A set of RT-PCR assays for detection of all known avian paramyxoviruses and application in surveillance of avian paramyxoviruses in China.

BACKGROUND: Avian paramyxoviruses (APMVs), also termed avian avulaviruses, are of a vast diversity and great significance in poultry. Detection of all known APMVs is challenging, and distribution of APMVs have not been well investigated. METHODS: A set of reverse transcription polymerase chain reaction (RT-PCR) assays for detection of all known APMVs were established using degenerate primers targeting the viral polymerase L gene. The assays were preliminarily evaluated using in-vitro transcribed double-stranded RNA controls and 24 known viruses, and then they were employed to detect 4,346 avian samples collected from 11 provinces. RESULTS: The assays could detect 20-200 copies of the double-stranded RNA controls, and detected correctly the 24 known viruses. Of the 4,346 avian samples detected using the assays, 72 samples were found positive. Of the 72 positives, 70 were confirmed through sequencing, indicating the assays were specific for APMVs. The 4,346 samples were also detected using a reported RT-PCR assay, and the results showed this RT-PCR assay was less sensitive than the assays reported here. Of the 70 confirmed positives, 40 were class I Newcastle disease virus (NDV or APMV-1) and 27 were class II NDV from poultry including chickens, ducks, geese, and pigeons, and three were APMV-2 from parrots. The surveillance identified APMV-2 in parrots for the first time, and revealed that prevalence of NDVs in live poultry markets was higher than that in poultry farms. The surveillance also suggested that class I NDVs in chickens could be as prevalent as in ducks, and class II NDVs in ducks could be more prevalent than in chickens, and class II NDVs could be more prevalent than class I NDVs in ducks. Altogether, we developed a set of specific and sensitive RT-PCR assays for detection of all known APMVs, and conducted a large-scale surveillance using the assays which shed novel insights into APMV epidemiology.

[Detection of an NA gene molecular marker in H7N9 subtype avian influenza viruses by pyrosequencing].

This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.

Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses.

Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.

[A review of research on Schmallenberg virus].

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.

[Establishment of reverse genetics system for class I NDV08-004 strain].

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.

[Detection of molecular markers of amantadine resistance in avian influenza viruses by pyrosequencing].

Mutation in any of five key amino acid residues (at positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses leads to resistance against the amantodine class of anti-influenza drugs. In this study, a pyrosequencing method was described to rapidly detect established five molecular markers of resistance to M2 blockers, amantadine. The residues L26, V27, A30, S31 and G34 in the M2 protein were targeted for pyrosequencing, and 94 avian influenza viruses were used to perform the amantadine resistance analysis. Our results showed that most of avian influenza viruses were amantadine resistant, Mutations V27I and S31N were founded in these isolates.

[Molecular epidemiological analysis of class I Newcastle disease virus isolated from China in 2008].

Thirteen isolates of Class I Newcastle disease virus obtained from healthy poultry in China during 2008 were characterized genotypically in this study. All the isolates were proved to be lentogenic strains based on the deduced amino acid sequence of the Fusion protein gene. Molecular epidemiological analysis showed that 13 isolates could be subdivided into 2 distinct genotypes, 11 isolates belonged to genotype 2, and other 2 isolates belonged to genotype 3. Results indicated two genotypes of Class I Newcastle disease virus might widely exist in domestic poultry in China.

[PCR site-directed mutagenesis of avian influenza virus hemagglutinin gene in vitro and expression in 293T cell].

The site-directed mutagenesis of HA gene was made by using PCR, and mismatches were introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the second PCR which contained the mutation site were cloned into the pcDNA3 vector, named pHAm. The sequencing analysis showed that the mutation site was correct. The amino acid sequence at the cleavage site of the HA protein was from RKKR decrease GLF to RSSR decrease GLF. The recombinant plasmid pHAm was transiently transfected into 293T cells by the calcium phosphate precipitation method. Indirect immunofluorescent assay (IFA), confirmed expression of the HA protein on the cell membrane, the mutant HA gene was a promising candidate for further studies.

[The prokaryotic expression and the establishment of the putative indirect ELISA assay for the HA gene for avian influenza virus (AIV) H5N1 subtype].

Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank, the HA1 gene of H5N1 subtype AIV was amplified with PCR method. The PCR product was cloned into pET-32a(+) to get a prokaryotic recombinant plasmid pET-HA1. The target gene was successfully expressed in the host cell BL21 (DE3) when induced with IPTG. The expression was optimized with proper inducing conditions of 0.8 mmol/L IPTG and 3 hours induction. The highest expression of the target protein added up to 32.7% of the total bacterial protein. Western blot analysis proved the recombinant protein has good reactive ability against H5N1 subtype AIV positive serum. The optional working circumstances for the iHA-ELISA assay (antigenicity concentration: 4 microg/mL; serum dilution: 1:200) was tried out with chess titration. The positive criterion of this ELISA assay is OD(the tested serum) > 0.5 and OD(the tested serum)/OD(the negative serum) > 2.0.

[Characterization of murine leukemia virus recombinants that express H5N1 subtype avian influenza virus hemagglutinin glycoproteins].

One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F-3. In order to study the viral entry mechanisms, the hemagglutinin (HA) gene of H5N1 subtype AIV isolate was amplified by RT-PCR, and then cloned into pGEM-T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405. The HA gene of F-3 had a complete open reading frame (ORE) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA-HA can express the HA glycoprotein. Co-transfected pcDNA-HA, pHIT60 (include Murine Leukemia Virus structural genes, namely gag and pol) and pHIT111 (retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post-transfection, filtered through 0.45 micromol/L filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T, NIH3T3 and COS-7, all the three kinds of cells infected were lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.

Electrophysiological effects of resveratrol on guinea pig papillary muscles.

The purpose of this study was to investigate the electrophysiological effects of resveratrol on guinea pig papillary muscles and the underlying mechanism. Action potentials were recorded by using intracellular microelectrode technique. The results obtained are as follows: (1) In normal papillary muscles, resveratrol (30, 60, and 120 micromol/L) shortened the duration of action potential (APD) in a concentration-dependent manner. (2) In partially depolarized papillary muscles, resveratrol (60 micromol/L ) not only shortened APD, but also decreased the amplitude of action potential (APA), overshoot (OS) and maximal rate of depolarization in phase 0 (Vmax). (3) Perfusion with Ca2+-free K-H solution, completely abolished the effects of resveratrol (60 micromol/L) on papillary muscles. (4) Application of potassium channel blocker tetraethylammonium chloride (TEA, 20 mmol/L) did not prevent the effect of resveratrol (60 micromol/L) on action potential. (5) Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), a nitric oxide (NO) synthase inhibitor, failed to abolish the effect of resveratrol (60 micromol/L). All these results indicate that the electrophysiological effects of resveratrol on guinea pig papillary muscles are likely due to the reduction of calcium influx, which might not be mediated by NO.