Zengqi Yang

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.

Phylogenetic characterization and virulence of two Newcastle disease viruses isolated from wild birds in China

Wild birds are considered as a natural reservoir of Newcastle disease virus (NDV). However, there is no information about genotype IX NDV from wild birds, especially from Columbiformes. In this study, two genotype IX NDV viruses were isolated from wild birds. One was from Eurasian Blackbird, while the other was from Spotted-necked dove. After purification by plaque technique, complete genomes of both viruses were sequenced. Phylogenetic analysis of partial fusion (F) gene and complete genome indicated both strains belonged to genotype IX. Based on intracerebral pathogenicity index (ICPI), the virus from Eurasian Blackbird was velogenic virus, while the strain from Spotted-necked dove was lentogenic virus. However, both strains showed one of velogenic cleavage sites. In addition, the strain from Eurasian Blackbird showed greater replication ability and generated larger fusion foci in vitro than that of strain from Spotted-necked dove. Comparing all the corresponding protein sequences of both strains, there were only 9 different amino acid residues between them. Furthermore, after analysis of these differences, the information about lentogenic NDV with multi-basic cleavage site was presented.

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.

Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2).

Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×106 cfu/3 µg and 2×109 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy.

Emergency vaccination alleviates highly pathogenic porcine reproductive and respiratory syndrome virus infection after contact exposure

BackgroundTo assess the effectiveness of emergency vaccination for reducing the contact-induced infection and pathological damage caused by the highly pathogenic porcine reproductive and respiratory syndrome virus (HPPRRSV), Twenty pigs were equally divided into four groups. Groups 1, 2 and 3 were housed in one unit, whereas Group 4 was separately housed. Group 1 was challenged with HPPRRSV on day 0. Group 2 and 4 did not receive treatment and were used as the contact-infected and uninfected controls, respectively. Group 3 was treated with the attenuated vaccine at 0 days post-inoculation. The rectal temperatures, clinical signs, pathologic lesions and viraemia of the piglets were detected and evaluated.ResultsThe vaccinated pigs in Group 3 showed less clinical morbidity, viraemia, temperature fluctuations and lung lesions at 14 days post-inoculation, as compared with the contact-infected (Group 2) and experimentally infected (Group 1) pigs. Higher serum IFN-γ levels were detected among the pigs that received emergency immunisation. Thus, IFN-γ may be involved in immunity against HPPRRSV infection.ConclusionsThese results indicated that emergency vaccination could effectively alleviate HPPRRSV infection during experimental contact exposure. Our findings provide a novel and useful strategy for controlling clinical HPPRRSV.

Oral administration of attenuated Salmonella typhimurium containing a DNA vaccine against rabbit haemorrhagic disease

The use of attenuated Salmonella typhimurium as a bactofection vehicle for the oral delivery of a DNA vaccine against rabbit haemorrhagic disease virus (RHDV) was investigated. The DNA vaccine plasmid pcDNA3.1-VP60, which encodes the viral capsid protein VP60, was transformed into the attenuated S. typhimurium strain SL7207. The resulting recombinant bacteria, named as SL/pcDNA3.1-VP60, were orally used to immunise rabbits. The successful delivery of the DNA plasmid was confirmed by the detected VP60 transcription in the rabbit intestines through the reverse transcription polymerase chain reaction. In addition, the RHDV-specific humoral and cell-mediated immune response that was induced by SL/pcDNA3.1-VP60 was detected by the enzyme-linked immunosorbent assay as well as the assays for T lymphocyte proliferation and cytokines secretion. The significant protection of immunised rabbits against the RHDV strain XA/China/2010 at 42 d post-immunisation was demonstrated. This study is the first report about the efficient usage of attenuated Salmonella as a live vector for the oral delivery of a DNA vaccine against RHDV.

Adenovirus-based oral vaccine for rabbit hemorrhagic disease

Vaccine antigens for rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from the livers of experimentally infected rabbits or from several recombinant immunogens. However, the application of these vaccine antigens has been restricted because of biosecurity and immunity characteristics. In the current study, a recombinant adenovirus expressing the RHDV capsid protein (VP60) was constructed and the expression of the recombinant protein was identified through western blot analysis using RHDV-positive rabbit sera. Eighteen rabbits were immunized by injection, direct oral instillation, or using bait. They were challenged with RHDV isolate three weeks after boost immunization. In all cases, the rabbits immunized with the recombinant adenovirus developed RHDV-specific antibodies and cell immune response. The rabbits injected with the recombinant adenovirus were completely protected against RHDV challenge. The adenovirus expression system may provide a strategy for the immunization of rabbits, particularly for the control of RHDV in wild rabbits.

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.

Antigenic characteristics of glycosylated protein 3 of highly pathogenic porcine reproductive and respiratory syndrome virus

Highly pathogenic (HP)-porcine reproductive and respiratory syndrome virus (PRRSV) emerged in 2006 and has now become a global threat to pig farms. Despite extensive characterization of HP-PRRSV proteins by direct analysis and comparison with typical PRRSV, immune recognition remain poorly understood. Glycosylated protein 3 (GP3) has an important function in inducing protective immune response. To analyze the antigenic character of HP-PRRSV GP3, a total of 217 peptides were printed on a chip and used to react with HP-PRRSV specific serum. The reactions of these peptides to HP-PRRSV specific pig serum were scanned and quantified using the software PepSlide Analyzer by fluorescence intensity. The intensity plots showed various reactions in different parts of GP3. The highest reaction intensity value reached 29,184.5 with the peptide sequence of CSENDHDELGFMVPP. Conversely, 88 peptides showed no reaction with 0 florescence intensity. A further analysis based on the result of the peptide microarray revealed an antigen reaction active region (AR) from Y(51) to S(106) in GP3. The AR had four parts of variation that may be a significant mutation of the typical PRRSV to HP-PRRSV. Acquired data may be useful for understanding HP-PRRSV variation and its GP3 immune recognition.

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.