Sa Xiao

Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.

Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus

BackgroundNewcastle disease (ND), which is caused by the Newcastle disease virus (NDV), is one of the most important avian diseases in poultry. Since its discovery in 1926, ND has caused great economic losses to the world poultry industry and remains a threat to chickens and wild birds. Although a stringent vaccination policy is widely adopted to control ND, ND outbreaks still occur, and virulent NDV is sporadically isolated from chickens and wild birds. To study the pathogenesis of ND and provide tools to prevent its prevalence, novel antibody fragments should be developed. The variable domains of the heavy chain of the heavy-chain antibodies (VHH) are the smallest naturally occurring antibodies derived from camelid heavy-chain antibodies. The comparatively small size, high affinity, high solubility, low immunogenicity and ability to bind epitopes inaccessible to conventional antibodies of VHH make them ideal candidates for a considerable number of therapeutic and biotechnological applications. However, an anti-NDV VHH has not been reported to date.ResultsIn this study, a VHH yeast two-hybrid library was constructed from NDV vaccine immunized C. bactrianus, and seven VHH fragments to the haemagglutinin-neuraminidase (HN) protein of NDV were successfully screened and characterized for the first time. These selected VHH clones were all expressed as soluble protein in E. coli. ELISA, dot blot, immunocytochemistry and pull down results showed that the screened VHHs could interact with NDV virion, among which five had neutralizing activity. In addition, the seven VHHs could inhibit the haemagglutination activity of different NDV strains.ConclusionsWe constructed an NDV-immunized VHH yeast two-hybrid library and screened and characterized seven VHHs targeting NDV HN protein for the first time. The seven VHHs may have great potential for NDV diagnosis, pathogenesis and therapeutics.

Re-evaluation the immune efficacy of Newcastle disease virus vaccine in commercial laying chickens

Newcastle disease virus (NDV) infection causes serious problems in laying chickens, like reducing egg production, increasing rate of abnormal eggs in spite of strict vaccination in layer farms program. A new evaluation system is needed to show complete protection of the immunization in laying chickens based on the egg-laying performance, rather than clinical signs of the disease. In this study, laying chickens with different anti-NDV HI (hemagglutination-inhibition) antibody titer after vaccination were divided into different groups. These chickens were then challenged with field isolated highly virulent NDV strains. Results showed that the chickens in low HI titers group (5log2 to 8log2) and medium HI titers group (9log2 to 11log2) had atypical symptoms, produced abnormal eggs, and shed virus. Whereas, with HI titers≥12log2, the chickens were completely protected, and did not show symptoms, or produce abnormal eggs or shed virus. Morbidity, positive viral shedding rate and abnormal egg-rate decreased with increase in pre-challenge HI antibody titer. Our result suggested that 12log2 is the threshold of the HI antibody in providing complete protection to laying chickens under field condition, and protective efficacy is correlated with HI antibody titer. This study provides a valuable reference for the vaccination and control of ND in poultry.

Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication.

Comprehensive analysis of amino acid sequence diversity at the F protein cleavage site of Newcastle disease virus in fusogenic activity

Newcastle disease virus (NDV) is a contagious agent of Newcastle disease in avian species and seriously affects the poultry industry. The cleavage site of the viral F protein (Fcs) is a key determinant of membrane fusion and viral virulence. In this study, we investigated the precise effect of variable amino acid sequences of the Fcs on fusogenic activity. Based on viral pathogenicity, the Fcs sequences of natural isolates (n = 1572) are classified into eight types of virulent Fcs (VFcs) with the motif “G/R/K-R-Q/R/K-R/K-R↓F” and ten types of the avirulent Fcs (AFcs) with the motif “G/R/E-R/K/Q-Q-G/E-R↓L”. The VFcs is only found in the Class II cluster of viral classification and not in Class I. The AFcs exists in both Class I and II isolates. The VFc and AFc types present an evolutionary relationship with temporal distribution and host species. Using a fusion assay in vitro, VFcs-1 “RRQKR↓F” and VFcs-2 “RRQRR↓F” show the highest efficiency in triggering membrane fusion. The neutral residue Q at the P3 position of the VFcs plays an enhancing role compared to effect of the basic residues R and K. A single residue K at P3 or P5 is less efficient of the fusogenic activity in the VFcs with all basic residues. Moreover, the cleavage efficiencies of F0 proteins with different types of Fcs motifs do not appear to affect membrane fusion. Our findings offer insight into the effect of amino acid variation of the Fcs on the fusion triggered by NDV.

Genomic characterization of a wild-bird-origin pigeon paramyxovirus type 1 (PPMV-1) first isolated in the northwest region of China

Pigeon paramyxovirus type-1(PPMV-1) is an enzootic in pigeon flocks and causes severe economic losses in the poultry industry in many countries. A PPMV-1 isolate, abbreviated as PPMV-1/QL-01/CH/15, was isolated from a great spotted woodpecker in the northwest region of China in 2015. The complete genome was sequenced, and the results showed that the virus genome was 15,192 nt in length, in the gene arrangement 3′-NP-P-M-F-HN-L-5′. Several amino acid mutations were identified in the functional domains of the F and HN proteins. The pathogenicity index of the isolate was evaluated, and the mean death time (MDT) was 72 h and the intracerebral pathogenicity index (ICPI) was 0.925, indicating that this isolate was mesogenic. Sequencing results showed that it had a virulent Newcastle disease virus cleavage motif 112R-R-Q-K-R-F117 at the fusion protein cleavage site. Morbidity and mortality were 70% and 50%, after inoculation of pigeons, whereas this virus was nonpathogenic in chickens. Different immune responses of pigeons and chickens were induced in vivo, which led to different HI serum antibody titers. The results of phylogenetic and evolutionary distance analysis showed that this PPMV-1 strain belonged to sub-genotype VIa in class II. To our knowledge, this is the first identification and analysis of PPMV-1 co-circulation among wild birds and domestic pigeon flocks in China. The data from this study highlight the important role of wild birds in the dissemination of PPMV-1 and provide useful references for improving our understanding of the distribution and evolution of PPMV-1 in China.

Dynamic distribution and tissue tropism of avian encephalomyelitis virus isolate XY/Q-1410 in experimentally infected Korean quail

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). However, to date, the dynamic distribution of AEV in quails has not been well described. Quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) assays were used to investigate the dynamic distribution and tissue tropism of AEV in experimentally infected Korean quail. AEV was detected in the cerebrum, cerebellum, proventriculus, intestine, liver, pancreas, spleen, bursa, lung and kidney as early as 3 days post-infection (dpi). The viral loads in the proventriculus, intestine, spleen and bursa were relatively higher than in other tissues. According to the qPCR results, AEV XY/Q-1410 infection lasted for at least 60 days in infected Korean quail. Immunohistochemistry-positive staining signals of AEV antigen were analysed by Image-Pro Plus software. A positive correlation between qPCR and IHC results was identified in most tissues. Our results provide an insight into the dynamic distribution of AEV in various tissues after infection. The distinct dynamic distribution of the viral genome in Korean quail in the early and late stages of infection suggests that AEV replication is affected by antibody levels and the maturity of the immune system of the host.

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

ABSTRACT Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148 h. The isolate possessed a motif of -112E-R-Q-E-R-L117- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.

Newcastle Disease Virus V Protein Promotes Viral Replication in HeLa Cells through the Activation of MEK/ERK Signaling

Newcastle disease virus (NDV) can infect a wide range of domestic and wild bird species. The non-structural V protein of NDV plays an important role in antagonizing innate host defenses to facilitate viral replication. However, there is a lack of knowledge related to the mechanisms through which the V protein regulates viral replication. The extracellular signal-regulated kinase (ERK) signaling pathway in the host is involved in a variety of functions and is activated by several stimuli, including viral replication. In this study, we show that both the lentogenic strain, La Sota, and the velogenic strain, F48E9, of NDV activate the mitogen-activated protein kinase (MEK)/ERK signaling pathway. The pharmacological inhibition of ERK1/2 phosphorylation using the highly selective inhibitors U0126 and SCH772984 resulted in the reduced levels of NDV RNA in cells and virus titers in the cell supernatant, which established an important role for the MEK/ERK signaling pathway in NDV replication. Moreover, the overexpression of the V protein in HeLa cells increased the phosphorylation of ERK1/2 and induced the transcriptional changes in the genes downstream of the MEK/ERK signaling pathway. Taken together, our results demonstrate that the V protein is involved in the ERK signaling pathway-mediated promotion of NDV replication and thus, can be investigated as a potential antiviral target.

Newcastle disease virus V protein inhibits apoptosis in DF-1 cells by downregulating TXNL1

Many viral proteins are related to suppressing apoptosis in target cells and are hence beneficial to viral replication. The V protein of Newcastle disease virus (NDV) is one such protein that plays an important role in inhibiting apoptosis in a species-specific manner. However, to date, there have been no reports clarifying the antiapoptotic mechanisms of the V protein. The present study was undertaken to determine the apoptotic potential of the V protein in a chicken embryo fibroblast cell line (DF-1 cell) and to elucidate its molecular mechanisms of action. Here, a yeast two-hybrid system was used to screen the host proteins that interact with the V protein and identified thioredoxin-like protein 1 (TXNL1) as a potential binding partner. Immuno-colocalization of V protein and TXNL1 protein in DF-1 cells further verified the interaction of the two proteins. Through the overexpression of TXNL1 protein and knockdown of TXNL1 protein in DF-1 cells, the effects of NDV replication and cell apoptosis were examined. Cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2 and Caspase-3 were detected by quantitative real-time PCR (Q-PCR) and Western blotting. NDV expression was detected by Q-PCR and plaque assay. The results revealed that the TXNL1 protein induced apoptosis and inhibited NDV replication in DF-1 cells. Furthermore, the Western blot and Q-PCR results suggested that TXNL1 induced cell apoptosis through a pathway involving Bcl-2\Bax and Caspase-3. Finally, this work provides insight into the mechanism by which the V protein inhibits apoptosis.