Strain VR-2332 Research Articles

Comparison of host immune responses to homologous and heterologous porcine reproductive and respiratory syndrome virus (PRRSV) challenge

Porcine reproductive and respiratory syndrome (PRRS) is a high‐consequence animal disease worldwide. The objective of this study was to identify immune responses that are predictive of protection against heterologous PRRSV challenge. Using MLV IngelVac PRRSV vaccine, its parental strain VR2332, and K‐LI, we compared immune responses induced by a single vaccination and by experimental infection of two different PRRSV isolates. Four groups of pigs were utilized in this study in which groups 1 & 2 were vaccinated with MLV PRRSV and groups 3 & 4 were not vaccinated. 28 days post vaccination (DPV), pigs in groups 1 & 3 were challenged with VR2332, and groups 2 & 4 were challenged with K‐LI. The results shown that lung pathology score of group 1 pigs was significantly lower than that of groups 2, 3, & 4 pigs 14 DPC, confirming the vaccination‐induced homologous protection. Serum neutralizing antibody against K‐LI was detected only in group 2 pigs 14 DPC. ELISpot assay indicated that VR2332 is a stronger inducer of IFNr‐secreting PBMCs than K‐LI in all pigs, and group 2 pigs possessed more PRRSV‐specific IFNr‐secreting PBMCs. This study indicates that heterologous challenge induces a higher level of cellular immune response against PRRSV in vaccinated pigs, but only serum neutralizing antibody titer is associated with PRRSV vaccination‐induced protection against homologous and heterologous challenge.

Genetic Diversity of the ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Isolates in Southwest China from 2007 to 2009

To gain insight into the molecular epidemiology and possible mechanisms of genetic variation of porcine reproductive and respiratory syndrome (PRRS) in Yunnan Province of China, the ORF5 gene of 32 PRRSV isolates from clinical samples collected from 2007 to 2009 were sequenced and analyzed. Nucleotide and amino acid analyses were carried out on 32 isolates and representative strains of the North American genotype, European genotype and two representative Chinese isolates. Results revealed that these isolates share 86.9–99.0% nucleotide and 87.5–98.0% amino acid identity with VR-2332 the prototypical North American PRRSV, 61.7–62.9% and 54.3–57.8% with Lelystad virus (LV) the representative strain of European genotype, 91.2–95.4% and 90.0–94.5% with CH-1a that was isolated in mainland China in 1996, 88.1–99.3% and 85.5–99.0% with JX-A1 the representative strain of High pathogenic PRRSV in China, and 86.2–99.8% and 85.5–100.0% between isolated strains of different years, respectively. Phylogenetic analysis revealed that all 32 PRRSV isolates belonged to the North American genotype and were further divided into two different subgenotypes. Subgenotype 1 comprised twenty two Yunnan isolates which divided into two branches. Subgenotype 2 comprised ten isolates which closely related to the RespPRRS vaccine and its parent strain VR-2332. The functional domains of GP5 such as the signal peptide, ectodomain, transmembrane regions and endodomain were identified and some motifs in GP5 with known functions, such as primary neutralizing epitope (PNE) and decoy epitope were also further analyzed. Our study shown the great genetic diversity of PRRSV in southwest China, rendering the guide for control and prevention of this disease.

A survey of porcine reproductive and respiratory syndrome among wild boar populations in Korea

No information is currently available on porcine reproductive and respiratory syndrome virus (PRRSV) infection in wild boars (Sus scrofa) in Korea. In this study, the status of PRRS in wild boars was investigated. Blood samples were collected from 267 wild boars from eight provinces in Korea. Four of the samples tested (1.5%) were positive for PRRSV antibodies and eight (3.0%) were positive for antigens. Of the virus-positive samples, three and five samples were typed as containing European (EU, type 1) or North American (NA, type 2) viruses, respectively. Two amplicons (one from type 1 and one from type 2) were used to analyze the PRRSV open reading frame 7 (ORF7) sequence. The nucleotide sequences of type 1 PRRSV ORF7 had identities between 96.1% and 98.4% with PRRSVs from domestic pigs in Korea. The sequences of type 2 PRRSV ORF7 had identities of 100% with the PRRSV strain VR-2332, which was prototypic North American strain. These results show that PRRSVs are present in wild boars in Korea, and effective PRRSV surveillance of the wild boar population might therefore be useful for disease control.

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

Fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolate genomes were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200, and 403 amino acid deletions in the second hypervariable region of nsp2, were found to be stable in the nsp2 coding region after in vivo infection ( Faaberg et al., 2010). Furthermore, 3 of 4 mutants achieved replication kinetics similar to wt virus by study end. We hypothesized that other mutations elsewhere in the virus may have contributed to their replication fitness in swine. To further assess the stability of the engineered viruses, all sequenced genomes were compared and contrasted. No specific mutations occurred in all nsp2 deletion mutant genomes that were not also seen in the parent genome of Type 2 PRRSV strain VR-2332. Second site (non-nsp2) deletions and/or insertions were not evident after replication in swine. The number of point mutations seen increased slightly with deletion size, but even the largest deletion (403 aa) had very few consensus mutations. Thus, our findings provide further substantiation that the nsp2 deletion mutant genomes were genetically stable after in vivo passage.

Inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer

Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the swine industry in many countries, and current control strategies are inadequate. Previously, we explored the strategy of using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to inhibit PRRS virus (PRRSV) replication. PPMOs are nuclease-resistant and single-stranded DNA analogs containing a modified backbone conjugated to a cell-penetrating peptide and can act as antisense agents through steric blockade of complementary messenger RNA. A PPMO (designated 5UP2) targeting highly conserved sequence in the 5′-terminal region of the PRRSV genome was found to produce multi-log10 inhibition of PRRSV replication in cultured cells. To evaluate 5UP2 in vivo, we here administrated the PPMO to 3-week-old piglets via intranasal instillation at 24 h before, and 2 and 24 h after infection with PRRSV (strain VR2385). Blood samples were collected at 6, 10 and 14 days post-infection (dpi) for detection of PRRSV RNA and antibodies. Necropsy was performed at 14 dpi. Monitoring weight gain in all piglet groups throughout the experiment indicated that PPMO was well tolerated at the doses used. PPMO 5UP2 treatment significantly reduced PRRSV viremia at 6 dpi. On day 14, piglets receiving 5UP2 had significantly less interstitial pneumonia and lower level of anti-PRRSV antibodies than untreated piglets. In alveolar macrophages isolated at the time of necropsy, the expression of antiviral genes in PPMO-treated piglets was elevated in comparison with untreated. This study provides further data indicating that the 5UP2 PPMO can be considered a candidate component for a novel PRRS control strategy.

Cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate ( Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.

Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus that emerged in the late 1980s in both Europe and North America as the causative agent of porcine reproductive and respiratory syndrome (PRRS), now the most important disease of swine worldwide. Despite extensive characterization of PRRSV proteins by direct analysis and comparison with other arteriviruses, determinants of virulence, pathogenesis and protective immune recognition remain poorly understood. Thus, we hypothesized that additional ORFs are present in the PRRSV genome that may contribute to its biological properties, and so we screened highly purified virions of strain VR2332, the prototype type 2 PRRSV, for evidence of novel polypeptides. A 51 aa polypeptide was discovered that is encoded by an alternative ORF of the subgenomic mRNA encoding the major envelope glycoprotein, GP5, and which is incorporated into virions. The protein, referred to as ORF5a protein, is expressed in infected cells, and pigs infected with PRRSV express anti-ORF5a protein antibodies. A similar ORF is present as an alternative reading frame in all PRRSV subgenomic RNA5 genes and in all other arteriviruses, suggesting that this ORF5a protein plays a significant role in arterivirology. Its discovery also provides a new potential target for immunological and pharmacological intervention in PRRS.

Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China

Porcine reproductive and respiratory syndrome (PRRS) has a major negative economic impact on the swine industry worldwide. During the investigation of PRRS virus (PRRSV) in mainland China, European genotype (EU, type 1) PRRSV isolates were detected in swine herds both with and without clinical symptoms. Two complete genome sequences for Chinese type 1 PRRSV isolates were identified from viruses isolated from lung tissue and sera. The two viruses, designated BJEU06-1 and NMEU09-1, produced cytopathic effects in primary porcine alveolar macrophages but not in Marc-145 cells, and had a mean diameter of 55 nm, as measured by transmission electron microscopy . Comparative sequence analysis revealed that they shared 87.0-91.5% and 58.0-58.2% identity with the EU and North American genotype (NA, type 2) prototypic strains LV and VR-2332, respectively. Remarkably, these isolates, characterized by concomitant deletions within non-structural protein 2 (Nsp2) and ORF3 hypervariable regions, have never been described. Phylogenetic trees showed that all of the novel Chinese isolates of European genotype are in the pan-European subtype 1 that is predominant in Europe. However, they evolved from different ancestors. These novel viruses are predicted to be products of the divergent evolution of ancestor PRRSV isolates introduced from Europe. This is the first report of type 1 PRRSV wild isolates being in mainland China. Our findings confirm that the Chinese type 1 PRRSV isolates originated from diverse progenitors and the type 1 and type 2 PRRSV isolates, having different biological properties, have coexisted on the Chinese mainland for several years.

Sequence Analysis and Expression of ORF5 in Korean Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure and respiratory disorders in pigs. The viral genome consists of eight overlapping open reading frames (ORFs). ORF5 encodes one of the major glycoproteins and is known as an immunologically important structural protein associated with virus neutralization. The ORF5 gene of the Korean PRRSV isolate, CNV-1, was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide and amino acid sequences of CNV-1 ORF5 shared 91% and 83% identity, respectively, with the American isolate (VR2332 strain) and 57% and 49% identity with the European isolate. For the expression and easy purification of ORF5, the cDNA containing the complete ORF5 sequence fused in-frame with sequence encoding glutathione S-transferase (GST) was cloned into a baculovirus transfer vector and transfected into Sf9 cells. The GST-ORF5 fusion protein produced in Sf9 cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Sequencing results confirmed that the recombinant baculovirus from Sf9 cells contains the complete ORF5 gene. Further studies in this direction will address whether ORF5 can be a good candidate for a subunit vaccine against PRRSV in Korea.

Pathogenicity and Molecular Characterization of Emerging Porcine Reproductive and Respiratory Syndrome Virus in Vietnam in 2007

In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the 'porcine high fever disease' that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7-99.4% identity to Chinese strain HUN4, 68-69% identity to strain VR-2332 and 58-59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re-isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re-isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.

Susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and CD163

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.ResultsDetailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHOSn-CD163 and PK15Sn-CD163 cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line. Only 2 out of 5 PRRSV strains replicated well in CHOSn-CD163 cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15Sn-CD163 cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15Sn-CD163 cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes.ConclusionsA PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.

Prevalence and phylogenetic analysis of the isolated type I porcine reproductive and respiratory syndrome virus from 2007 to 2008 in Korea

The first Korean strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated in 1997, and it exhibited high similarity to strain VR-2332 (type II PRRSV; North American type). Recently, however, infection with type I PRRSV (European type) has also been reported in Korea. To date, preliminary data about type I PRRSV prevalence in Korea have not been reported. Here, using reverse transcriptase (RT)-PCR, we analyzed 383 archived field samples from 101 pig farms in Korea that were collected from 2007 to 2008. We identified 155 samples from 68 farms that were positive for PRRSV. Fifty-one samples (51/155; 32.9%) and 20 farms (20/68; 29.4%) were type I PRRSV-positive/type II PRRSV-negative. Furthermore, we tried to isolate the type I PRRSV from positive samples and seven type I PRRSV were isolated using PAM. The phylogenetic analysis using the type I PRRSV isolates (7 isolates) was performed based on open reading frame (ORF)5 (accession numbers GU325642 to GU325648) and ORF7 (accession numbers GU325635 to GU325641). In the phylogenetic study, seven type I PRRSV isolates were closely related with panEuropean based on ORF7, while they were genetically distinct from Lelystad virus and made a unique clade based on ORF5. The results of this study demonstrate that infection with type I PRRSV is not uncommon in Korean pig farms, which suggests that diagnosis and control of type I PRRSV should be considered in Korea. A new approach to vaccination against, and epidemiological analysis of, Korean PRRSV is urgently needed.

Porcine reproductive and respiratory syndrome viruses predominant in southeastern China from 2004 to 2007 were from a common source and underwent further divergence

The full-length glycoprotein 5 (GP5) gene and a partial nonstructural protein 2 (NSP2) gene fragment of 46 porcine reproductive and respiratory syndrome viruses (PRRSV) from pig farms in southeastern China between 2004 and 2007 were sequenced for phylogenetic analysis. All of the PRRSV isolates in this study were of the North American type, and the majority of them were clustered in subgroup II and had 84.1-89.1% amino acid sequence identity to those of subgroup I including the North American strain VR-2332. Three variable regions containing epitopes A and B in the N-terminal region were identified and found to be under positive selection. Several additional mutations, which were also located in the variable regions, were seen in isolates from the years 2006 and 2007 in subgroup II, as compared with those of earlier years (2004-2005) in the same group. Further analysis revealed that the majority of the subgroup II PRRSV isolates prevalent in the region since 2004 had thirteen mutation sites that distinguished them from subgroup I strains, indicating a possible introduction of a certain strain from the same source in the region or elsewhere well before 2004. A 29-aa deletion in the NSP2 fragment was found in PRRSV isolates as early as in 2005, one year earlier than the virulent PRRSV with the same deletion became dominant in China. Taken together, this study shows that subgroup II PRRSV strains with a partial deletion of nsp2 are currently prevailing in southeastern China.

Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China

Herds of pigs in the Mid-Eastern region of China have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (PRRS) characterised by high fever and morbidity and mortality in animals of different ages. Eighty-one herds were diagnosed with PRRS virus (PRRSV) infection from June to December 2006 on the basis of clinical signs, pathological findings and reverse transcriptase polymerase chain reaction (RT-PCR). Twenty strains of PRRSV were isolated from 20 herds in six provinces. Following experimental inoculation, one isolate, designated SY0608, caused 100% morbidity and 25–50% mortality in 30-, 65- and 105-day-old pigs and the birth of stillborn and weak piglets from affected sows.The ORF5 gene had 99.5–99.8% nucleotide and 99–100% derived amino acid sequence identities among SY0608 and five other field isolates, but only 89.4% and 88.6% identities, respectively, with VR-2332, the prototypic North American isolate. The 2850bp Nsp2 gene of SY0608 had 79.4% nucleotide and 74.9% derived amino acid sequence identities with VR-2332; deletions of 1 and 29 amino acids corresponded to positions 480 and 531–559 of strain VR-2332, respectively. These findings demonstrated that a new highly pathogenic Northern American type PRRSV has spread widely in the Mid-Eastern region of China.

Identification of Nonessential Regions of the nsp2 Replicase Protein of Porcine Reproductive and Respiratory Syndrome Virus Strain VR-2332 for Replication in Cell Culture

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is a multidomain protein and has been shown to undergo remarkable genetic variation, primarily in its middle region, while exhibiting high conservation in the N-terminal putative protease domain and the C-terminal predicted transmembrane region. A reverse genetics system of PRRSV North American prototype VR-2332 was developed to explore the importance of different regions of nsp2 for viral replication. A series of mutants with in-frame deletions in the nsp2 coding region were engineered, and infectious viruses were subsequently recovered from transfected cells and further characterized. The results demonstrated that the cysteine protease domain (PL2), the PL2 downstream flanking sequence (amino acids [aa] 181 to 323), and the putative transmembrane domain were critical for replication. In contrast, the segment of nsp2 preceding the PL2 domain (aa 13 to 35) was dispensable for viral replication, and the nsp2 middle hypervariable region (aa 324 to 813) tolerated 100-aa or 200-aa deletions but could not be removed as a whole; the largest deletion was about 400 aa (nsp2Delta324-726). Characterization of the mutants demonstrated that those with small deletions possessed growth kinetics and RNA expression profiles similar to those of the parental virus, while the nsp2Delta324-726 mutant displayed decreased cytolytic activity on MARC-145 cells and did not develop visible plaques. Finally, the utilization of the genetic flexibility of nsp2 to express foreign genes was examined by inserting the gene encoding green fluorescent protein (GFP) in frame into one nsp2 deletion mutant construct. The recombinant virus was viable but impaired and unstable and gradually gained parental growth kinetics by the loss of most of the GFP gene.

Genetic Variation of Chinese PRRSV Strains Based on ORF5 Sequence

Thirteen isolates of porcine reproductive and respiratory syndrome virus (PRRSV) from different provinces of China were studied and compared with several PRRSV isolates from other countries. Phylogenetic analysis shows that all Chinese isolates of PRRSV in this study belong to the American genotype, except for one strain, B13, which clustered as a European genotype. Sequence analysis revealed that PRRSV Chinese isolates of the American genotype were highly similar in the ORF5 sequence and could be classified into two subclades. One contains PRRSV isolates that are more closely related to the American vaccine strain MLV Resp and its parent strain VR-2332, and the other contains ones only distantly related to them. Within the Chinese isolates slight genetic variation occurred, and some strains may originate directly from the vaccine virus.

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1β, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66–70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324–434, 486 and 505–523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].

Neutralizing Antibody Responses of Pigs Infected with Natural GP5 N-Glycan Mutants of Porcine Reproductive and Respiratory Syndrome Virus

In order to assess the effect of the N-glycans associated with the GP5 neutralization epitope of porcine reproductive and respiratory syndrome virus (PRRSV) on the neutralizing antibody (Ab) response of swine, groups of young pigs were infected with PRRSV strains differing in N-glycosylation pattern. The humoral immune response to strain VR-2332, harboring four potential N-glycan sites, was compared to that of two natural field isolates carrying mutations either abolishing the N-glycosylation site at position 44 (N44) or the two N-glycosylation sites in the hypervariable region upstream of the neutralization epitope (HV-1). The pigs were bled at intervals and their sera were assayed for neutralizing Abs by indirect and competition ELISAs using peptides containing the GP5 neutralization epitope, and selectively for infectivity neutralization of a number of PRRSV strains. In addition, viremia was monitored by quantitative RT-PCR, and anti-N-protein Ab formation was measured by HerdChek ELISA. The neutralizing Ab responses as measured by peptide ELISA varied greatly between individual pigs infected with each PRRSV strain. Some pigs generated high titers of peptide binding Abs between 7 and 28 days post infection (p.i.), whereas other pigs had not generated a response by 90 days p.i. However, the HV-1-infected pigs generated Abs to the neutralization epitope more rapidly and to a 5-10 times higher level than VR-2332-infected pigs, and the Abs neutralized the homologous HV-1 virus 10-20 times more efficiently than PRRSV strains VR-2332, N44, MN184, or SDSU73. In contrast, most N44-infected pigs generated neutralizing Abs only after 42 days p.i. and only to low levels. The results suggest that the deletions of the N-glycans or other amino acid substitutions in the GP5 ectodomains of the mutants affect the immunogenicity of the neutralization epitope and the specificity of the Abs raised to it but not the sensitivity of the virions to Ab neutralization.

Polyclonal Hypergammaglobulinemia and Formation of Hydrophobic Immune Complexes in Porcine Reproductive and Respiratory Syndrome Virus-Infected and Uninfected Pigs

Infection of young conventional, domestic pigs with porcine reproductive and respiratory syndrome virus (PRRSV) strains VR2332 and JA142 resulted in a rapid, progressive increase in serum IgG reaching maximum levels of 20-30 mg/mL at about 3 weeks post infection (p.i.), which were maintained until at least 63 days p.i., whereas the level of serum IgG remained at 4-6 mg/mL in sham infected pigs. In most of the VR2332 and JA142-infected pigs hypergammaglobulimenia was associated with the formation of hydrophobic, 150-300-kDa IgG-containing immune complexes that bound in the presence of 0.1% Tween 20 to ELISA plates that were not coated with any antigen. The ELISA plate-binding activity remained low in most infected pigs, but reached high levels in some JA142-infected pigs. Binding of the immune complexes was also observed, but at a lower level, to uncoated ELISA plates in the peptide ELISA for anti-PRRSV Abs. The immune complexes bound to uncoated ELISA plates with a much lower affinity than Abs to plates coated with peptides containing the appropriate epitopes. The immune complexes also bound to HerdChek ELISA plates, but because of low binding affinity for these plates, the bound complexes were removed by the repeated washes with Tween 20 solution. Overall the PRRSV-induced hypergammaglobulinemia and generation of ELISA plate-binding immune complexes resembled those observed in mice infected with the closely related lactate dehydrogenase-elevating virus (LDV) and thus, like the latter, seem a result of a polyclonal activation of B cells. We also found that sera of a group of older sows possessed high levels of IgG as well as of ELISA plate-binding immune complexes, in spite of being PRRSV infection negative by all criteria presently available. On the other hand, sera from wild hogs contained no ELISA plate-binding IgG in spite of possessing high total serum IgG levels.

Variation analysis of the ORF5 gene of Porcine reproductive and respiratory syndrome virus

AbstractThree Porcine reproductive and respiratory syndrome virus (PRRSV) isolates (HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002) were obtained from pig farms in Hebei and Jiangxi provinces, China. The complete ORF5 gene of the isolates was amplified using RT-PCR and sequenced. It was shown that ORF5 genes of all isolates encoded 200 amino acids. Comparing ORF5 genes of the three isolates and published sequences for five other PRRSV isolates in China, variation analysis showed that all of the isolates were of the American genotype, with 88.2–99.0% amino acid identity. ORF5 genes among BJ-4, S1 and J1 had higher similarity, sharing 98–99% identity of the deduced amino acids. HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002 and CH-1a presented 92–96% identity among their ORF5 genes. Phylogenetic analysis revealed that these isolates could be divided into two subgroups based on the genetic distance of their ORF5 gene: the first subgroup comprised BJ-4, S1 and J1 and was closer to VR2332 and vaccine strains; the second included HB-1(sh)/2002, HB-2(sh)/2002, JX-1/2002 and CH-1a.