Genome characterization of two NADC30-like porcine reproductive and respiratory syndrome viruses in China.

BACKGROUND: The recent emergence of NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) in vaccinated pigs arose more attentions for the high incidents of mutation and recombination of PRRSVs. FINDINGS: In this study, we determined full-length genome sequences of two NADC30-like PRRSV isolates from recent PRRSV outbreaks in China. Phylogenetic analysis showed that these two isolates were clustered in an independent branch together with NADC30, an American isolate in 2008. Genetically, HNjz15 shared 95.6 % nucleotide similarity to NADC30 without any exotic gene insertion. By contrast, HNyc15 shared 93.8 % similarity to NADC30 with recombination with VR-2332 and CH-1a. Two more previously reported NADC30-like PRRSVs were also analyzed and had exotic gene insertions with different PRRSV strains in their nonstructural protein genes. CONCLUSIONS: The above results showed the increased mutation and recombination rates of NADC30-like PRRSV under current vaccination pressure and a more pressing situation for the PRRSV eradication and control in China.

Complete Genome Sequence of an NADC30-Like Porcine Reproductive and Respiratory Syndrome Virus Characterized by Recombination with Other Strains.

We report here the complete genome sequence of an NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), HNyc15, which was characterized by recombination with VR-2332 and CH-1a PRRSV strains in open reading frames (ORFs) 2 to 4.

Complete Genome Sequence of an NADC30-Like Strain of Porcine Reproductive and Respiratory Syndrome Virus in China.

The most recently emergent porcine reproductive and respiratory syndrome virus strains in China are characterized by 393 nucleotide deletions in the nonstructural protein 2 (NSP2) region and are known as NADC30-like strains. Here, we report the complete genome sequence of the NADC30-like HNjz15 strain that was isolated in 2015.

Complete Genome Sequence of a Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus That Has a Further Deletion in the Nsp2 Gene.

Here, we report the complete genome of a Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) characterized by a further 29-amino acid (87 nucleotides) deletion in its Nsp2-coding region compared to the prototype of the HP-PRRSV JXA1 strain.

The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets.

A severe porcine epidemic diarrhea (PED) epizootic has been affecting pigs of all ages that are characterized by high mortality among suckling piglets in China since late 2010, causing significant economic losses. Obtaining a current-circulating PEDV variant isolate that can grow efficiently in cell culture is prerequisite for the development of efficient vaccines. In this study, PEDV strain HN1303 was isolated successfully on Vero cells with supplemental trypsin, and the isolate has been serially propagated in cell culture for over 95 passages. The infectious titers of the virus during the first 10 passages ranged from 10(2.6) to 10(5.8) 50% tissue culture infective doses (TCID50)/ml, and the titers of 20-95 passages ranged from 10(6.2) to 10(8.0)TCID50/ml. The growth curve of Vero cell-adapted HN1303 in cell culture was determined, and dynamics of virus production was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analysis based on spike gene indicate that the HN1303 strain belongs to genotype IIa. In addition, the fourth passage cell-culture HN1303 was subjected to 2-day old piglets. All piglets orally inoculated developed severe watery diarrhea and vomiting within 24 hours post-inoculation (hpi) and died within 72 hpi. The results of animal experiments reveal that this strain is highly pathogenic to 2-day old piglets.

Replacement of the heterologous 5' untranslated region allows preservation of the fully functional activities of type 2 porcine reproductive and respiratory syndrome virus.

The 5' untranslated region (UTR) is believed to be vital for the replication of porcine reproductive and respiratory syndrome virus (PRRSV), yet its functional mechanism remains largely unknown. In this study, to define the cis-acting elements for viral replication and infectivity, The 5' UTR swapping chimeric clones pTLV8 and pSHSP5 were constructed based on two different genotypes full-length infectious cDNA clone pAPRRS and pSHE backbones. Between them, vTLV8 could be rescued from pTLV8 and had similar virological properties to vAPRRS, including phenotypic characteristic and RNA synthesis level. However, pSHSP5 exhibited no evidence of infectivity. Taken together, the results presented here demonstrate that only the 5' UTR of type 1 PRRSV did not affect the infectivity and replication of type 2 PRRSV in vitro. The 5' UTR of type 2 PRRSV could be functionally replaced by its counterpart from type 1.

Immunization of pigs with a type 2 modified live PRRSV vaccine prevents the development of a deadly long lasting hyperpyrexia in a challenge study with highly pathogenic PRRSV JX143.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been confirmed to be the underlying cause of the so-called 'porcine high fever disease' (PHFD), a disease that emerged in China in 2006 and subsequently spread over South East Asia. The aim of this study was to investigate whether animals challenged with the Chinese highly pathogenic PRRSV JX143 would be protected by vaccination with single dose of a type 2 modified live virus (MLV) vaccine. Forty-four pigs 17-19 days of age were weighed and randomly assigned to either vaccination with subsequent challenge (V/C, n=20), challenge only (NV/C, n=12) and no vaccination and no challenge (strict controls, n=12). Pigs of the challenged groups (V/C and NV/C) were inoculated intranasally 27 days post-vaccination with PRRSV JX143. Animals were monitored during the subsequent 21 days post challenge and were necropsied at the end of the experiment on day 49. Observations and measurements included body temperature, clinical scores for behavior/general condition, cough and breathing pattern, mortality, serological response and PRRSV viremia via RNA detection. Challenge in the NV/C pigs resulted in 100% morbidity and 67% mortality whereas all vaccinated pigs survived. There was a close association between hyperpyrexia (fever over 41°C) and incidence in mortality, which was completely prevented by vaccination. Clinical symptoms were less severe, and of transient nature only, in the vaccinated pigs. Vaccination did not prevent infection, but reduced the impact of clinical disease and prevented hyperpyrexia associated mortality.

Disulfide linkages mediating nucleocapsid protein dimerization are not required for porcine arterivirus infectivity.

The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3' untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N-ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly.

Identification of non-essential regions in nucleocapsid protein of porcine reproductive and respiratory syndrome virus for replication in cell culture.

Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) plays a central role in virus replication. In this study, serial N- and C-terminal truncations of N protein were performed in the context of type 2 PRRSV infectious cDNA clone, and our results revealed that a stretch of inter-genotypic variable N terminal residues aa 5-13 ((5)NGKQQKKK(13)K) and the last four inter-genotypic variable aa residues ((120)SPS(123)A) at the C terminus of N protein were dispensable for type 2 PRRSV infectivity. All the recovered deletion mutant viruses had spontaneous mutations in the N coding region, including substitution, deletion and insertion. We re-engineered the additional internal deletion with or without the original C-terminal deletion back into wild-type APRRS and found that the internal domain spanning the inter-genotypic variable residues 39-42 ((39)KGP(42)G) and conserved residues 48-52 ((48)KNPE(52)K), respectively, were dispensable for type 2 PRRSV viability. These results demonstrated that N protein contains non-essential regions for virus viability in cell culture. Such dispensable regions could be utilized as insertion site for foreign tag expression and the rescued viruses could be the candidates for marker vaccine.

Chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is classified into two genotypes, type 1 and type 2, which share only about 60% genetic identity. Here, we report viable chimeric viruses in which the envelope protein genes from ORF2a to ORF5 of vSHE (type 1) were swapped into the genetic backbone of vAPRRS (type 2). We found that the envelope proteins of genotype 1 were fully functional in genotype 2 PRRSV, and the rescued chimeric progeny viruses showed robust genetic stability and similar replication properties to the parental strains in vitro. To our knowledge, this is the first study to report the substitution of complete ORFs between different genotypes of porcine arterivirus. These findings pave the way to further elucidate the structure-function relationship of PRRSV envelope proteins, and may enable the development of novel marker vaccines that can be used to differentiate vaccinated from infected animals.

Baculovirus expression of cloned porcine arterivirus generates infectious particles in both insect and mammalian cells.

Studies on several viral pathogens have been hampered by the lack of appropriate in vitro systems for their propagation and amplification. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus containing a single-stranded positive-sense RNA genome (∼15kb), was served as a model virus and its genomic cDNA was recombinated into baculovirus. We investigated whether infectious virus particles could be generated by expression of the full-length cloned genome from the modified baculovirus vector. The recombinant baculovirus, AcAPRRS, was used to infect sf9 cells. Immunofluorescence assay demonstrated the presence of PRRSV nonstructural protein (nsp) 2 and nucleocapsid (N) protein and electron microscopy revealed PRRSV particles in the culture supernatant. Infectious PRRSV particles were also produced in susceptible MARC-145 cells inoculated with AcAPRRS, and the growth characteristics of the PRRSV generated were similar to those of the parental PRRSV strain. Infectious PRRSV particles were also generated following AcAPRRS transduction of BHK-21 cells and Vero cells that are not sensitive to PRRSV. Titers of PRRSV obtained from BHK-21 and Vero cells were up to 10(4.05)TCID(50)/ml. These findings open a new route to the propagation of the virus in vitro and will be of utility in vaccine development.