Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Emergence of human-like H3N2 influenza viruses in pet dogs in Guangxi, China.

BACKGROUND: After the 1968 H3N2 pandemic emerged in humans, H3N2 influenza viruses continuously circulated and evolved in nature. An H3N2 variant was circulating in humans in the 1990s and subsequently introduced into the pig population in the 2000s. This virus gradually became the main subtype of swine influenza virus worldwide. However, there were no reports of infections in dogs with this virus. FINDINGS: In 2013, 35 nasal swabs from pet dogs were positive for Influenza A virus by RT-PCR. Two viruses were isolated and genetically characterized. In the phylogenetic trees of all gene segments, two H3N2 canine isolates clustered with Moscow/10/99 and most H3N2 swine influenza viruses. These results indicated that two H3N2 CIVs possessed high homology with human/swine influenza viruses, which at the same time exhibited some amino acid substitutions in NA, polymerase basic protein 1 (PB1), and nucleoprotein (NP), which probably were related to the interspecies transmission. CONCLUSIONS: These two viruses share the highest homology with swine H3N2, Moscow/99-like viruses, which indicated that these viruses might originate from swine viruses.

Molecular epidemiology and phylogenetic analysis of diverse bovine astroviruses associated with diarrhea in cattle and water buffalo calves in China.

Astroviruses are the principal causative agents of gastroenteritis in humans and have been associated with diarrhea in other mammals as well as birds. However, astroviral infection of animals had been poorly studied. In the present study, 211 rectal swabs collected from cattle and water buffalo calves with mild to severe diarrhea were tested for bovine astrovirus (BAstV) by RT-PCR. Results: 92/211 (43.6%) samples were positive for BAstV, at a rate of 46.10% (71/154) in cattle and 36.84% (21/57) in water buffalo. Phylogenetic analysis based on the partial and full-length of 25 ORF2 amino acid sequences obtained in this study classified the Guangxi BAstVs isolates into five subgroups under the genus of Mamastrovirus, genotype MAstV33, which suggested that the water buffalo was a new host of this genogroup that previously included only cattle and roe deer. Despite the origin of the host, the Guangxi BAstV isolates were closely related to the BAstV Hong Kong isolates (B18/HK and B76-2/HK), but highly divergent from the BAstV NeuroS1 isolate previously associated with neurologic disease in cattle in the U.S.A. Nucleotide sequence-based characterization of the ORF1b/ORF2 junction and corresponding overlapping regions showed distinctive properties, which may be common to BAstVs. Our results suggested that cattle and water buffalo are prone to infection of closely related astroviruses, which probably evolved from the same ancestor. The current study described astroviruses in water buffalo for the first time and is thus far among the largest epidemiological investigations of BAstV infection in cattle conducted in China.

High prevalence of torque teno sus virus in China and genetic diversity of the 5' non-coding region.

Members of the family Anelloviridae are emerging circular DNA viruses infecting many species of vertebrates including pigs. To date, members of two distinct genera, Iotatorquevirus, including torque teno sus virus 1a and torque teno sus virus 1b (TTSuV1a and TTSuV1b), and Kappatorquevirus, including torque teno sus virus k2a and torque teno sus virus k2b (TTSuVk2a and TTSuVk2b), have been identified in domestic pigs and wild boars. The goal of this study was to evaluate the prevalence and genetic diversity of these viruses based on 5' non-coding genes in Chinese swine herds experiencing clinical symptoms. One hundred eighty-five clinical samples from 11 different regions, collected during 2008-2009, were analyzed using a PCR method, and the results revealed a high TTSuV-positive rate of 78.9 % (146/185) in pigs. Moreover, we detected co-infection with multiple TTSuV strains in the same pig. Nucleotide sequencing results revealed greater genetic diversity within the genus Kappatorquevirus than within the genus Iotatorquevirus. In addition, TTSuVk2b, a novel virus discovered in New Zealand in 2012, was also identified in this study. In summary, the present work helps us obtain more knowledge about the epidemiology and genetic diversity of TTSuVs.

Co-existence of multiple strains of porcine circovirus type 2 in the same pig from China.

Pigs are often co-infected by different viral strains from the same virus. Up to now, there are few reports about co-existence of different porcine circovirus type 2 (PCV2) strains in China. The aim of this study was to evaluate it in Chinese swine herds. 118 PCV2 positive DNAs isolated from diseased pigs identified by classic PCR were re-detected using a modified differential PCR assay. The results indicated that co-existence rates of PCV2 were 32.2% (38/118) in diseased pigs and 0% (0/41) in asymptomatic pigs. Four PCV2 complete genomes were cloned from two co-infected samples and their nucleotide (nt) identities were 95%-97.3%. The phylogenetic analysis showed that four PCV2 strains were divided into different genotypes, PCV2a, PCV2b, PCV2d and PCV2e, respectively. In addition, co-existence were not detected in 41 serum samples from healthy pigs but PCV2 single infection (31.7%, 13/41) existed. These data revealed that the co-existence of different strains of PCV2 might contribute to the development of more severe clinical symptoms for pigs. This is the first report confirming the co-existence of different PCV2 strains in Chinese swine herds. Meanwhile, this study could help us to understand new infection and prevalence forms of PCV2 clinically.