Hanchun Yang

The 30-Amino-Acid Deletion in the Nsp2 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China Is Not Related to Its Virulence

ABSTRACT During the past 2 years, an atypical clinical outbreak, caused by a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with a unique 30-amino-acid deletion in its Nsp2-coding region, was pandemic in China. In this study, we generated four full-length infectious cDNA clones: a clone of the highly virulent PRRSV strain JXwn06 (pWSK-JXwn), a clone of the low-virulence PRRSV strain HB-1/3.9 (pWSK-HB-1/3.9), a chimeric clone in which the Nsp2 region containing the 30-amino-acid deletion was replaced by the corresponding region of the low-virulence PRRSV strain HB-1/3.9 (pWSK-JXwn-HB1nsp2), and a mutated HB-1/3.9 clone with the same deletion in Nsp2 as JXwn06 (pWSK-HB1-ND30). We also investigated the pathogenicities of the rescued viruses (designated RvJXwn, RvJXwn-HB1nsp2, RvHB-1/3.9, and RvHB1-ND30, respectively) in specific-pathogen-free piglets in order to determine the role of the 30-amino-acid deletion in the virulence of the highly pathogenic PRRSV. All the rescued viruses could replicate stably in MARC-145 cells. Our findings indicated that RvJXwn-HB1nsp2 retained high virulence for piglets, like RvJXwn and the parental virus JXwn06, although the survival time of piglets infected with RvJXwn-HB1nsp2 was obviously prolonged. RvHB1-ND30 exhibited low virulence for piglets, like RvHB-1/3.9 and the parental virus HB-1/3.9. Therefore, we conclude that the 30-amino-acid deletion is not related to the virulence of the highly pathogenic PRRSV emerging in China.

Porcine reproductive and respiratory syndrome in China.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important viral disease for the pig industry worldwide. This disease has brought great losses to the Chinese pig production in recent years, particularly following the emergence of the highly pathogenic PRRS virus (PRRSV), and has become an intractable problem for the development of pig industry in China. This paper will review the history of PRRS, the epidemic of atypical PRRS caused by the highly pathogenic virus, and the molecular characteristics of the Chinese highly pathogenic PRRSV, and the development of vaccines against PRRS in China, as well as current control status and perspective of PRRS in China.

Genomic characterization of two Chinese isolates of Porcine respiratory and reproductive syndrome virus

Summary.The genomes of two isolates of Porcine respiratory and reproductive syndrome virus (PRRSV) from China, designated HB-1(sh)/2002 and HB-2(sh)/2002, were sequenced and analyzed. The size of the genomes of HB-1(sh)/2002 and HB-2(sh)/2002 were 15,411 and 15,373 nucleotides respectively, excluding the poly(A) tails. Comparative analysis with the genomic sequences of another Chinese isolate (BJ-4) and North American (VR2332) and European (Lelystad virus, LV) viruses revealed that HB-1(sh)/2002 shared 89.8% identity with BJ-4 and VR2332, but only 54.7% with LV; while HB-2(sh)/2002 shared 89.4% and 89.5% identity with BJ-4 and VR2332 respectively and 54.3% with LV, indicating that the two new Chinese isolates were related to the North American PRRSV genotype. Phylogenetic analysis based on the nucleotide sequence of the structural protein ORF’s showed that the two new Chinese isolates belong to same genetic subgroup. HB-2(sh)/2002 additionally exhibited variations in the NSP2 nonstructural protein encoded by ORF1 and the structural protein GP3 encoded by ORF3 in comparison with other North American PRRSV isolates, namely a 12 amino acids deletion in Nsp2 and one amino acid deletion in GP3 were found in HB-2(sh)/2002. Therefore, HB-2(sh)/2002 was a novel strain with unique deletions.

Genetic variation analysis of Chinese strains of porcine circovirus type 2.

Forty Chinese PCV2 strains collected between 2004 and 2008 were sequenced and their genetic variations were analyzed together with nine previous PCV2 isolates. Phylogenetic analysis indicated that these Chinese PCV2 strains could be divided into four genotypes (PCV-2a, PCV-2b, PCV-2d and PCV-2e), and the genotype PCV-2c defined in Denmark was not found. PCV-2d and PCV-2e were two genotypes firstly determined in our study. Variation analysis of amino acids of capsid protein revealed that Chinese PCV2 strains clustered within PCV-2d had four amino acid marker positions and the isolates within PCV-2e had seven unique amino acid mutations. Our analysis also showed that PCV-2b became dominating in China in recent years. These data contribute to the understanding of PCV2 molecular epidemiology.

Molecular variation analysis of porcine reproductive and respiratory syndrome virus in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. The objective of the present study was to analyze the molecular variation and evolution of porcine reproductive and respiratory syndrome virus in China based on the complete genomic sequences of three highly pathogenic Chinese PRRSV strains isolated in 2006 and the sequences of the amplified Nsp2, ORF5 and ORF7 genes from clinical specimens during 2006-2008. Full-length genome sequencing and phylogenetic analysis showed that the three strains (JXwn06, BJsy06 and NX06) had a unique 30-amino-acid discontinuous deletion in Nsp2, and were classified into the same subgroup that consisted of the most Chinese strains isolated during 2006-2007, the pandemic period of atypical PRRS. The evolution analysis suggested that the emergence of the highly pathogenic PRRSV in China experienced a gradual variation and evolution accumulation progress from Chinese domestic virus. The variation analysis of the amplified 41 Nsp2, 59 ORF5 and 59 ORF7 genes indicated that the diversity of PRRSV strain existed in the field, and the highly pathogenic PRRSV strain with the 30-amino-acid deletion in Nsp2 was the dominating virus in China in recent years. Our data contribute to the understanding of molecular variation and epidemiology surveillance of PRRSV in China.

Complete sequence of a duck astrovirus associated with fatal hepatitis in ducklings.

Duck astroviruses (DAstVs) are known to cause duck viral hepatitis; however, little is known regarding their molecular biology. Here, we report the complete sequence of a DAstV associated with a recent outbreak of fatal hepatitis in ducklings in China. Sequence analyses indicated that the genome of DAstV possessed a typical astrovirus organization and also exhibited two unique features. The polyadenylated genome comprised 7722 nt, which is the largest among astroviruses sequenced to date. The ORF2 of DAstV was not in the same reading frame as either ORF1a or ORF1b, which was distinct from all other astroviruses. Sequence comparisons and phylogenetic analyses revealed that DAstV was more closely related to turkey astrovirus (TAstV) type 2, TAstV-3 and TAstV/MN/01 (a possible new TAstV serotype) than to TAstV-1 or other astroviruses. These findings suggest that astroviruses may transmit across ducks and turkeys.

Nsp9 and Nsp10 Contribute to the Fatal Virulence of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China

Atypical porcine reproductive and respiratory syndrome (PRRS), which is caused by the Chinese highly pathogenic PRRS virus (HP-PRRSV), has resulted in large economic loss to the swine industry since its outbreak in 2006. However, to date, the region(s) within the viral genome that are related to the fatal virulence of HP-PRRSV remain unknown. In the present study, we generated a series of full-length infectious cDNA clones with swapped coding regions between the highly pathogenic RvJXwn and low pathogenic RvHB-1/3.9. Next, the in vitro and in vivo replication and pathogenicity for piglets of the rescued chimeric viruses were systematically analyzed and compared with their backbone viruses. First, we swapped the regions including the 5′UTR+ORF1a, ORF1b, and structural proteins (SPs)-coding region between the two viruses and demonstrated that the nonstructural protein-coding region, ORF1b, is directly related to the fatal virulence and increased replication efficiency of HP-PRRSV both in vitro and in vivo. Furthermore, we substituted the nonstructural protein (Nsp) 9-, Nsp10-, Nsp11- and Nsp12-coding regions separately; or Nsp9- and Nsp10-coding regions together; or Nsp9-, Nsp10- and Nsp11-coding regions simultaneously between the two viruses. Our results indicated that the HP-PRRSV Nsp9- and Nsp10-coding regions together are closely related to the replication efficiency in vitro and in vivo and are related to the increased pathogenicity and fatal virulence for piglets. Our findings suggest that Nsp9 and Nsp10 together contribute to the fatal virulence of HP-PRRSV emerging in China, helping to elucidate the pathogenesis of this virus.

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence.

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.

Recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. In this study, five SPF pigs were co-infected with two strains of PRRSV (JXwn06-81c and HB-1/3.9c), and 352 viruses were cloned by plaque assay from the sera of the infected pigs on days 3, 5, 7, 10, 14, 21 postinfection (pi), and the recombinant events between the two viruses were systematically investigated by sequencing the ORF5, ORF3 and Nsp2 genes of each cloned virus and using SimPlot and Genetic Algorithm for Recombination Detection (GARD) analysis. Totally, 133 recombinant viruses out of the plaque viruses were acquired from four of five infected pigs during days 7-21pi upon co-infection with JXwn06-81c and HB-1/3.9c. The intragenic recombination and intergenic fragment exchange of the ORF5, ORF3 and Nsp2 genes between the two viruses exhibited different patterns, and the recombination for ORF5 gene and Nsp2 occurred as early as on day 7pi. The recombination between the ORF5, ORF3 or Nsp2 gene resulted in the generation of chimeric GP5, GP3 or Nsp2. Of the three genes, Nsp2 gene exhibited more complicated recombination situation. Meanwhile, the putative recombination breakpoints and hotspots for the three genes were analyzed. Our findings not only provide valuable evidences for understanding that recombination is an important genetic mechanism contributing to the variation and evolution of PRRSV, but also suggest that extensive use of attenuated vaccine of PRRSV undoubtedly contributes to the increased diversity of PRRSV in field.

Porcine circovirus type 2 and its associated diseases in China

Porcine circovirus type 2 (PCV2) has been recognized as an important vial pathogen for global swine industry. The virus is associated with postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as porcine circovirus-associated disease (PCVAD). PCV2 infection and PMWS have caused great impact on the pig production in China during the past 10 years. This review will involve in the history of PCVAD, serological epidemiology of PCV2 infection, and clinical aspect of PCVAD, and the genomic characteristics, variation and genotyping of PCV2, as well as control situation of PCVAD in China.