Development and evaluation of a C-ELISA for rapid detection of antibody to AIV- H7.

Since 2013, the H7 subtype avian influenza virus (AIV-H7) has seriously endangered human life and health, and has had a serious impact on the poultry industry in China. A competitive enzyme-linked immunosorbent assay (C-ELISA) which detects the antibody for AIV-H7 was developed, basing on a monoclonal antibody (mAb) against the neutralizing epitopes on hemagglutinin (HA)gene. Twelve hybridoma cell lines were screened by cell fusion. Hemagglutination inhibition (HI) assay and indirect ELISA were used to identify the competitive effect of the mAbs. High-affinity mAb 1H11 was selected as a competitive antibody. The reaction conditions for the C-ELISA were optimized for AIV-H7 antibody detection. The cross-reactivity of the C-ELISA was determined by AIV-(H1H15), NDV, IBV and IBDV positive serum. A total of 1294 field samples (chicken (462), duck (318), goose (219), quail (203) and pigeon (92) were simultaneously detected by C-ELISA and HI assay. The C-ELISA was found to have a high specificity of 93.23% and a sensitivity of 96.24%. These results reveal a positive coincidence between C-ELISA and HI assay at a coincidence rate of 97.52%. In addition, It confirmed that this method can be used for the diagnosis of AIV-H7 antibodies from chicken, ducks, goose, quail and pigeons.

Propidium Monoazide Combined With RT-qPCR Detects Infectivity of Porcine Epidemic Diarrhea Virus.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.

Detection of suid herpesvirus 1 infectivity in pigs by propidium monoazide-qPCR.

At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50-100 µM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.

Evolutionary characterization of influenza virus A/duck/Hubei/W1/2004 (H9N2) isolated from central China.

Full-length eight gene segments of avian influenza virus A/duck/Hubei/W1/2004(H9N2) (Dk/Hub/W1/04) were amplified by RT-PCR and completely sequenced. Phylogenetic analysis revealed that Dk/Hub/W1/04 was derived from A/Duck/HongKong/Y280/97, not displaying direct evolutional relationship with A/Quail/HongKong/G1/97 or Hubei H5N1 viruses. Meanwhile, Dk/Hub/W1/04 was found highly related to recent three chicken isolates. The connecting peptide of HA and the deletion in NA stalk were consistent with three chicken isolates, and the number of potential glycosylation site on the HA and NA also was similar or identical to the three chicken isolates. These findings suggested that Dk/Hub/W1/04 is likely to transmit back to ducks from chickens.

Inhibition of Japanese encephalitis virus NS1 protein expression in cell by small interfering RNAs.

Japanese encephalitis virus (JEV), a serious mosquitoborne flavivirus, causes an acute infection of the central system resulting in encephalitis of humans and many kinds of animals. A high proportion of the survivors exhibit neurogical and psychiatric sequelae. NS1 is one of important non-structural proteins, which was found to be associated with viral RNA replication. To inhibit NS1 expression, four small interfering RNAs (siRNAs) expression plasmids (pS-NS1A, pS-NS1B, pS-NS1C and pS-NS1D) were generated to target four different coding regions of the NS1 gene, and were separately co-transfected into Vero cells with an NS1-EGFP fusion expression plasmid pNS1-EGFP. NS1 expression was evaluated by fluorescence microscope, flow cytometry assay, Western blot and RT-PCR. The results revealed that pS-NS1B, pS-NS1C and pS-NS1D could effectively and specifically inhibit NS1 expression in Vero cells. Our data suggested that these siRNAs could be used to inhibit JEV replication by silencing NS1 protein expression in further study.

Construction of recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis (SA14-14-2) virus and its safety and immunogenicity.

The bivalent genetic engineering vaccine of Japanese encephalitis (JE) and Aujeszkj disease (AD) was developed to provide a novel approach to prevent and control these two diseases. NS1 gene of Japanese encephalitis virus (JEV) SA14-14-2 strain was produced by reverse transcriptase-mediated PCR (RT-PCR) and was cloned into vector pUSK to form recombinant plasmid (designed as pUSK-NS1). A co-transfection experiment was performed in porcine kidney (PK-15) cells with pUSK-NS1 and the genome of the vector virus (PRV TK(-)/gG(-)/LacZ(+) mutant). By plaque purification, PCR detection and southern hybridization, recombinant pseudorabies virus (PRV) expressing NS1 protein of JEV was acquired and named TK(-)/gG(-)/NS1(+). Western blot analysis and ELISA demonstrated the NS1 protein expression. To evaluate the recombinant virus's potential application, we characterized the safety and immune responses in Balb/c mice and swine. The safety test indicated that, when receiving the recombinant virus at a concentration of 10(6.0)pfu, no virulence of the recombinant virus to the mice, piglets and pregnant sows was observed. The vaccinated animals could acquire protective immunity against lethal challenge of the virulent PRV Ea strain and develop a good humoral and cellular immune response against JEV. The above results revealed that the recombinant virus could be a suitable candidate vaccine strain for developing a novel genetic vaccine to combat pseudorabies and Japanese encephalitis in the pig industry.

[Construction and characterization of a pseudorabies virus TK-/gG- mutant].

To construct a TK-/gG- mutant of pseudorabies virus, the gG-detected transfer vector pUSKKBB and genomic DNA of pseudorabies virus TK-/gG-/LacZ+ were co-transfected into IBRS-2 cells. Transfection progeny were plated onto PK-15 cells and incubated for 2 days under methylcellulose. Then the overlay was removed and replaced by 1% low melting point agarose in DMEM supplemented with 150 microg/mL X-gal. After 2 days, white plaques were screened for and purified 4 times. By PCR amplification of gG-deleted gene and LacZ gene, a recombinant virus with TK-/gG- phenotype was confirmed. Sequence of the PCR product revealed that there were 1,176 bp detection in gG gene of the PRV TK-/gG- mutant. Amplifying the gG-deleted gene of different generations of the TK-/gG- mutant showed that the mutant was stable within PK-15 cells. TCID50 assay indicated that the recombinant virus grows well on PK-15 cells. The mice immunized with the TK-/gG- virus showed no sign of abnormality. As a control, all mice inoculated with PRV strain died from the infection. All mice that received TK-/gG- survived after a lethal PRV challenge. However none of the mice injected with phosphate-buffer saline (PBS) survived from the challenge. The above results demonstrated that the recombinant virus could be a candidate marker vaccine strain for eradicating pseudorabies in pig herds.