Minze Zhang

Avian-origin H3N2 canine influenza virus circulating in farmed dogs in Guangdong, China.

Since 2006, more and more cases of the infectious H3N2 canine influenza virus (CIV) in pet dogs have been reported in southern China. However, little is known about the prevalence situation of H3N2 CIV infections in farmed dogs in China. This is the first systematic epidemiological surveillance of CIV in different dog populations in southern China. Two virus strains A/Canine/Guangdong/1/2011(H3N2) and A/canine/Guangdong/5/2011(H3N2) were isolated from canine nasal swabs collected at one dog farm in Guangzhou and the other farm in Shenzhen. Sequence and phylogenetic analysis of eight gene segments of these viruses revealed that they were most similar to the newly isolated canine H3N2 viruses in dogs and cats from Korea and China, which originated from avian strain. This indicates that H3N2 CIV may be a common pathogen for pet and farmed dog populations in southern China at present. Serological surveillance has shown that the infection rate of this avian-origin canine influenza in farmed dogs and in pet dogs were 12.22% and 5.3%, respectively; as determined by the ELISA. The data also suggested that transmission occurred, most probably by close contact, between H3N2 CIV infected dogs in different dog populations in recently years. As H3N2 outbreaks among dogs continue in the Guangdong province (located very close to Hong Kong), the areas where is densely populated and with frequent animal trade, there is a continued risk for pets H3N2 CIV infections and for mutations or genetic reassortment leading to new virus strains with increased transmissibility among dogs. Further in-depth study is required as the H3N2 CIV has been established in different dog populations and posed potential threat to public health.

Genetic evolution and phylogenetic analysis of porcine circovirus type 2 infections in southern China from 2011 to 2012

Porcine circovirus type 2 (PCV2), the primary causative agent of porcine circovirus-associated diseases (PCVADs), is a serious economic problem for the swine industry worldwide. Three major PCV2 genotypes (PCV2a, PCV2b, and PCV2c), have been identified. To explore the prevalence of different subgroups of PCV2 in southern China, 66 PCV2 isolates collected during 2011-2012 were analyzed. PCV2b was the predominant genotype circulating in southern China from 2011 to 2012. Moreover, subtype 1C was the predominant subtype. Comparisons of the complete ORF2 nucleotide sequence revealed 89.3-100% homology and 87.2-100% amino acid sequence identities. The deletion at position 1042 and two nucleotide substitutions at positions T1035A or T1033C were important for the PCV2 evolution. Base-by-base of ORF2 comparison showed that the PCV2 evolution trace was PCV2a to PCV2b-1A1B to PCV2b-1C. These results contribute to the understanding of PCV2 epidemiology in southern China.

Epidemiological and evolutionary characteristics of the PRRSV in Southern China from 2010 to 2013

In 2006, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in China and caused lasting damage to the swine industry. To analyze the genetic variation of PRRSV in Southern China, 126 tissue samples were collected; 41 ORF5 and partial Nsp2 genes were sequenced and analyzed. The results showed that the PRRSV positive rate was 32.54% over the last four years, that there are two main subgenotypes in Southern China, and that the dominant strain is HP-PRRSV. An amino acid analysis of Nsp2 showed that 40 strains contained a 30-amino acid deletion in the hypervariable region. However, the 13YJ6-8 mutant exhibited a unique amino acid deletion at positions 508-514 of Nsp2. A phylogenetic analysis of ORF5 revealed that this mutant and five other strains, belong to an intermediate subgenotype (inter-subgenotype), which is characterized by extensive mutations, especially in the signal peptide and N-glycosylation sites. The results of this study demonstrate the genetic diversity of PRRSV in Southern China and provide basic knowledge of the PRRSV epidemic in this region.

Molecular epidemiology of PRRSV in South China from 2007 to 2011 based on the genetic analysis of ORF5

Porcine reproductive and respiratory syndrome virus (PRRSV) has proven to be highly genetically variable; however, comprehensive information regarding the viruss genetic diversity in South China is limited. In this study, a total of 3199 clinical samples were collected from 267 pig farms suspected of PRRSV infection between 2007 and 2011. The ORF5 genes of 51 PRRSV-positive samples were sequenced and analyzed. The 51 study strains were divided into three primary subgenotypes. Fourty-five of the strains belonged to subgenotype I and were closely related to the highly pathogenic PRRSV (HP-PRRSV) strains. The subgenotype I strains were generally clustered into genetically similar groups by year. Only one of the strains belonged to subgenotype II, clustering with the classical North American type, VR2332. Five of the strains were grouped into subgenotype III, which occupied a separate branch and was closely related to the recently isolated novel field strains, QYYZ and GM2. The 5 subgenotype III strains shared an amino acid identity with the remaining 46 study strains ranging from 79.6%-83.6%. Amino acid analysis showed extensive mutations in subgenotype III; the diverse genetic mutations of these novel strains are of great concern.

Complete Genome Sequence of Duck Tembusu Virus, Isolated from Muscovy Ducks in Southern China

ABSTRACT We report here the complete genomic sequence of the duck Tembusu virus (DTMUV) WJ-1 strain, isolated from Muscovy ducks. This is the first complete genome sequence of DTMUV reported in southern China. Compared with the other strains (TA, GH-2, YY5, and ZJ-407) that were previously found in eastern China, WJ-1 bears a few differences in the nucleotide and amino acid sequences. We found that there are 47 mutations of amino acids encoded by the whole open reading frame (ORF) among these five strains. The whole-genome sequence of DTMUV will help in understanding the epidemiology and molecular characteristics of duck Tembusu virus in southern China.

Genetic characterization of avian-origin H3N2 canine influenza viruses isolated from Guangdong during 2006–2012

Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory disease in dogs. In 2006, the H3N2 canine influenza virus was first identified in dogs from Guangdong province in China. Up to now, nine CIVs have been isolated from different populations in Guangdong. The nine isolates were grouped together with the canine H3N2 viruses isolated from dogs and felines in Korea, when the eight phylogenetic trees constructed were compared. These findings emphasize the importance of CIV surveillance in this region for understanding the genesis of this virus, and it is important to remain aware of the potential of H3N2 CIV to be transmitted from dogs to the human population.

Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspβ) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What’s more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.

Inhibition of porcine reproductive and respiratory syndrome virus by specific siRNA targeting Nsp9 gene

To screen siRNAs for effectively inhibiting the replication of porcine reproductive and respiratory syndrome virus (PRRSV). Four pairs of siRNA targeting Nsp9 gene of PRRSV and one non-efficient pair used as control were designed, synthesized and cloned into pSilencer4.1-CMV neo, designated as pSi-294, pSi-367, pSi-409, pSi-1488, pSi-Ctr. The recombinant plasmids were transfected into Marc-145 cells and infected with PRRSV 24h post transfection. Subsequently, IFA, real-time PCR, TCID50 and western blot were used for evaluating the inhibitory effect of the siRNA. IFA and western-blot results showed that pSi-294, pSi-1488 can effectively inhibit the expression of Nsp9 and M protein of PRRSV, real-time PCR result showed that the expression of Nsp9 gene were decreased from 86.56% to 93.66% compared to the negative control. siRNAs can be used as candidates for basic research of PRRSV.

Expression and Antibody Preparation of GP5a Gene of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most important infectious diseases to affect the swine industry and characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The GP5a gene, encoding RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. It plays an important role in the process of duplication and transcription carried out by Porcine reproductive and respiratory syndrome virus (PRRSV). We firstly expressed and purified the GP5a protein of PRRSV. This provides a good method for the purification of expressed proteins and the preparation of the corresponding antibodies.

Short communication: isolation and phylogenetic analysis of an avian-origin H3N2 canine influenza virus in dog shelter, China

A H3N2 canine influenza virus, A/canine/Guangdong/3/2011 (H3N2), was isolated from roaming dogs in rural China. Sequence and phylogenetic analysis of eight gene segments revealed that the A/canine/Guangdong/3/2011 (H3N2) was most similar to a recent H3N2 canine influenza virus isolated in cats from South Korea, which originated from an avian strain. To our knowledge, this is the first report of an avian-origin H3N2 CIV which was isolated from roaming dogs in China. The epidemiologic information provided herein suggests that continued study is required to determine if this virus could be established in the roaming dog population in rural China and pose potential threats to public health.