Wild-type Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Unfolding of an RNA G-quadruplex motif in the negative strand genome of porcine reproductive and respiratory syndrome virus by host and viral helicases to promote viral replication.

G-quadruplex (G4) is a unique secondary structure formed by guanine-rich nucleic acid sequences. Growing studies reported that the genomes of some viruses harbor G4 structures associated with viral replication, opening up a new field to dissect viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV), a representative member of Arteriviridae, is an economically significant pathogen that has devastated the swine industry worldwide for over 30 years. In this study, we identified a highly conserved G-rich sequence with parallel-type G4 structure (named PRRSV-G4) in the negative strand genome RNA of PRRSV. Pyridostatin (PDS), a well-known G4-binding ligand, stabilized the PRRSV-G4 structure and inhibited viral replication. By screening the proteins interacting with PRRSV-G4 in PRRSV-infected cells and single-molecule magnetic tweezers analysis, we found that two helicases, host DDX18 and viral nsp10, interact with and efficiently unwound the PRRSV-G4 structure, thereby facilitating viral replication. Using a PRRSV reverse genetics system, we confirmed that recombinant PRRSV with a G4-disruptive mutation exhibited resistance to PDS treatment, thereby displaying higher replication than wild-type PRRSV. Collectively, these results demonstrate that the PRRSV-G4 structure plays a crucial regulatory role in viral replication, and targeting this structure represents a promising strategy for antiviral therapies.

Infection dynamics and incidence of wild-type porcine reproductive and respiratory syndrome virus in growing pig herds in the U.S. Midwest.

Porcine reproductive and respiratory syndrome virus (PRRSV) infections greatly impact the health and productivity of growing pigs. The introduction and persistence of wild-type PRRSV (WT-PRRSV) strains in growing pig populations is poorly understood. In an observational prospective cohort study, we monitored and surveyed 63 wean-to-finish (WTF) herds across 10 companies located in medium to high pig dense areas in the U.S. Midwest. All herds received weaned pigs from PRRSV-negative or positive-stable breeding herds. Herds were monitored monthly using oral fluids collected following a fixed spatial sampling regime and samples were tested by PRRSV ELISA, RT-PCR and ORF5 sequencing. In most (90%) of the herds, pigs were vaccinated with PRRSV modified-live vaccines either at processing, weaning or shortly after weaning. Wild type PRRSV (WT-PRRSV) infections were defined by the criterion of having more than 2% nucleotide differences in the ORF-5 region compared with reference vaccine strain sequences. Wild type PRRSV was detected in 42% of the herds with infections being more prevalent in the mid to late growing period, with a mean of 20 weeks post placement. Nineteen distinct WT-PRRSV were identified in seven out of 10 production companies with an average of 3 distinct WT-PRRSV strains per company. Vaccinated WTF herds with and without WT-PRRSV detection were compared to each other showing different PCR and ELISA infection patterns. Close-out mortality in vaccinated herds with WT-PRRSV was numerically higher (6.5%) than mortality in those sites where WT-PRRSV was not detected (5.0%) (p=0.07). Mortality was also higher (10.5%) when WT-PRRSV was detected earlier at eight weeks post-placement compared to late finishing at 20 and 25 weeks post-placement, 2.9% and 4.5% respectively (p=0.017). Overall, this study sheds light on WT-PRRSV infection dynamics in vaccinated populations of growing pigs, reinforces the importance of biosecurity practices in this phase of production and calls for better understanding of risk factors associated with PRRSV introductions in growing pig sites.

Comparison of virus detection, productivity, and economic performance between lots of growing pigs vaccinated with two doses or one dose of PRRS MLV vaccine, under field conditions

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a significant economic impact on swine production. It has been demonstrated that PRRS modified-live virus (MLV) vaccination of pigs, with one full dose, significantly reduces clinical consequences of wild-type PRRSV infection compared to non-vaccinates. However, there is limited information about the effect that two doses of PRRSV MLV vaccine have on the performance of growing pigs, compared to vaccination with a single dose. This study was conducted with the objectives to compare (a) the wild-type PRRSV detection in oral fluids over time, (b) key closeout productivity indicators, and (c) economic performance between lots of growing pigs vaccinated with two doses of Ingelvac PRRS® MLV vaccine and lots vaccinated with a single dose of the same vaccine. This randomized field trial included 15 lots of growing pigs from PRRSV positive-unstable sow farms and 66 lots from PRRSV positive-stable sow farms, according to the American association of swine veterinarians’ terminology. All pig lots received the first vaccination either around processing or weaning age. Lots allocated in the two doses group received the second vaccination three to four weeks after the first vaccination. The pig lots were monitored for PRRSV detection over time. Six oral fluids samples were collected in three weeks intervals and were tested for wild-type PRRSV-2 RNA by RT-qPCR and open reading frame 5 (ORF)− 5 sequencing. Regression models were used to compare wild-type PRRSV detection dynamics on oral fluids samples and to compare key closeout performance indicators between one dose group and two doses group. Additionally, a benefit-cost ratio analysis compared economic performance between one dose group and two doses group. The proportion of wild-type PRRSV detection on oral fluids samples and the log counts of viral RNA per ml of oral fluids from the two doses group was lower than the one dose group on lots originated from PRRSV positive-stable sow farms, with a risk ratio of 1.24 and a rate ratio of 1.17, respectively. The two doses group had a significantly lower mortality rate than the one dose group, with a rate ratio of 1.21. The effect size increased on lots originated from PRRSV positive-unstable sow farms, and on lots with higher frequency and diversity of wild-type PRRSV detection during the growth phase. No differences in growth performance were detected between two doses group and one dose group. The second MLV vaccination dose had a benefit-cost ratio of 1.83. For lots originated from PRRSV positive-unstable farms, the benefit-cost ratio was 4.45, and for lots originated from PRRSV positive-stable farms, the benefit-cost ratio was 0.45. Under study conditions, vaccinating growing pig lots with two doses of PRRS MLV vaccine was a useful strategy to immunize growing pigs against PRRSV, lowering the wild-type PRRSV detection, lowering mortality rate, and increasing profitability, compared to lots of growing pigs that received a single dose of the same vaccine.

Implementing a user-friendly format to analyze PRRSV next-generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events.

The open reading frames (ORF)5 represents approximately 4% of the porcine reproductive and respiratory syndrome virus (PRRSV)-2 genome (whole-PRRSV) and is often determined by the Sanger technique, which rarely detects>1 PRRSV strain if present in the sample. Next-generation sequencing (NGS) may provide a more appropriate method of detecting multiple PRRSV strains in one sample. This work assessed the effect of PRRSV genetic variability and recombination events, using NGS, on the time-to-low prevalence (TTLP) and total losses in breeding herds (n 20) that detected a PRRSV outbreak and adopted measures to eliminate PRRSV. Serum, lung or live virus inoculation material collected within 3-weeks of outbreak, and subsequently, processing fluids (PFs) were tested for PRRSV by RT-qPCR and NGS. Recovered whole-PRRSV or partial sequences were used to characterize within and between herd PRRSV genetic variability. Whole-PRRSV was recovered in five out of six (83.3%) lung, 16 out of 22 (72.73%) serum and in five out of 95 (5.26%) PF. Whole-PRRSV recovered from serum or lung were used as farm referent strains in 16 out of 20 (80%) farms. In four farms, only partial genome sequences were recovered and used as farm referent strains. At least two wild-type PRRSV strains (wt-PRRSV) were circulating simultaneously in 18 out of 20 (90%) and at least one vaccine-like strain co-circulating in eight out of 20 (40%) farms. PRRSV recombination events were detected in 12 farms (59%), been 10 out of 12 between wt-PRRSV and two out of 12 between wt-PRRSV and vaccine-like strains. Farms having ≥3 strains had a 12-week increase TTLP versus herds ≤2 strains detected. Farms with ≤2 strains (n 10) had 1837 and farms with no recombination events detected (n 8) had 1827 fewer piglet losses per 1000 sows versus farms with ≥3 PRRSV strains (n 8) or detected recombination (n 10), respectively. NGS outcomes and novel visualization methods provided more thorough insight into PRRSV dynamics, genetic variability, detection of multiple strains co-circulating in breeding herds and helped establish practical guidelines for using PRRSV NGS outputs.

A Molecular and Epidemiological Description of a Severe Porcine Reproductive and Respiratory Syndrome Outbreak in a Commercial Swine Production System in Russia.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating disease of swine in many parts of the world. Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 is endemic in Europe, and prevalence of the subtypes differ spatially. In this study, we investigated a severe PRRS outbreak reported in 30 farms located in eastern Russia that belong to a large swine production company in the region that was also experiencing a pseudorabies outbreak in the system. Data included 28 ORF5 sequences from samples across 18 of the 25 infected sites, reverse transcriptase real-time polymerase chain reaction (RT-qPCR) results from diagnostic testing, reports of clinical signs, and animal movement records. We observed that the outbreak was due to two distinct variants of wildtype PRRSV type 1 subtype 1 with an average genetic distance of 15%. Results suggest that the wildtype PRRSV variants were introduced into the region around 2019, before affecting this production system (i.e., sow farms, nurseries, and finisher farms). Clinical signs did not differ between the variants, but they did differ by stage of pig production. Biosecurity lapses, including movement of animals from infected farms contributed to disease spread.

Next Generation of Voluntary PRRS Virus Regional Control Programs.

Porcine reproductive and respiratory syndrome virus (PRRSV) became pandemic in the 1980's and today remains one of the most significant pathogens of the global swine industry. At the herd level, control of PRRSV is complicated by its extreme genetic diversity and its ability to persist in pigs, despite an active immune response. Ultimately, PRRSV control or elimination requires the coordination and active cooperation of producers and veterinarians at the regional level. Early voluntary PRRSV regional control programs focused on routine diagnostic testing and voluntary data-sharing regarding the PRRSV status of participants' herds, but no pre-defined action plans or decision trees were developed to secure project successes (or recover from failures). Given that control of PRRSV is paramount to producer profitability, we propose a coordinated approach for detecting, controlling, and ultimately eliminating wild-type PRRSV from herds participating in regional projects. Fundamental to project success is real-time, multi-platform communication of all data, information, and events that concern the regional project and project participants. New to this approach is the concept of agreed-upon action plans to be implemented by project participants in response to specific events or situations. The simultaneous and coordinated implementation of these strategies allows for early detection of wild-type PRRSV virus introductions and rapid intervention based on agreed-upon response plans. An example is given of a project in progress in the Midwest USA.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody

The ontogeny of PRRSV antibody in oral fluids has been described using isotype-specific ELISAs. Mirroring the serum response, IgM appears in oral fluid by 7days post inoculation (DPI), IgA after 7 DPI, and IgG by 9 to 10 DPI. Commercial PRRSV ELISAs target the detection of IgG because the higher concentration of IgG relative to other isotypes provides the best diagnostic discrimination. Oral fluids are increasingly used for PRRSV surveillance in commercial herds, but in younger pigs, a positive ELISA result may be due either to maternal antibody or to antibody produced by the pigs in response to infection. To address this issue, a combined IgM-IgA PRRSV oral fluid ELISA was developed and evaluated for its capacity to detect pig-derived PRRSV antibody in the presence of maternal antibody. Two longitudinal studies were conducted. In Study 1 (modified-live PRRS vaccinated pigs), testing of individual pig oral fluid samples by isotype-specific ELISAs demonstrated that the combined IgM-IgA PRRSV ELISA provided better discrimination than individual IgM or IgA ELISAs. In Study 2 (field data), testing of pen-based oral fluid samples confirmed the findings in Study 1 and established that the IgM-IgA ELISA was able to detect antibody produced by pigs in response to wild-type PRRSV infection, despite the presence of maternal IgG. Overall, the combined PRRSV IgM-IgA oral fluid ELISA described in this study is a potential tool for PRRSV surveillance, particularly in populations of growing pigs originating from PRRSV-positive or vaccinated breeding herds.

Vaccination mitigates the impact of PRRSv infection on the pharmacokinetics of ceftiofur crystalline-free acid in pigs.

The pharmacokinetics of intramuscularly administered ceftiofur crystalline-free acid (CCFA) were determined in pigs that were clinically healthy (n=8), vaccinated with a Porcine reproductive and respiratory syndrome modified live virus (PRRS MLV) (n=10), challenged with wild-type porcine reproductive and respiratory syndrome virus (PRRSv) VR-2385 (n=10), or vaccinated with PRRS MLV and later challenged with wild-type PRRSv VR-2385 (n=10). Animals were given a single dose of CCFA intramuscularly at 5mg/kg body weight. Blood was collected at 0 (pretreatment), 0.25, 0.5, 1, 6, 12, 24, 48, 96, 144, 192, and 240h postinjection. Plasma was analyzed using liquid chromatography-mass spectrometry. Plasma concentration-time curves for each group were evaluated with noncompartmental modeling. When compared to control animals, those receiving the PRRSv wild-type challenge only had a lower AUC0-last , higher Cl/F, and higher Vz/F. The PRRSv wild-type challenge only group had the longest T1/2λ . The Cmax did not differ among all four treatments. Control animals had no statistically significant differences from animals vaccinated with PRRS MLV alone or animals vaccinated with PRRS MLV and later challenged with wild-type PRRSv. Our results suggest that PRRSv wild-type infection has the potential to alter CCFA pharmacokinetics and PRRS MLV vaccination may attenuate those changes.

A Synthetic Porcine Reproductive and Respiratory Syndrome Virus Strain Confers Unprecedented Levels of Heterologous Protection.

Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine. The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This concept may be significant for the development of vaccines against genetically variable viruses that require active viral replication in order to achieve complete immune protection.

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry worldwide. Although inactivated and live vaccines are commercially available for the control of PRRS, both types of vaccine have not always proven successful in terms of generating a protective immune response, particularly in the case of inactivated vaccines. In this study, we tested whether an inactivated vaccine could induce a humoral immune response to PRRS during a homologous challenge. Amino acid substitutions were introduced into glycoprotein (GP) 5 of the FL12 strain of the PRRS virus (PRRSV) using site-directed mutagenesis with a pFL12 infectious clone. The substitutions led to double deglycosylation in the putative glycosylation moieties on GP5. The mutant virus was subsequently inactivated with binary ethylenimine. The efficacy of the inactivated mutant virus was compared with that of the inactivated wild-type PRRSV. Only the inactivated mutant PRRSV induced serum neutralizing antibodies at six weeks post-vaccination. The group that was administered the inactivated mutant virus twice exhibited a significantly increased neutralizing antibody titer after a challenge with the virulent homologous strain and exhibited more rapid clearing of viremia compared to other groups, including the groups that were administered either the inactivated mutant or wild-type virus only once and the group that was administered the inactivated wild-type virus twice. Histopathological examination of lung tissue sections revealed that the group that was administered the inactivated mutant virus twice exhibited significantly thinner alveolar septa, whereas the thickness of the alveolar septa of the other groups were markedly increased due to lymphocyte infiltration. These results indicated that the deglycosylation of GP5 enhanced the immunogenicity of the inactivated mutant PRRSV and that twice administrations of the inactivated mutant virus conferred better protection against the homologous challenge. These findings suggest that the inactivated PRRSV that expresses a hypo-glycosylated GP5 is a potential inactivated vaccine candidate and a valuable tool for controlling PRRS for the swine industry.

Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus

Synthetic attenuated virus engineering (SAVE) is an emerging technology that enables rapid attenuation of viruses. In this study, by using SAVE we demonstrated rapid attenuation of an arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV). The major envelope GP5 gene of PRRSV was codon-pair deoptimized aided by a computer algorithm. The codon-pair deoptimized virus, designated as SAVE5 with a deoptimized GP5 gene, was successfully rescued in vitro. The SAVE5 virus replicated at a lower level in vitro with a significant decrease of GP5 protein expression compared to the wild-type PRRSV VR2385 virus. Pigs experimentally infected with the SAVE5 virus had significantly lower viremia level up to 14 days post-infection as well as significantly reduced gross and histological lung lesions when compared to wild-type PRRSV VR2385 virus-infected pigs, indicating the attenuation of the SAVE5 virus. This study proved the feasibility of rapidly attenuating PRRSV by SAVE.

Antibody repertoire development in fetal and neonatal piglets. XXIII: Fetal piglets infected with a vaccine strain of PRRS Virus display the same immune dysregulation seen in isolator piglets

The Ig levels and antibody repertoire diversification in fetal piglets infected with an attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) were measured. Serum Ig levels were greatly elevated in PRRSV-infected fetuses; IgG was elevated >50-fold, IgM>5–15-fold and IgA>2-fold compared to control fetuses. Their IgM to IgG to IgA profile was the same as that in isolator piglets infected for the same period with wild-type PRRSV. Fetal animals showed less repertoire diversification than even isolator piglets that were maintained germfree (GF) while the repertoire diversification index (RDI) for PRRSV-infected isolator piglets was 10-fold higher and comparable to littermates infected with swine influenza (S-FLU). However, when expressed as the RDI:Ig ratio, infected fetuses appeared 10-fold less capable of repertoire diversification than uninfected littermates and GF isolator piglets. Compared to S-FLU isolator piglets that resolve the infection, the RDI:Ig of PRRSV-infected isolator piglets was 100-fold lower. Overall, infection of fetuses with an attenuated virus shows the same immune dysregulation seen postnatally in wild type infected isolator piglets, indicating that: (a) attenuation did not alter the ability of the virus to cause dysregulation and (b) the isolator infectious model reflects the fetal disease.

Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth

The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is known to possess the properties of an ion-channel protein, and in the present study we show that the PRRSV E protein is N-terminal myristoylated. The PRRSV E protein contains the consensus motif of 1MGxxxS 6 for myristoylation, and in the presence of 2-hydroxymyristic acid, the virus titer decreased by 2.5 log TCID 50 and the level of viral RNA was reduced significantly. When the glycine at position 2 was mutated to alanine (G2A) using an infectious cDNA clone, a viable virus was recoverable and a mutant PRRSV was obtained. The titers of G2A mutant virus were 2.0 × 10 4 and 1.0 × 10 6 TCID 50/ml for ‘passage-2’ and ‘passage-3’ viruses, respectively, in PAM cells, and these titers were significantly lower than those of wild-type PRRSV. When treated with the myristoylation inhibitor, the G2A mutant virus was resistant to the drug. The data show that the PRRSV E protein myristoylation is non-essential for PRRSV infectivity but promotes the growth of the virus.

Function of PRRSV GP5 envelope protein by using pseudotyped virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus. Virions of PRRSV contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2, GP3, GP4, and E. The GP5 is the major envelope proteins, which was involved in the formation and infectivity of PRRSV by coaction with other membrane proteins. Here, to determine the function of alone GP5 envelope protein in viral entry, we investigated the formation and infectivity of GP5-pseudotyped virus particles. By co-transfection of GP5 expression plasmids with murine leukemia virus (MuLV) based retroviral vectors (pHIT60, encoding MuLV Gag-Pol; pHIT111, encoding an MuLV genome with a β-galactosidase reporter gene) into 293 T cells and analysis of the culture medium using ultracentrifugation, Western blot, and infection assay. We observed that the GP5 envelope protein was incorporated into the MuLV retroviral vectors to generate an pseudotyped murine leukemia virus, which was infectious to PAM and Mack-145 target cells and displayed the same host range with wild-type PRRSV. The infection of the pseudotyped virus on PAM target cells is effectively neutralized by polyclonal antibodies specific for PRRSV or GP5. The results suggested that the GP5 protein may play a key role in the viral entry by interacting with the host cell receptor. The GP5-pseudotyped virus will be useful in the identification of the cellular receptor binding with GP5 protein.

Effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs

To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. Four hundred forty 6- to 8-week-old PRRSV-naïve pigs. Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.

Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from Yorkshire, Hampshire, and Landrace boars.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.

Heteroclite Subgenomic RNAs Are Produced in Porcine Reproductive and Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame 1a nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5′–3′ junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNAs in arteriviruses and we have termed them heteroclite (meaning “deviating from common forms or rules”) subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (e) little apparent interference with parental virus replication. These subgenomic RNAs may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite subgenomic RNAs.

Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction

Porcine reproductive and respiratory syndrome virus (PRRSV) is in boar semen for extended periods of time as determined by reverse transcription-nested polymerase chain reaction (RT-nPCR) assay. The concentration of PRRSV RNA in semen and the biological significance of the detection level, however, remain to be resolved. In order to determine the concentration of PRRSV VR-2332 (a prototypic strain of North American isolates) in semen following infection, we established a ‘standard curve’-quantitative competitive (SC-QC)-RT-nPCR assay as well as an equimolar QC-RT-nPCR assay. A deletion-type competitor RNA derived from the Lelystad virus, a European strain of PRRSV, ORF-7 gene standard which shares the nested sets of primer recognition sequences with the VR-2332 ORF-7 gene was used as an internal standard. The equimolar QC-RT-nPCR assay results revealed that the number of copies of PRRSV RNA in 1 TCID 50/ml of virus derived from CL-2621 cell culture supernatants varied depending upon the type of samples in which virus was added; 143±24.0 and 266.5±48.5 copies in serum and semen samples spiked with PRRSV VR-2332, respectively. For the establishment of SC-QC-RT-nPCR assay, a standard curve was generated from band intensity ratios versus a series of known initial numbers of wild-type RNA copies which were quantified by the equimolar QC-RT-nPCR assay. Various initial numbers of copies of wild-type PRRSV RNA and each band intensity ratio with 1000 copies of competitor RNA were well correlated within the range of 100 to 200 000 copies ( R 2=0.947). A ‘standard curve’ quantitation assay using competitive single-tube RT-nPCR will offer a rapid and reliable way to quantify low concentrations of PRRSV RNA in semen.

Effects of a modified-live virus vaccine against porcine reproductive and respiratory syndrome in boars

Abstract Objectives To determine whether vaccine virus is found in serum and semen of vaccinated boars, whether vaccination prevents subsequent shedding of wild-type virus after challenge exposure, and whether semen and blood variables are altered after vaccination or challenge exposure with wild-type virus, or both. Design Throughout the 50-day postvaccination period, serum and semen from exposed boars were evaluated for the presence of porcine reproductive and respiratory syndrome virus (PRRSV). All boars were then challenge-exposed with PRRSV isolate VR-2332 and evaluated for an additional 27 days. Semen quality variables, serostatus, and blood variables were monitored. Animals 7 PRRSV-seronegative adult boars. Procedure Semen was collected 3 times weekly and evaluated by use of a nested reverse-transcriptase polymerase chain reaction for detection of PRRSV RNA. Serum was obtained weekly and evaluated by nested reverse-transcriptase polymerase chain reaction, virus isolation, and PRRSV ELISA. Semen quality variables were evaluated 3 times weekly, and CBC was performed weekly. Results Vaccine virus was shed in the semen of all vaccinated boars, but shedding was of shorter duration in 4 of 5 vaccinated boars than that generally observed after exposure to wild-type virus. After challenge exposure, shedding of wild-type virus in semen was shortened or eliminated in 4 of 5 vaccinated boars. Percentage of forward movement and normal spermatozoal morphology and motility were significantly reduced in vaccinated boars after challenge exposure. Conclusions Vaccine virus was shed in semen of vaccinated boars, but vaccination generally reduced or eliminated shedding of wild-type PRRSV after challenge exposure. Semen quality appeared to be less than optimal, particularly after vaccination and subsequent challenge exposure with wild-type virus. Clinical Relevance Extra-label use of the PRRSV vaccine in boars remains controversial because some boars may still shed wild-type virus in semen after challenge exposure at postvaccination day 50. Semen quality also appeared to be altered after vaccination and subsequent challenge exposure. (Am J Vet Res 1997;58:40–45)