North American Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Phylogenetic analysis of porcine reproductive and respiratory syndrome virus in Vietnam, 2021.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes more economic losses in the swine industry than any other virus. This study aimed to investigate the genetic diversity of PRRSV to assist in evaluating the effectiveness of PRRS vaccines. Twenty-eight samples from clinical cases were collected from 19 farms in seven provinces of Vietnam in 2021. Full-length PRRSV ORF5 genes from the 19 samples were amplified, sequenced, and compared to the corresponding sequences of referenced PRRSV strains from Genbank. The genetic analysis showed that 12 isolates were the highly pathogenic PRRSV subtype (HP—PRRSV) lineage 8, sublineage 8.7; six isolates were the classical North American PRRSV subtype (US-PRRSV), NADC-like group, lineage 1, sublineage 1.4, which were reported in Vietnam for the first time; and the final isolate was a vaccine-like strain. The field isolates of HP-PRRSV had relatively higher genetic diversity with US-PRRSV vaccine strains (84.0–94.5%) than HP-PRRSV vaccine strains (95.3–98.6%). Meanwhile, the six NADC-like isolates had low nucleotide similarity with US-PRRSV and HP-PRRSV vaccine strains (83.4–85.4% and 83.2–84.0%, respectively). Many amino acid substitutions were found in antigenic regions of GP5 involved in response to early antibody production, neutralizing antibodies, and viral immune evasion between these field strains and PRRSV vaccine strains. These findings provide insights into the molecular characteristics, genetic diversity, antigenicity, and evolution of PRRSV strains in Vietnam and postulate a compelling explanation for the limitations of current vaccination efforts.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-022-01912-w.

Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05349-8.

Novel ORF5 deletion of NADC30-like porcine reproductive and respiratory syndrome viruses circulating in northern China from 2016 to 2018.

North American porcine reproductive and respiratory syndrome virus (NA-PRRSV), especially NADC30-like PRRSV, has evolved and is prevalent in China. We collected 503 samples from pig breeding farms across 4 provinces in northern China from 2016 to 2018. The samples were screened by PCR testing with specific primers that could differentiate groups of NA-PRRSV; phylogenetic trees were constructed and analyzed. Overall, 175 of 503 (34.8%) samples were positive for NA-PRRSV. Dual (NADC30-like and highly pathogenic [HP]-PRRSV; NADC30-like and typical PRRSV; HP and typical PRRSV) and triple (NADC30-like, HP, and typical PRRSV) infections (92 of 175, 52.6%) were common in coinfections by NADC30-like and HP-PRRSV. Notably, 18 of 125 (14.4%) semen samples were positive for PRRSV, and 17 of the 18 positive semen samples contained NADC30-like PRRSV. Phylogenetic analysis based on GP5 amino acids revealed that the novel NADC30-like PRRSV with a unique single amino acid deletion at position 34 has become widespread and has evolved into a new subgroup.

Identification of a linear B-cell epitope on glycoprotein (GP) 2a of porcine reproductive and respiratory syndrome virus (PRRSV)

Glycoprotein (GP) 2a was a minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV) and was one of crucial proteins for PRRSV to bind cell receptor, which indicated that there were neutralizing epitopes on GP2a. In the present work, we used mouse anti-GP2a41–208aa serum and one GP2a41–208aa specific monoclonal antibody (McAb) to identify B-cell epitopes of GP2a by peptide-based ELISA. A liner B-cell epitope F194PTPGSRPKLHDFQQ208 was identified. However, the results of virus neutralization experiment showed that the McAb could not reduce the titers of PRRSV, which indicated that the identified epitope was not the neutralizing epitope of PRRSV. While the amino acid sequence of this epitope was conserved in North American (type 2) PRRSV, which suggested that this epitope might be diagnostic potential for type 2 PRRSV strains. In conclusion, our present work identified a new epitope on GP2a and this epitope might be diagnostic potential for type 2 PRRSV strains.

S0119 CD163 A gatekeeper for susceptibility to porcine reproductive and respiratory syndrome virus

It is estimated that in North America and Europe, porcine reproductive and respiratory syndrome virus (PRRSV) results in combined losses of $6 million per day. The virus replicates in macrophages, induces prolonged viremia, and animals become persistently infected. Infection by PRRSV predisposes animals to other bacterial and viral pathogens. Young and growing pigs exhibit pneumonia, diarrhea, and mortality of 12 to 15%. Sows and gilts have reproductive failure, late abortion, early farrowing, increased number of mummies, and decreased litter size. Boars have low libido, fever, and low sperm count. Vaccines have not effectively controlled the virus. As with many pathogens, numerous candidates have been proposed to be the receptor for viral entry into the host cell. The most popular receptor candidates for PRRSV have included heparin sulfate, SIGLEC1, VIM, CD151, CD209, and CD163. To determine if these candidate receptors were indeed involved, we first created SIGLEC1−/− animals by homologous recombination and somatic cell nuclear transfer, and challenged them with a Type II (North American) PRRSV. The SIGLEC1−/− animals were not resistant to infection. Next, we created CD163−/− pigs by CRISPR/Cas9 gene editing and challenged these pigs with a different Type II PRRSV strain. In contrast to SIGLEC1−/− animals, the CD163−/− pigs were resistant to the challenge. When the CD163−/− animals were created, other edits in additional animals were introduced that were predicted to modify the structure of CD163. We are working toward in vitro and in vivo challenge experiments with these additional genotypes, with both Type I (European) and Type II strains of PRRSV. This line of research has identified a gatekeeper for PRRSV infection (CD163) and possibly provided an on-farm solution to an otherwise incurable and economically devastating disease.

Complete genomic characterization of a porcine reproductive and respiratory syndrome virus isolate in Xinjiang province of China.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. In the present study, one strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated in Xinjiang province, Northwest China, designated XJu-1. The full-length genome of XJu-1 was found to be 14,987 nucleotides in length, including the poly(A) tail. Comparative analysis with the genomic sequences of type 2 isolates revealed that XJu-1 shared 87.2-99.2 % identity with these isolates, but only 60.4 % with the type 1 virus-Lelystad Virus, indicating that this new Chinese isolate was closely related to the North American PRRSV genotype. XJu-1 was a novel strain with unique deletions in NSP2 region, namely that 150-amino acid deletion in NSP2. The genomic variations of XJu-1 strain provided the basis for further studies of virulence determinants and evolution for PRRSVs.

Genetic analysis of ORF5 porcine reproductive and respiratory syndrome virus isolated in Vietnam

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008-2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8-100.0% nt identity and 94.0-100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR-2332; nt 87.8-89.3%, aa 87.5-90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1-61.9%, aa 55.1-57.0%). There was greater similarity with QN07 (nt 96.5-98.5%, aa 96.0-99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH-1a (nt 93.2-95.1%, 91.5-93.5%) isolated in China in 1995 and JXA1 (nt 96.5-98.6%, aa 95.0-98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1-R (nt 96.5-98.6%, aa 95.0-98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL-PS (nt 87.2-89.0%, aa 86.0-89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub-lineage 8.7. This sub-lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam.

Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1β subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1β. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1β. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection.

Effect of the Modified Live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine on European and North American PRRSV Shedding in Semen from Infected Boars

The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 10(0.1) to 10(1.0) viral genome copies per ml and 3.63 to 10(1.1) 50% tissue culture infective doses (TCID(50))/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 10(0.2) to 10(4.7) viral genome copies per ml and 1.14 to 10(3.07) TCID(50)/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 10(0.1) to 10(4.57) viral genome copies per ml and 1.66 to 10(3.10) TCID(50)/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 10(0.3) to 10(5.14) viral genome copies per ml and 1.69 to 10(3.17) TCID(50)/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge.

Complete genomic characterization of a Chinese isolate of porcine reproductive and respiratory syndrome virus

The genome of one isolate of porcine reproductive and respiratory syndrome virus (PRRSV) from China, designated GDQY2, was sequenced and analyzed. The full length of GDQY2 was 15,215 nucleotides, excluding the poly(A) tail. Comparative analysis with the genomic sequences of numerous worldwide North American isolates revealed that GDQY2 shared 85.0–98.9% identity with these isolates, but only 60.9% with the European virus—LV (Lelystad Virus), indicating that this new Chinese isolate was closely related to the North American PRRSV genotype. Phylogenetic analysis based on the nucleotide sequences of the full length and ORF5 showed that this new isolate belong to the same genetic group with all other Chinese isolates. Comparison with North American PRRSV isolates revealed that GDQY2 exhibited variations in the non-structural protein 2 (NSP2) encoded by ORF1a, namely that an additional 35-amino acid deletion in NSP2 was found in GDQY2. Therefore, GDQY2 was a novel strain with unique deletions. Furthermore, our study demonstrated that North American genotype PRRSVs in China have evolved independently from other countries, indicating that geographic separation might be one factor influencing the molecular evolution of PRRSV.

Phylogenetic analysis and molecular characteristics of seven variant Chinese field isolates of PRRSV

BackgroundPorcine reproductive and respiratory syndrome (PRRS) has now been widely recognized as an economically important disease. The objective of this study was to compare the molecular and biological characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) field isolates in China to those of the modified live virus (MLV) PRRS vaccine and its parent strain (ATCC VR2332).ResultsFive genes (GP2, GP3, GP4, GP5 and NSP2) of seven isolates of PRRSV from China, designated LS-4, HM-1, HQ-5, HQ-6, GC-2, GCH-3 and ST-7/2008, were sequenced and analyzed. Phylogenetic analyses based on the nucleotide sequence of the ORF2-5 and NSP2 showed that the seven Chinese isolates belonged to the same genetic subgroup and were related to the North American PRRSV genotype. Comparative analysis with the relevant sequences of another Chinese isolate (BJ-4) and North American (VR2332 and MLV) viruses revealed that these isolates have 80.8-92.9% homology with VR-2332, and 81.3-98.8% identity with MLV and 80.7-92.9% with BJ-4. All Nsp2 nonstructural protein of these seven isolates exhibited variations (a 29 amino acids deletion) in comparison with other North American PRRSV isolates. Therefore, these isolates were novel strain with unique amino acid composition. However, they all share more than 97% identity with other highly pathogenic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and the Nsp2 protein when compared with the previous isolates.ConclusionsThese results might be useful to study the genetic diversity of PRRSV in China and to track the infection sources as well as for vaccines development.

Phages Harboring Specific Peptides That Recognize the N Protein of the Porcine Reproductive and Respiratory Syndrome Virus Distinguish the Virus from Other Viruses

The aim of the current study was to develop a novel diagnostic test for detecting porcine reproductive and respiratory syndrome virus (PRRSV) using phage display technology. The N gene of PRRSV isolate HH08 was cloned following reverse transcription-PCR. Sequence comparison indicated that the N gene shared 96.4% homology to that of North American PRRSV (isolate VR2332) and 35.5% with that of European PRRSV (isolate LV), indicating that the PRRSV isolate was related to the North American PRRSV genotype. The bacterially expressed N protein was used as a target in a biopanning process using a phage display random peptide library. Seven phages expressing different peptides had a specific binding activity with the N protein. The putative binding motifs were identified by DNA sequencing. More importantly, the selected phages harboring specific peptides that recognize the N protein of PRRSV were able to efficiently distinguish PRRSV from other viruses in enzyme-linked immunosorbent assays.

Detection of Asymptomatic Antigenemia in Pigs Infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by a Novel Capture Immunoassay with Monoclonal Antibodies against the Nucleocapsid Protein of PRRSV

Routine surveillance for porcine reproductive and respiratory syndrome virus (PRRSV) infections is crucial for the epidemiological control of this disease. Antibody tests are widely used but cannot differentiate between vaccination and reinfection. We developed a PRRSV antigen capture enzyme-linked immunosorbent assay (ELISA) using well-characterized monoclonal antibodies (MAbs) raised against the nucleocapsid (N) protein of North American and European PRRSV. This antigen assay detected purified N protein from both genotypes at levels as low as 0.4 and 0.8 ng, respectively. The specificity and sensitivity of the N antigen assay were evaluated with ground lung tissues from 8 PRRSV-infected and 16 healthy swine, and culture supernatants from six PRRSV isolates as well as other swine viruses were confirmed by reverse transcriptase PCR (RT-PCR). Antigen assays were positive in all eight infected tissues and with six different PRRSV isolates, with no false positives among healthy tissues and other swine viruses (i.e., pseudorabies and foot and mouth disease viruses). A number of sera, field collected from 466 vaccinated and asymptomatic pigs in Guangdong, China, between 2008 and 2009, tested positive by the N antigen assay (12.45%), RT-PCR (15.02%), and a commercial test for antibodies against PRRSV (78.97%). Of the 466 sera, 47 were positive by both antigen and RT-PCR tests, 11 by antigen test only, and 23 by RT-PCR only; the two assays had an overall agreement of 92.7%, indicating a significant percentage of active PRRSV in asymptomatic pigs despite previous immunization. These findings suggest that the antigen assay is a valuable field tool for the epidemiological control of PRRSV that can be used for rapid screening, particularly in asymptomatic animals.

Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is characterized by late-term abortions in sows and respiratory disease in young pigs. Using an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Upon transfection of cells with this plasmid, PRRSV infection was initiated and progeny virus "P129-GFP" was obtained. Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS) and inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". To determine the immunogenicity of the recombinant viruses, pigs were immunized intramuscularly with P129-WT (wild-type), P129-GFP, or P129-PCV2. By 5 weeks post-infection, specific antibody responses to GFP and PCV2 capsid were elicited. This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.

Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers

Porcine reproductive and respiratory syndrome (PRRS) has caused heavy economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) can be an effective antiviral against the PRRS virus (PRRSV). PPMO is structurally similar to DNA with modified backbone and is resistant to nuclease. This study was designed to examine increasing inhibitory effect of PPMO combination. Two pairs of PPMOs were identified to have enhanced suppression of PRRSV replication in cell culture, while individual constituents did not work under the same testing conditions. PPMO 5UP1 that is complementary to 5′ terminus of PRRSV genome was paired with 4P1 or 7P1 that are complementary to sequence in the translation initiation regions of ORFs 4 and 7, respectively. The PPMO combination also inhibited replication of heterologous strains in the North American PRRSV genotype. Treatment of the cells with the combinations reduced PRRSV RNA and protein levels. In cell-free or cell-based luciferase reporter assays, the PPMO combination suppressed target mRNA translation more effectively than individual constituents, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for PRRS control.

Detection of Porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: a longitudinal study under experimental conditions

Isolation of Porcine reproductive and respiratory syndrome virus (PRRSV) from oral fluids was first reported in 1997. The objective of the present study was to determine whether PRRSV and/or anti-PRRSV antibodies were present in oral fluids at diagnostic levels. The level and duration of PRRSV and anti-PRRSV antibodies in serum and oral fluids was evaluated in 3 age groups of pigs (4, 8, or 12 weeks of age) inoculated with a type 2 (North American) PRRSV isolate. Serum, buccal swabs, and pen-based oral fluid samples were collected for 63 days following inoculation. Specimens were assayed for PRRSV by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-PRRSV antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). Porcine reproductive and respiratory syndrome virus was detected by real-time qRT-PCR in serum for approximately 5 weeks and in oral fluids for approximately 4 weeks postinoculation. Pig age at the time of inoculation had no effect on the quantity or duration of virus in oral fluid samples. Low levels of anti-PRRSV antibody were detected in oral fluid samples by ELISA and IFAT. Although the approach remains to be validated in the field, the results of this experiment suggest that pen-based oral fluid sampling could be an efficient, cost-effective approach to PRRSV surveillance in swine populations.

Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green

The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03 TCID 50/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature ( T m) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T m values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.

GP5 ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain Lelystad virus

Sera from pigs infected with the European porcine reproductive and respiratory syndrome virus (PRRSV), strain Lelystad virus (LV), were screened by indirect ELISA for antibodies that bound to a series of overlapping synthetic peptides covering amino acids (AA) 19–60 of the primary envelope glycoprotein (GP)5. Antibodies were detected that bound to an epitope(s) located in an ectodomain segment composed of AA 38–54. The antibodies strongly cross-reacted with peptides specific for the North American PRRSV VR2332. Antibodies with the same specificity were present in sera of pigs infected in the US with a European-like PRRSV. Competitive ELISA using overlapping 8–10-AA-long peptides confirmed that the epitope recognized by the antibodies from LV-infected pigs is located in the same segment as the neutralization epitopes previously identified for PRRSV VR2332 and the closely related arterivirus, lactate dehydrogenase-elevating virus (LDV). No antibodies were detected that bound to synthetic peptides representing further upstream GP5 segments that have been reported to carry neutralization or non-neutralization epitopes of some PRRSV isolates.

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b.

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2.