Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

Emerging multiple reassortant H5N5 avian influenza viruses in ducks, China, 2008.

Three highly pathogenic H5N5 avian influenza viruses (HPAI), A/duck/Guangdong/wy11/2008 (WY11), A/duck/Guangdong/wy19/2008 (WY19), and A/duck/Guangdong/wy24/2008 (WY24) were isolated from ducks in southern China in April 2008. Here, we characterized these viruses by performing sequencing and phylogenetic analyses of their viral genes, assessing their virulence in ducks and mice, and performing cross-protection experiments in chickens. Sequence analysis revealed that the HA genes of these H5N5 viruses showed 97.1-97.8% homology to A/wild duck/Hunan/211/2005 (H5N1) influenza virus and that their NA genes showed 96.4-96.8% nucleotide identity to the NA gene of A/duck/Hunan/5613/2003 (H6N5) influenza virus, which belongs to the Eurasian lineage. Genotypic analysis indicated that these H5N5 viruses were multiple reassortants among H5N1, H5N2, H6N2, and H6N5 viruses. The analysis of HA clade showed that these H5N5 viruses are clustering into clade 2.3.4. In animal experiments, these H5N5 viruses caused 50% mortality in ducks and 100% mortality in chickens. In cross-protection experiments, the clade 2.3.2 avian influenza vaccine could provide only 75% protection with chickens against H5N5 virus challenge. Moreover, the H5N5 virus replicated efficiently in the lungs of mice, which suggested that the H5N5 viruses have the potential to infect mammalian hosts. Since ducks have served as reassortant vessels, playing pivotal roles in the generation of new subtypes of influenza viruses, it is important to monitor the emergence of this novel subtype of influenza viruses in waterfowl to understand their ecology and evolution and to control the spread of new viruses.

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.

Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.

[Cloning and phylogenetic analysis of the entire gene of an H1N1 subtype swine influenza virus isolated from Guangdong Province].

To study the genetic variation and evolutionary characteristics of H1N1 swine influenza virus, all the eight genes of LM were amplified by RT- PCR, cloned into pMD18-T vector and sequenced respectively. The results showed that neither insertion nor deletion was observed in nucleotides of LM. The amino acids sequence of cleavage site of HA is IPSIQSR decrease G, suggesting that LM did not have the molecular characteristics of high pathogen. HA had highly conservative N-glycosylation site at position 11, 23, 87 and 276 sites of HA1, and two more at position 154 and 213 sites of HA2. NA had highly conservative N-glycosylation site at position 58, 63, 68, 88, 146, and two more at position 44 and 235 sites, which might be one molecular characteristics of H1N1 subtype of SIV. The results of Bast showed HA gene had high homology to the strain of 'human-like' SIV (99%), while others had high homology to the 'classical' SIV. So it is inferred that HA of LM might originate from human-like linage swine influenza virus, while others might originate from 'classical' swine influenza virus.

[Generation of cell culture high-yield recombinant H3N2 subtype swine influenza vaccine candidate by reverse genetics].

High-yield H3N2 subtype swine influenza virus for large-scale vaccine production in cell culture was generated by reverse genetics. The rescued H3N2 (rH3N2) candidate virus contained hemagglutinin (HA) and neuraminidase (NA) genes derived from a field isolate A/Swine/Henan/S4/01 (H3N2), PB2 gene from A/PR/8/34, and the other five internal genes from A/Goose/Dalian/3/01 (H9N2). The rH3N2 virus titer in MDCK cell culture were measured by hemagglutination assay and the maximum virus titre of 1:512 hemagglutination unit was obtained after infection of MDCK cell for 60 h. The results of the present study indicated that rH3N2 virus was suitable for growth in MDCK cell culture and is feasible to be used for the production of cell grown influenza vaccine.