Determination of the frequency of individuals with broadly cross-reactive neutralizing antibodies against PRRSV in the sow population under field conditions.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a significant swine pathogen, yet the immune response components contributing to protection remain incompletely understood. Broadly reactive neutralizing antibodies (bNAs) may play a crucial role in preventing reinfections by heterologous viruses, although their occurrence is considered low under both field and experimental conditions. This study aimed to assess the frequency of sows exhibiting bNAs against PRRSV under field conditions and to analyze the epidemiological factors influencing the occurrence of these elite neutralizers. Blood samples were collected from breeding sows across eleven unrelated pig farms, with samples categorized by parity. Serum obtained was utilized in virus neutralization assays (VNs) against six PRRSV field isolates and two MLV strains. RESULTS: Approximately 7% of the sows exhibited neutralization activity against all viruses in the panel, with a geometric mean of the titer (GMT) of NAs at or exceeding 4 log2. Exclusion of the PRRSV-2 isolate from the panel increased the proportion of elite neutralizers to around 15%. Farm-specific analysis revealed significant variations in both GMT of NAs and proportion of elite neutralizers. PRRSV unstable farms and those with a PRRS outbreak in the last 12 months displayed higher GMT of NAs compared to stable farms without recent outbreaks. The GMT of NAs showed a gradual, albeit moderate, increase with the parity of the sows. Parity's impact on bNA response was consistently observed in stable farms but not necessarily in unstable farms or those with recent outbreaks. Finally, the results indicated that vaccinated animals had higher NA titers against the vaccine virus used in the farm than against field viruses. CONCLUSION: bNAs against heterologous isolates induced by PRRSV infection under field conditions are generally low, often falling below titers necessary for protection against reproductive failure. However, a subset of sows (approximately 15%) can be considered elite neutralizers, efficiently recognizing various PRRSV strains. Repeated exposures to PRRSV play a crucial role in eliciting these bNAs, with a higher frequency observed in unstable farms and those with recent outbreaks. In stable farms, parity only marginally influences bNA titers, highlighting its limited role compared to the impact of PRRSV exposure history.

Predicting Antigenic Distance from Genetic Data for PRRSV-Type 1: Applications of Machine Learning.

The control of porcine reproductive and respiratory syndrome (PRRS) remains a significant challenge due to the genetic and antigenic variability of the causative virus (PRRSV). Predominantly, PRRSV management includes using vaccines and live virus inoculations to confer immunity against PRRSV on farms. While understanding cross-protection among strains is crucial for the continued success of these interventions, understanding how genetic diversity translates to antigenic diversity remains elusive. We developed machine learning algorithms to estimate antigenic distance in silico, based on genetic sequence data, and identify differences in specific amino acid sites associated with antigenic differences between viruses. First, we obtained antigenic distance estimates derived from serum neutralization assays cross-reacting PRRSV monospecific antisera with virus isolates from 27 PRRSV1 viruses circulating in Europe. Antigenic distances were weakly to moderately associated with ectodomain amino acid distance for open reading frames (ORFs) 2 to 4 (ρ < 0.2) and ORF5 (ρ = 0.3), respectively. Dividing the antigenic distance values at the median, we then categorized the sera-virus pairs into two levels: low and high antigenic distance (dissimilarity). In the machine learning models, we used amino acid distances in the ectodomains of ORFs 2 to 5 and site-wise amino acid differences between the viruses as potential predictors of antigenic dissimilarity. Using mixed-effect gradient boosting models, we estimated the antigenic distance (high versus low) between serum-virus pairs with an accuracy of 81% (95% confidence interval, 76 to 85%); sensitivity and specificity were 86% and 75%, respectively. We demonstrate that using sequence data we can estimate antigenic distance and potential cross-protection between PRRSV1 strains. IMPORTANCE Understanding cross-protection between cocirculating PRRSV1 strains is crucial to reducing losses associated with PRRS outbreaks on farms. While experimental studies to determine cross-protection are instrumental, these in vivo studies are not always practical or timely for the many cocirculating and emerging PRRSV strains. In this study, we demonstrate the ability to rapidly estimate potential immunologic cross-reaction between different PRRSV1 strains in silico using sequence data routinely collected by production systems. These models can provide fast turn-around information crucial for improving PRRS management decisions such as selecting vaccines/live virus inoculation to be used on farms and assessing the risk of outbreaks by emerging strains on farms previously exposed to certain PRRSV strains and vaccine development among others.

The Ability of Porcine Reproductive and Respiratory Syndrome Virus Isolates to Induce Broadly Reactive Neutralizing Antibodies Correlates With In Vivo Protection.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most relevant diseases of swine. The condition is caused by PRRS virus (PRRSV), an extremely variable virus of the Arteriviridae family. Its heterogeneity can be responsible, at least partially, of the poor cross-protection observed between PRRSV isolates. Neutralizing antibodies (NAs), known to play a role in protection, usually poorly recognize heterologous PRRSV isolates, indicating that most NAs are strain-specific. However, some pigs develop broadly reactive NAs able to recognize a wide range of heterologous isolates. The aim of this study was to determine whether PRRSV isolates that induce broadly reactive NAs as determined in vitro are able to confer a better protection in vivo. For this purpose two in vivo experiments were performed. Initially, 40 pigs were immunized with a PRRSV-1 isolate known to induce broadly reactive NAs and 24 additional pigs were used as controls. On day 70 after immunization, the pigs were divided into eight groups composed by five immunized and three control pigs and exposed to one of the eight different heterologous PRRSV isolates used for the challenge. In the second experiment, the same experimental design was followed but the pigs were immunized with a PRRSV-1 isolate, which is known to generate mostly strain-specific NAs. Virological parameters, specifically viremia and the presence of challenge virus in tonsils, were used to determine protection. In the first experiment, sterilizing immunity was obtained in three groups, prevention of viremia was observed in two additional groups, although the challenge virus was detected occasionally in the tonsils of immunized pigs, and partial protection, understood as a reduction in the frequency of viremia compared with controls, was recorded in the remaining three groups. On the contrary, only partial protection was observed in all groups in the second experiment. The results obtained in this study confirm that PRRSV-1 isolates differ in their ability to induce cross-reactive NAs and, although other components of the immune response might have contributed to protection, pigs with cross-reactive NAs at the time of challenge exhibited better protection, indicating that broadly reactive NAs might play a role in protection against heterologous reinfections.

Interaction of PRRS virus with bone marrow monocyte subsets.

PRRSV can replicate for months in lymphoid organs leading to persistent host infections. Porcine bone marrow comprises two major monocyte subsets, one of which expresses CD163 and CD169, two receptors involved in the entry of PRRSV in macrophages. In this study, we investigate the permissiveness of these subsets to PRRSV infection. PRRSV replicates efficiently in BM CD163+ monocytes reaching titers similar to those obtained in alveolar macrophages, but with a delayed kinetics. Infection of BM CD163- monocytes was variable and yielded lower titers. This may be related with the capacity of BM CD163- monocytes to differentiate into CD163+ CD169+ cells after culture in presence of M-CSF. Both subsets secreted IL-8 in response to virus but CD163+ cells tended to produce higher amounts. The infection of BM monocytes by PRRSV may contribute to persistence of the virus in this compartment and to hematological disorders found in infected animals such as the reduction in the number of peripheral blood monocytes.

Splenic CD163+ macrophages as targets of porcine reproductive and respiratory virus: Role of Siglecs.

CD169 and CD163 have been involved in the process of PRRS virus attachment and infection in macrophages, although recent studies have challenged the requirement for CD169. In addition to CD169, macrophages express other siglecs, whose role in PRRS virus infection is so far unknown. Splenic CD163+ macrophages express Siglec-3 and Siglec-5 but almost undetectable levels of CD169. Hence, we considered this cell population appropriate for analysing the role of these siglecs in the attachment and internalization of PRRS virus into macrophages. PRRS virus replicated efficiently in these macrophages, yielding even higher titres than in alveolar macrophages. Besides, a recombinant protein consisting in the ectodomain of porcine Siglec-3 fused to the Fc fragment of human IgG1 (Siglec3-Fc) was able to bind PRRS virus, while binding to Siglec-5-Fc was inconsistent. Antibodies to CD169 but not to Siglec-3 or Siglec-5 blocked the binding and infection of PRRS virus on alveolar macrophages. Unexpectedly, our antibody to CD169 also blocked the binding of PRRS virus to splenic CD163+ macrophages, whereas antibodies to Siglec-3 or Siglec-5 had no effect. These results show that very low levels of CD169 expression are enough to support the attachment and internalization of PRRS virus into macrophages, whereas Siglec-3 and Siglec-5 do not seem to contribute to the virus entry in these cells.

Safety of Porcine Reproductive and Respiratory Syndrome Modified Live Virus (MLV) vaccine strains in a young pig infection model.

The objective of this study was to compare the safety of all modified live virus vaccines commercially available in Europe against Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) under the same experimental conditions. For this purpose, one hundred and twenty three-week-old piglets, divided into five groups, were used. On day 0 of the experiment, nine pigs per group were removed and the remaining fifteen were vaccinated with the commercial vaccines Ingelvac PRRS MLV, Amervac PRRS, Pyrsvac-183 and Porcilis PRRS by the IM route or were mock vaccinated and used as controls. On day 3, the nine unvaccinated pigs were re-introduced into their respective groups and served as sentinel pigs. Clinical signs were recorded daily and lung lesions were determined on days 7, 14 and 21, when 5 vaccinated pigs per group were euthanized. Blood samples and swabs were taken every three days and different organs were collected at necropsy to determine the presence of PRRSV. None of the vaccines studied caused detectable clinical signs in vaccinated pigs although lung lesions were found. Altogether, these results indicate that all vaccines can be considered clinically safe. However, some differences were found in virological parameters. Thus, neither Pyrsvac-183 nor Porcilis PRRS could be detected in porcine alveolar macrophage (PAM) cultures or in lung sections used to determine PRRSV by immunohistochemistry, indicating that these viruses might have lost their ability to replicate in PAM. This inability to replicate in PAM might be related to the lower transmission rate and the delay in the onset of viremia observed in these groups.

Porcine Reproductive and Respiratory Syndrome Virus isolates differ in their susceptibility to neutralization.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is highly heterogenic. This heterogeneity has an effect on antigenic composition of PRRSV and might create differences in sensitivity to neutralization between isolates. The sensitivity to neutralization could be an important feature of PRRSV isolates because it is likely that isolates resistant to neutralization pose a significant challenge for the development of vaccines that elicit broad protective immunity. Nonetheless, little information is available for understanding or categorizing the viral neutralization phenotype of PRRSV isolates. Consequently, the main purpose of this study was to determine whether PRRSV isolates differ in their susceptibility to neutralization and if they can be classified in different categories based on their neutralization phenotype. For this purpose, a panel of 39 PRRSV isolates and a set of 30 hyperimmune monospecific sera were used in cross-neutralization assays. The results of this study indicate that PRRSV isolates differ in their sensitivity to neutralization and k-means clustering system allowed classifying the isolates in four different categories according to their neutralization phenotype: highly sensitive, sensitive, moderately sensitive and resistant to neutralization. Further analyses using two additional clustering systems that considered individual data for the classification of the isolates confirmed that classification obtained by k-means is accurate in most cases and that only in a few instances classification is less stringent. Sequences of GP3, GP4 and GP5 were analyzed but no correlation could be found between the sequence of previously identified neutralizing epitopes or the number of N-linked glycosylation sites in different proteins and the neutralization phenotype of the isolates. These data provide the first systematic assessment of overall neutralization sensitivities of a panel of diverse PRRSV isolates. The classification of the isolates provides a useful tool to facilitate the systematic characterization of neutralizing antibody production elicited by new vaccine candidates.

Immunisation of pigs with a major envelope protein sub-unit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) results in enhanced clinical disease following experimental challenge.

Disease exacerbation was observed in pigs challenged with virulent porcine reproductive and respiratory syndrome virus (PRRSV) following immunisation with a recombinant GP5 sub-unit PRRSV vaccine (rGP5) produced in E. coli. Eighteen animals were divided into three experimental groups: group A were immunised twice IM with rGP5, 21 days apart; group B acted as positive controls (challenged but not immunised); and group C were negative controls. Pigs in groups A and B were challenged 21 days after the second immunisation of the group A animals. Following challenge, three pigs given rGP5 exhibited more severe clinical signs than the positive controls, including respiratory distress and progressive weight-loss. Although not statistically significant, the more severe disease exhibited by group A animals may suggest previous immunisation as a contributory factor. The mechanisms of these findings remain unclear and no association could be established between the severity of disease, non-neutralising antibody concentrations and tissue viral loads.

Influence of time on the genetic heterogeneity of Spanish porcine reproductive and respiratory syndrome virus isolates.

The aim of the present study was to establish the degree of diversity of porcine reproductive and respiratory virus (PRRSV) isolates that circulate in the same geographical area in different years. Nucleotide sequences of open reading frame (ORF) 5 were determined for 28 Spanish field PRRSV isolates from different years and three European-type modified live virus vaccines. Sequences were aligned using Clustal W software and a phylogenetic tree constructed using the neighbour joining method. The results of pairwise homology comparisons of nucleotide and deduced amino acid sequences of these PRRSV isolates indicate a tendency for heterogeneity to increase with time. The study of the phylogenetic tree revealed that Spanish PRRSV isolates constitute two well-defined clades and a group of unrelated sequences. The observed heterogeneity does not appear to be due to temporal evolution exclusively. Early and recent isolates group themselves into different clusters independently of the time of isolation, indicating the co-circulation of different variants and the maintenance of variants of the original isolates in the field.

Similarity of European porcine reproductive and respiratory syndrome virus strains to vaccine strain is not necessarily predictive of the degree of protective immunity conferred.

The objective of this study was to determine the degree of protection conferred by a Lelystad-like modified live virus (MLV) vaccine against a heterologous wild-type porcine reproductive and respiratory syndrome virus (PRRSV) isolate of the same cluster. For this purpose, fourteen 3-week-old piglets were divided into three groups: Group A pigs were vaccinated with a modified live virus vaccine, Group B pigs were used as positive controls, and Group C pigs as negative controls. Twenty-eight days after the last dose of vaccine, all pigs in Groups A and B were inoculated with the Spanish PRRSV strain 5710. To evaluate efficacy, clinical signs were recorded and the presence of challenge virus was determined by virus isolation in blood samples and nasal swabs collected at various time points post-challenge (p.c.) and in tissue samples collected at necropsy 24 days p.c. After challenge, moderate clinical signs were observed in pigs from Groups A and B. In addition, all vaccinated pigs were viremic at least once, although viremia tended to be more sporadic in this group than in Group B pigs. PRRSV was detected in at least one tissue sample from four out of five pigs from Group A and in all pigs from Group B. The results indicate that the protection conferred by the MLV vaccine used in this study against a closely related virulent strain was only partial. The findings suggest that the degree of genetic homology of ORF5 between MLV vaccine and challenge isolate is not a good predictor of vaccine efficacy.

Reproductive performance of gilts following vaccination and subsequent heterologous challenge with European strains of porcine reproductive and respiratory syndrome virus.

The objective of this study was to evaluate the efficacy of two commercially available modified live virus vaccines for preventing the reproductive and early postnatal consequences of infecting (challenging) pregnant gilts with virulent porcine reproductive and respiratory syndrome virus (PRRSV). For this purpose 21 crossbred gilts were allocated to one or another of four groups (Groups A-D). Group A comprised four gilts neither vaccinated nor challenged; Group B comprised five gilts that were challenged but not vaccinated; Group C comprised seven gilts that were vaccinated (AmervacPRRS) and challenged; Group D comprised five gilts that were vaccinated (Pyrsvac-183) and challenged. Vaccination was 24 days before conception, and challenge was at 90 days of gestation. Both vaccine viruses and the challenge virus were European strains but differed in part from one another on the basis of their genetic (nucleotide) sequence. After challenge PRRSV was isolated from five (100%), four (57%), and two (40%) of the gilts of Groups B, C and D, respectively. Although vaccination failed to prevent a detectable viremia in all of the gilts of Groups C and D after they were challenged (or congenital infection of some of their pigs), it did provide a statistically significant level of protection in regard to the incidence of congenital infection, reproductive performance, and pig health and viability. Namely, for Groups C and D the numbers of liveborn pigs/litter and healthy pigs/litter throughout the early postnatal period were similar to those of Group A (nonvaccinated and nonchallenged) and far exceeded those of Group B (nonvaccinated and challenged).

Effects of two commercial European modified-live vaccines against porcine reproductive and respiratory syndrome viruses in pregnant gilts.

The purpose of this study was to assess the effects of two currently available commercial European-type modified-live virus (MLV) vaccines against porcine reproductive and respiratory syndrome in a reproductive pig model. Sixteen 90-day pregnant gilts were divided into four groups and allocated to one of the following intranasal treatments: group A gilts served as negative controls; group B gilts were exposed to a virulent European field strain; group C gilts were exposed to vaccine strain VP046 Bis and group D gilts to vaccine strain All-183. The results indicated that MLV strains can replicate in breeding animals and have the ability to cross the placenta. In particular, viraemia was detected in all gilts in group C and 2/4 gilts in group D, at least at one time point. In addition, transplacental infection was demonstrated in 3/4 gilts in group C and 2/4 gilts in group D. However, congenital and early postnatal infection did not have a marked detrimental effect on piglet performance when compared to negative controls, and no statistically significant differences were observed in most cases. Conversely, the reproductive performance of gilts in group B was significantly worse than that of the other groups. Specifically, the number of born-alive piglets, the survival rate of piglets during lactation and the mean weight of weaned pigs were significantly lower. It was concluded that the two commercial European-type MLV vaccines tested had no marked detrimental effects in pregnant gilts, although the MLV strains can cross the placenta leading to the birth of congenitally infected piglets.

Porcine reproductive and respiratory syndrome virus infection in the boar: a review.

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus, which, like other members of the Arterividae family, has the ability to infect macrophages and to persist in tissues for at least several months after the acute stage of infection subsides. As a consequence, PRRS has a complex epidemiologic profile and has been especially difficult to control under the usual conditions of commercial swine production. Although vaccines are commonly used, vaccination is only one of several approaches to be considered in designing a control strategy. At least equally important are procedures developed on the basis of a thorough understanding of the epidemiology of the disease. The objective of this review is to summarize current knowledge in relation to PRRS virus (PRRSV) infection in the boar. The information available related to this topic will be summarized and discussed, and the implications for the control of the condition highlighted. The main emphasis will be on questions about the pathogenesis of infection, including duration of viremia and the origin of PRRSV found in semen; the clinical signs associated with the disease, paying special attention to the effects on seminal quality; the epidemiology of the condition, with special emphasis on the duration of PRRSV shedding in semen and the implications that this may have on venereal transmission, as well as the role that other potential routes of shedding may have on the dissemination of PRRSV.

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.