscope, MTT cell viability and DNA agarose gels electrophoresis were used to evaluate the effect of HSV-1 infection on the neurons.

RESULTSAt 24, 48, 72 hours post infection the viability of the neurons decreased by 25%, 56%, 97% as compared with the control group. Morphological changes and DNA agarose gel electrophoresis showed a necrotic effect.

CONCLUSIONSHSV-1 infection induced a remarkable decrease on the viability of neurons by necrosis rather than apoptosis.

Animals ; Cercopithecus aethiops ; Chick Embryo ; Herpes Simplex ; pathology ; virology ; Herpesvirus 1, Human ; In Vitro Techniques ; Necrosis ; Neurons ; pathology ; Telencephalon ; cytology ; Vero Cells ; Virus Cultivation