RESUMEN
Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.
RESUMEN
BACKGROUND: Newcastle disease (ND) is an economically significant poultry disease worldwide. During field surveillance for ND in 2010 in Iran, a backyard chicken flock showed clinical signs of ND with 100% mortality. OBJECTIVES: We aimed to characterise genetically, biologically and epidemiologically an exotic virulent ND virus (NDV) detected in Iran. METHODS: After observing high mortality, dead birds were sampled and then disposed of by burial, and the chicken house was disinfected. Tissue samples were molecularly tested for NDV. The genetic homogeneity of the isolate RT30/2010 was tested by plaque assay, and then a large virus plaque was used for the second step of plaque purification. Fusion and matrix complete genes were sequenced and used for genotyping and epidemiological tracing. We tested biological pathotypes using mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. RESULTS: The isolate formed heterogeneous plaques in chicken embryo fibroblast cells. The second step of plaque purification produced homogeneous and large plaques. Phylogenetic analysis using both genes classified the virus into sub-genotype XIII.2.1. Nucleic acid and amino acid identities of RT30/2010 fusion gene with the closest available isolate SPVC/Karachi/NDV/43 are 97.95% and 98.73%, respectively. Isolate has 112 RRRKRF117 motif at the fusion cleavage site, and pathogenicity tests showed MDT of 56.4 h and ICPI of 1.85. CONCLUSIONS: This study presents the first detection and characterisation of a velogenic NDV of sub-genotype XIII.2.1 from Iran. Our follow-up surveillance for ND shows that timely virus detection and carcass management have led to the cessation of virus transmission in Iran.
Asunto(s)
Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Animales , Embrión de Pollo , Pollos , Filogenia , Irán/epidemiología , Enfermedad de Newcastle/epidemiología , GenotipoRESUMEN
Neutralizing, serotype-specific, and hemagglutination-inhibiting antibodies against infectious bronchitis virus (IBV) are induced by epitopes in the S1 protein. Most changes in the virus genome due to mutation and recombination during serial passaging in embryonated chicken eggs occur in the S1 gene. In the current study, we tried to predict the potential linear B-cell epitopes of the S1 subunit of two Iranian 793/B isolates and then we analyzed their changes at passage level 90 due to mutations at this passage level. To predict linear B-cell epitopes of the S1 protein belonging to two Iranian 793/B isolates, we used two online epitope prediction programs called BepiPred and ABCpred. Some of the most important features of proteins including antigenicity, physicochemical properties, and secondary structure composition were analyzed. The predicted epitopes were studied between wild viruses and their passage level 90 viruses. We identified 15 potential linear B-cell epitopes among which six epitopes had the highest scores of physicochemical properties and antigenicity. Due to amino acid substitutions, seven predicted epitopes had different amino acid sequences at passage level 90. Among eight epitopes with no amino acid substitution at passage level 90, three epitopes had the highest scores. These three conserved epitopes including NH2-NQLGSCPLTGMI-COOH, NH2-GNFSDGFYPFTNSSLVKD-COOH, and NH2-GPIQGGC-COOH might be strategic and potential candidates for use in designing epitope-based vaccine researches. In conclusion, based on scores of physicochemical properties and antigenicity, it seemed that the sequence of most epitopes in wild viruses might be more antigenic and immunogenic compared to their sequence in viruses of passage 90.
RESUMEN
Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1ß, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1ß (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1ß proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1ß (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.