Dongmei Hu

Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark.

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called “high fever” disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the “high fever” disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40–42°C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.

Pathogenic pseudorabies virus, China, 2012.

In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic.

Identification of an emerging recombinant cluster in porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is continuously evolving through point mutation and genome recombination. In the present study, genetic affiliations of 40 PCV2 strains were determined by amplification, sequencing and phylogenetic analyses during the nationwide molecular epidemiology investigation from 2009 to 2010 in mainland China. The results revealed that PCV2b was the predominant genotype in mainland China from 2009 to 2010 and PCV2b-1C within PCV-2b genotype was an emerging predominant subtype. More interestingly, five strains (09HaiN-1, 09HaiN-2, 10AH, 10GX and 10QH) were classified into a novel cluster along with the two main PCV2 genotypes PCV2a and PCV2b. Further analyses revealed that this novel cluster arose from recombination between PCV2a and PCV2b stains within the ORF2 gene. Moreover, BLAST search on NCBI website revealed that PCV2 strains of the novel cluster also emerged in Thailand, Indonesia and Laos, indicating that the novel cluster of PCV2 has also been circulating in some other Asian countries. This study is the first time to perform comprehensive analyses to demonstrate a cluster of PCV2 strains derived from the same type of inter-genotypic recombination pattern. Our findings provide valuable information on PCV2 evolution.

Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR.

Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2TCID(50)/ml or 38 RNA copies/microl for C-US-PRRSV and 0.4 TCID(50)/ml or 14 RNA copies/microl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.

Genomic Sequencing Reveals Mutations Potentially Related to the Overattenuation of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to evolve when serially passaged in Marc-145 cells. In this study, we analyzed the genomic and antigenic variants of HP-PRRSV strain JXA1 during in vitro passage. Protective efficacies of JXA1 from passages 100, 110, 120, 140, and 170 against the high-virulence parental virus were evaluated by inoculating pigs with each of these viruses and then challenging with JXA1 from passage 5 at 28 days postimmunization. We found that the antigenicities of JXA1 from passages after 110 were significantly reduced. Inoculation with JXA1 from passages after 110 provided only insufficient protection against the parental strain challenge, indicating that the immunogenicity of JXA1 is significantly decreased when it is in vitro passaged for 110 times and more. To identify the genomic variants that emerged during the overattenuation, eight complete genomes of highly passaged JXA1 were sequenced. One guanine deletion in the 5′ untranslated region (UTR), two nucleotide substitutions in the 3′ UTR, and 65 amino acid mutations in nonstructural and structural proteins that accompanied with the attenuation and overattenuation were determined. Genomic sequencing of in vitro serially passaged HP-PRRSV first identified the mutations potentially correlated with the overattenuation of a HP-PRRSV strain. These results facilitate the research aimed at elucidating the mechanisms for PRRSV genomic and antigenic changes and may also contribute to developing a safe and effective PRRSV vaccine.

Complete Genome Sequence of Two Novel Chinese Virulent Porcine Reproductive and Respiratory Syndrome Virus Variants

ABSTRACT A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by a discontinuous 30-amino-acid deletion in its Nsp2-coding region, has been emerging in China since 2006. Here, we report the complete genomic sequence of two novel Chinese virulent PRRSV variants with additional NSP2-gene deletions, which will help us understand the molecular and evolutionary characteristics of PRRSV in Asia.

Assessment of the safety and efficacy of an attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus.

ABSTRACT The safety and efficacy of the JXA1-R vaccine, an attenuated strain of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), were examined using an intramuscular challenge model in piglets. The JXA1-R vaccine was obtained by passing HP-PRRSV JXA1 through Marc-145 cells (82nd passage). Genomic sequence comparisons showed that strain JXA1-R and its parental strain, JXA1, differ by 47 amino acids, and most of these differences are scattered throughout the PRRSV genome. Four-week-old PRRSV-free piglets were inoculated intramuscularly with JXA1-R vaccine (103.0, 104.0, 105.0, 106.0, and 107.0 50% tissue culture infective doses [TCID50]/ml for groups 1 to 5, respectively) and then challenged intramuscularly with the 5th passage virus of JXA1 virus (JXA1-F5, 3 ml × 104.5 TCID50/ml) 28 days after inoculation. The humoral immune response, swine growth, clinical signs, and differential organ lesions were monitored. The results showed that all vaccinated piglets had a perceptible humoral immune response to vaccination after day 7, which then promptly increased, almost reaching the maximum sample/positive (S/P) ratio value at 28 days postimmunization. Viremia detection indicated that the viral replication levels of the challenge virus in the immunized groups (immunization doses ≥104.0/ml) were significantly lower than that of the virus-challenged unvaccinated control group. Piglets in groups 2 to 5 were effectively protected against lethal HP-PRRSV infection and did not show any obvious changes in body temperature or clinical signs of disease at any point during the experiment. However, two of five piglets in group 1 showed mild pathological lesions and transitory high fever. These results suggest that JXA1-R (TCID50/ml ≥104.0) is sufficiently attenuated and can provide effective protection against the lethal wild-type HP-PRRSV.

Complete genome sequence of a European genotype porcine reproductive and respiratory syndrome virus in china.

ABSTRACT Here, we report a novel European genotype porcine reproductive and respiratory syndrome virus (PRRSV) strain with 15 nucleotide deletions in the nonstructural protein 2 region and 3 nucleotide deletions in the overlapping regions of the open reading frame 3 (ORF3) and ORF4 regions. This study will aid in further exploration of the genetic and antigenic diversity of the European genotype of the PRRSV in China.

A Novel Porcine Circovirus Type 2a Strain, 10JS-2, with Eleven-Nucleotide Insertions in the Origin of Genome Replication

ABSTRACT Here, we report a novel porcine circovirus type 2a (PCV2a) strain with 11 nucleotides (nt) inserted in the origin of genome replication (Ori). This is the first report of a PCV2a strain with nucleotide insertion in Ori. Our study will help further epidemiological studies and extend our knowledge of evolutionary characteristics of PCV2.