Persistent domestic circulation of African swine fever virus in Tanzania, 2015-2017.

BACKGROUND: African swine fever (ASF) is a highly fatal viral hemorrhagic disease of domestic pigs that threatens livelihoods and food security. In Africa, ASF virus (ASFV) circulates in sylvatic (transmission between warthogs and soft argasid ticks) and domestic (transmission between domestic pigs) cycles, with outbreaks resulting from ASFV spill-over from sylvatic cycle. A number of outbreaks were reported in different parts of Tanzania between 2015 and 2017. The present study investigated ASFV transmission patterns through viral DNA sequencing and phylogenetic analysis. A total of 3120 tissue samples were collected from 2396 domestic pigs during outbreaks at different locations in Tanzania between 2015 and 2017. Partial sequencing of the B646L (p72) gene was conducted for diagnostic confirmation and molecular characterization of ASFV. Phylogenetic analysis to study the relatedness of current ASFV with those that caused previous outbreaks in Tanzania and representatives of all known 24 ASFV was performed using the Maximum Composite Likelihood model with 1000 bootstrap replications in MEGA 6.0. RESULTS: ASFV was confirmed to cause disease in sampled domestic pigs. ASFV genotypes II, IX, and X were detected from reported outbreaks in 2015-2017. The current ASFV isolates were similar to those recently documented in the previous studies in Tanzania. The similarities of these isolates suggests for continuous circulation of ASFV with virus maintenance within the domestic pigs. CONCLUSIONS: Genetic analysis confirmed the circulation of ASFV genotypes II, IX, and X by partial B646L (p72) gene sequencing. The similarities of current isolates to previously isolated Tanzanian isolates and pattern of disease spread suggest for continuous circulation of ASF with virus' maintenance in the domestic pigs. Although certain viral genotypes seem to be geographically restricted into certain zones within Tanzania, genotype II seems to expand its geographical range northwards with the likelihood of spreading to other states of the East African Community. The spread of ASFV is due to breach of quarantine and transportation of infected pigs via major highways. Appropriate control measures including zoosanitary measures and quarantine enforcement are recommended to prevent ASF domestic circulation in Tanzania.

Implementation of full-length 16S nanopore sequencing for bacterial identification in a clinical diagnostic setting.

This study describes the implementation of 16S nanopore sequencing in a diagnostic lab for pathogen identification without prior enrichment. First, the universality of the test and taxonomic resolution was evaluated for 78 clinically relevant bacteria (69 known and 9 unknown bacterial cultures). Next, the diagnostic value of the test was evaluated based on clinical samples. It was shown that 16S sequencing can be used both for identification of unknown cultures and to find bacteria directly in the clinical sample without cultivation. All culture-positive samples (n=11) tested positive with 16S sequencing directly performed on the sample, but bacteria were found as well in 15/30 culture-negative samples. Pathogenic bacteria were found in a background of commensal flora, and even complex polymicrobial infections could be unraveled. This study demonstrates the feasibility of implementing 16S nanopore sequencing in a clinical diagnostic setting and demonstrates its value for the diagnosis of culture-negative and polymicrobial infections.

Comparison of the efficacy of autogenous inactivated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccines with that of commercial vaccines against homologous and heterologous challenges.

BACKGROUND: The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving pathogen of swine. At present, there is a high demand for safe and more effective vaccines that can be adapted regularly to emerging virus variants. A recent study showed that, by the use of a controlled inactivation procedure, an experimental BEI-inactivated PRRSV vaccine can be developed that offers partial protection against homologous challenge with the prototype strain LV. At present, it is however not known if this vaccine can be adapted to currently circulating virus variants. In this study, two recent PRRSV field isolates (07 V063 and 08 V194) were used for BEI-inactivated vaccine production. The main objective of this study was to assess the efficacy of these experimental BEI-inactivated vaccines against homologous and heterologous challenge and to compare it with an experimental LV-based BEI-inactivated vaccine and commercial inactivated and attenuated vaccines. In addition, the induction of challenge virus-specific (neutralizing) antibodies by the different vaccines was assessed. RESULTS: In a first experiment (challenge with 07 V063), vaccination with the experimental homologous (07 V063) inactivated vaccine shortened the viremic phase upon challenge with approximately 2 weeks compared to the mock-vaccinated control group. Vaccination with the commercial attenuated vaccines reduced the duration of viremia with approximately one week compared to the mock-vaccinated control group. In contrast, the experimental heterologous (LV) inactivated vaccine and the commercial inactivated vaccine did not influence viremia. Interestingly, both the homologous and the heterologous experimental inactivated vaccine induced 07 V063-specific neutralizing antibodies upon vaccination, while the commercial inactivated and attenuated vaccines failed to do so.In the second experiment (challenge with 08 V194), use of the experimental homologous (08 V194) inactivated vaccine shortened viremia upon challenge with approximately 3 weeks compared to the mock-vaccinated control group. Similar results were obtained with the commercial attenuated vaccine. The experimental heterologous (07 V063 and LV) inactivated vaccines did not significantly alter viremia. In this experiment, 08 V194-specific neutralizing antibodies were induced by the experimental homologous and heterologous inactivated vaccines and a faster appearance post challenge was observed with the commercial attenuated vaccine. CONCLUSIONS: The experimental homologous inactivated vaccines significantly shortened viremia upon challenge. Despite the concerns regarding the efficacy of the commercial attenuated vaccines used on the farms where the field isolates were obtained, use of commercial attenuated vaccines clearly shortened the viremic phase upon challenge. In contrast, the experimental heterologous inactivated vaccines and the commercial inactivated vaccine had no or only a limited influence on viremia. The observation that homologous BEI-inactivated vaccines can provide a more or less standardized, predictable degree of protection against a specific virus variant suggests that such vaccines may prove useful in case virus variants emerge that escape the immunity induced by the attenuated vaccines.

Pitfalls of SARS-CoV-2 antigen testing at emergency department.

BACKGROUND: Current method for diagnosis of SARS-CoV-2 infection is an RT-PCR test on the nasopharyngeal or oropharyngeal swab. Rapid diagnosis is essential for containing viral spread and triage of symptomatic patients presenting to hospital ER departments. As a faster alternative to RT-PCR, we evaluated a SARS-Cov-2 Rapid Antigen test in symptomatic patients presenting to hospital ER departments. METHODS: We evaluated the diagnostic performance of the Roche SARS-CoV-2 Rapid Antigen test (SD Biosensor) for detection of SARS-CoV-2 compared to RT-PCR. RESULTS: Our study showed inferior performance of the SARS-CoV-2 Rapid Antigen test for detection of SARS-CoV-2. Firstly, because of the lack of specificity, which is potentially life-threatening due to the association of nosocomial-acquired SARS-CoV-2 infection. Secondly, with a sensitivity of 45.5%, it is impossible to rule out SARS-CoV-2 infection, resulting in reflex PCR-testing. Comparison of viral load in RT-PCR positive samples with corresponding antigen results showed a significant difference between antigen positive and negative samples. COVID-19 infection will not be detected in patients admitted to the hospital in an early or late phase, typically associated with low viral loads. Sensitivity increases when testing within 5-7 symptomatic days, but the implementation of this cut-off is impractical in ER settings. However, diagnostic performance is better to detect high viral load (> = 5 log10 copies/mL) linked with contagiousness. CONCLUSION: Our study showed inferior performance of the Roche SARS-CoV-2 Rapid Antigen test (SD Biosensor) for detection of SARS-CoV-2 which limits its use as a diagnostic gatekeeper in ER departments, but is able to differentiate contagious individuals.

Cycle Threshold Probability Score for Immediate and Sensitive Detection of B.1.351 SARS-CoV-2 Lineage.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern associated with immune escape is important to safeguard vaccination efficacy. We describe the potential of delayed N gene amplification in the Allplex SARS-CoV-2 Assay (Seegene) for screening of the B.1.351 (20H/501.V2, variant of concern 2 [VOC.V2], South African SARS-CoV-2 variant) lineage. METHODS: In a study cohort of 397 consecutive polymerase chain reaction-positive samples genotyped by whole-genome sequencing, amplification curves of E/N/S-RdRP targets indicated delayedN vs E gene amplification characteristic of B.1.351. Logistic regression was used to calculate a VOC.V2 probability score that was evaluated as a separate screening test in an independent validation cohort vs sequencing. RESULTS: B.1.351 showed a proportionally delayed amplification of the  N vs E gene. In logistic regression, only N and E gene cycle thresholds independently contributed to B.1.351 prediction, allowing calculation of a VOC.V2 probability score with an area under the curve of 0.94. At an optimal dichotomous cutoff point of 0.12, the VOC.V2 probability score achieved 98.7% sensitivity at 79.9% specificity, resulting in a negative predictive value (NPV) of 99.6% and a positive predictive value of 54.6%. The probability of B.1.351 increased with an increasing VOC.V2 probability score, achieving a likelihood ratio of 12.01 above 0.5. A near-maximal NPV was confirmed in 153 consecutive validation samples. CONCLUSIONS: Delayed N vs E gene amplification in the Allplex SARS-CoV-2 Assay can be used for fast and highly sensitive screening of B.1.351.

Severity of COVID-19 among Hospitalized Patients: Omicron Remains a Severe Threat for Immunocompromised Hosts.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the general population in the context of a relatively high immunity gained through the early waves of coronavirus disease 19 (COVID-19), and vaccination campaigns. Despite this context, a significant number of patients were hospitalized, and identifying the risk factors associated with severe disease in the Omicron era is critical for targeting further preventive, and curative interventions. We retrospectively analyzed the individual medical records of 1501 SARS-CoV-2 positive hospitalized patients between 13 December 2021, and 13 February 2022, in Belgium, of which 187 (12.5%) were infected with Delta, and 1036 (69.0%) with Omicron. Unvaccinated adults showed an increased risk of moderate/severe/critical/fatal COVID-19 (crude OR 1.54; 95% CI 1.09-2.16) compared to vaccinated patients, whether infected with Omicron or Delta. In adults infected with Omicron and moderate/severe/critical/fatal COVID-19 (n = 323), immunocompromised patients showed an increased risk of in-hospital mortality related to COVID-19 (adjusted OR 2.42; 95% CI 1.39-4.22), compared to non-immunocompromised patients. The upcoming impact of the pandemic will be defined by evolving viral variants, and the immune system status of the population. The observations support that, in the context of an intrinsically less virulent variant, vaccination and underlying patient immunity remain the main drivers of severe disease.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites.

Reproductive failure due to porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by late-term abortions, early farrowing and an increase of dead and mummified fetuses and weak-born piglets. The mechanism of PRRSV-induced reproductive failure is poorly understood. Human pregnancies, complicated by some pathogens leading to reproductive disorders exhibit increased apoptosis in the fetal membranes. Because PRRSV-target cells are present in endometrium/fetal placentas from healthy sows and PRRSV-infected macrophages in other organs die by apoptosis, we hypothesized that PRRSV can replicate and induce apoptosis in the fetal implantation sites at the last stage of gestation. In the present study, identification, localization and quantification of the PRRSV-positive and apoptotic cells were performed in the fetal implantation sites. Three dams were inoculated intranasally with 10(5) TCID(50) PRRSV 07V063 at 90 days of gestation and sampled at 10 days post-inoculation. Two non-inoculated dams that were euthanized at 100 days of gestation served as control animals. Inoculation of the dams resulted in a viremia that lasted until the end of the study. Transplacental PRRSV spread was detected in all inoculated dams. Using immunofluorescence staining, single PRRSV-positive cells were found in the endometrial connective tissues adjacent to both PRRSV-positive and PRRSV-negative fetuses. In the fetal placental mesenchyme of the PRRSV-positive fetuses, infected cells were more abundant and spread focally. Double staining showed that all PRRSV-positive cells in the fetal implantation sites were positive for sialoadhesin and CD163. Apoptotic cells (TUNEL+) were detected in endometrium and fetal placentas of both non- and PRRSV-inoculated dams. The number of apoptotic cells was significantly higher in PRRSV-positive endometrium/fetal placentas. PRRSV caused apoptosis in infected cells since 20-61% of PRRSV-positive cells were apoptotic and in surrounding cells since 43-91% of the apoptotic cells were virus-negative. The main conclusion obtained from the present study is that PRRSV replicates in the fetal implantation sites and causes apoptosis in infected macrophages and surrounding cells at the last stage of gestation. The possible mode of PRRSV replication in the fetal implantation sites and the events that might contribute to the reproductive disorders are discussed.

Functional Analysis of Human and Feline Coronavirus Cross-Reactive Antibodies Directed Against the SARS-CoV-2 Fusion Peptide.

To face the continuous emergence of SARS-CoV-2 variants, broadly protective therapeutic antibodies are highly needed. We here focused on the fusion peptide (FP) region of the viral spike antigen since it is highly conserved among alpha- and betacoronaviruses. First, we found that coronavirus cross-reactive antibodies are commonly formed during infection, being omnipresent in sera from COVID-19 patients, in ~50% of pre-pandemic human sera (rich in antibodies against endemic human coronaviruses), and even in feline coronavirus-infected cats. Pepscan analyses demonstrated that a confined N-terminal region of the FP is strongly immunogenic across diverse coronaviruses. Peptide-purified human antibodies targeting this conserved FP epitope exhibited broad binding of alpha- and betacoronaviruses, besides weak and transient SARS-CoV-2 neutralizing activity. Being frequently elicited by coronavirus infection, these FP-binding antibodies might potentially exhibit Fc-mediated effector functions and influence the kinetics or severity of coronavirus infection and disease.

GP4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro.

Glycoprotein 4 (GP4) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a highly variable neutralizing epitope. The present study aimed to investigate whether this epitope is susceptible to immunoselection by antibodies in vitro. Cultivation of PRRSV in vitro in the continuous presence of neutralizing monoclonal antibodies (mAbs) directed against this epitope resulted in the selection of mAb-resistant PRRSV strains within five passages. Comparison of the GP4 amino acid (aa) sequence of the original PRRSV strain with the GP4 aa sequences of the mAb-resistant PRRSV strains revealed aa substitutions within this epitope. In conclusion, this study shows that the neutralizing epitope on GP4 is susceptible to immunoselection by antibodies in vitro.

Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is divided into a European and North American genotype. East European PRRSV isolates have been found to be of the European genotype, but form different subtypes. In the present study, PRRSV was isolated from a Belarusian farm with reproductive and respiratory failure and designated "Lena". Analyses revealed that Lena is a new East European subtype 3 PRRSV isolate. The main purpose of this investigation was to study the pathogenesis and antigenic characteristics of PRRSV (Lena). RESULTS: Obvious clinical and virological differences were observed between the animals inoculated with a recent European subtype 1 PRRSV isolate (Belgium A) and animals inoculated with PRRSV (Lena). Three out of six pigs inoculated with PRRSV (Belgium A) had anorexia and low fever at 3, 4 and 5 days post-inoculation (dpi). High fever, anorexia and depression were prominent signs in most pigs inoculated with PRRSV (Lena) between 2 and 28 dpi. Four pigs out of ten died during the experiment. Arcanobacterium pyogenes was isolated from lungs of one animal that died, and Streptococcus suis was isolated from lungs of one animal that was euthanized. The difference in viral titres in sera from PRRSV (Belgium A) and PRRSV (Lena)-infected pigs was statistically significant (p < 0.05) at 7, 10, 14 and 21 dpi. The highest viral titres in sera ranged from 10(4.8) to 10(6.1) TCID50/ml for PRRSV (Lena) whereas they ranged from 10(3.1) to 10(4.8) TCID50/ml for PRRSV (Belgium A).The replication of PRRSV (Lena) was further studied in depth. Viral titres ranged from 10(2.5) TCID50/100 mg to 10(5.6) TCID50/100 mg in nasal secretions between 3 and 14 dpi and from 10(2.8) TCID50/100 mg to 10(4.6) TCID50/100 mg in tonsillar scrapings between 3 and 21 dpi. High viral titres were detected in lungs (10(2.3)-10(7.7) TCID50/g tissue), tonsils (10(2.0)-10(6.2) TCID50/g tissue) and inguinal lymph nodes (10(2.2)-10(6.6) TCID50/g tissue) until 35, 28 and 35 dpi, respectively.To examine the antigenic heterogeneity between the East European subtype 3 isolate Lena, the European subtype 1 strain Lelystad and the North American strain US5, sets of monospecific polyclonal antisera were tested in immunoperoxidase monolayer assays (IPMAs) with homologous and heterologous viral antigens. Heterologous antibody titres were significantly lower than homologous titres (p = 0.01-0.03) for antisera against PRRSV (Lena) at all sampling time points. For antisera against PRRSV (Lelystad) and PRRSV (US5), heterologous antibody titres were significantly lower than homologous titres at 14 and 21 dpi (p = 0.01-0.03) and at 10 and 14 dpi (p = 0.04), respectively. CONCLUSIONS: Lena is a highly pathogenic East European subtype 3 PRRSV, which differs from European subtype 1 Lelystad and North American US5 strains at both the genetic and antigenic level.

Toxocara in sandpits of public playgrounds and kindergartens in Flanders (Belgium).

Belgium counts more than a million dogs and at least two million cats, of which many are carrying zoonotic nematodes of the genus Toxocara. Environmental contamination with worm eggs is considered the key transmission route from animals to humans, and mainly young children are at risk. Contamination of soil with Toxocara eggs has been reported from all over the world, but data are lacking for Belgium. In this study, faecal contamination and the presence of Toxocara eggs in sand were investigated in sandpits of public playgrounds and kindergartens in Flanders (Northern Belgium). Faeces, of which 85% originated from cats, were found in about one third of the public playgrounds and one fifth of the kindergartens. Toxocara eggs were found in 12% of the faecal samples, in 14% of the public sandpits, and in 2% of the sandpits of kindergartens. These data indicate that environmental contamination with Toxocara exists in urban areas in Belgium, and that cats are most likely the main source.

RNA-sequence analysis of primary alveolar macrophages after in vitro infection with porcine reproductive and respiratory syndrome virus strains of differing virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects porcine alveolar macrophages (PAMs), resulting in porcine reproductive and respiratory syndrome (PRRS) in pigs. Most of the transcriptomic studies on PAMs infected with PRRSV conducted thus far have made use of microarray technology. Here, we investigated the transcriptome of PAMs in vitro at 12 h post-infection with two European PRRSV strains characterized by low (Lelystad, LV) and high (Lena) virulence through RNA-Seq. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with the two strains. Gene ontology analysis confirmed that infection of PAMs with both the Lena and LV strains affected signaling pathways directly linked to the innate immune response, including interferon regulatory factors (IRF), RIG1-like receptors, TLRs and PKR pathways. The results confirmed that interferon signaling is crucial for transcriptional regulation during PAM infection. IFN-ß1 and IFN-αω, but not IFN-α, were up-regulated following infection with either the LV or Lena strain. The down-regulation of canonical pathways, such as the interplay between the innate and adaptive immune responses, cell death and TLR3/TLR7 signaling, was observed for both strains, but Lena triggered a stronger down-regulation than LV. This analysis contributes to a better understanding of the interactions between PRRSV and PAMs and outlines the differences in the responses of PAMs to strains with different levels of virulence, which may lead to the development of new PRRSV control strategies.

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains.

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10(4.3)TCID50, three with K39-10(4.3)TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10(4.3)TCID50, six with K2-low dose-10(2.3)TCID50 and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10(3.3)TCID50/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10(5.6)TCID50/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.

Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and respiratory disease in pigs of all ages. Despite the frequent use of vaccines to maintain PRRSV immunity in sows, little is known on how the currently used vaccines affect the immunity against currently circulating and genetically divergent PRRSV variants in PRRSV-immune sows, i.e. sows that have a pre-existing PRRSV-specific immunity due to previous infection with or vaccination against the virus. Therefore, this study aimed to assess the capacity of commercially available attenuated/inactivated PRRSV vaccines and autogenous inactivated PRRSV vaccines - prepared according to a previously optimized in-house protocol - to boost the antibody immunity against currently circulating PRRSV variants in PRRSV-immune sows. PRRSV isolates were obtained from 3 different swine herds experiencing PRRSV-related problems, despite regular vaccination of gilts and sows against the virus. In a first part of the study, the PRRSV-specific antibody response upon booster vaccination with commercial PRRSV vaccines and inactivated farm-specific PRRSV vaccines was evaluated in PRRSV-immune, non-pregnant replacement sows from the 3 herds. A boost in virus-neutralizing antibodies against the farm-specific isolate was observed in all sow groups vaccinated with the corresponding farm-specific inactivated vaccines. Use of the commercial attenuated EU type vaccine boosted neutralizing antibodies against the farm-specific isolate in sows derived from 2 farms, while use of the commercial attenuated NA type vaccine did not boost farm-specific virus-neutralizing antibodies in any of the sow groups. Interestingly, the commercial inactivated EU type vaccine boosted farm-specific virus-neutralizing antibodies in sows from 1 farm. In the second part of the study, a field trial was performed at one of the farms to evaluate the booster effect of an inactivated farm-specific vaccine and a commercial attenuated EU-type vaccine in immune sows at 60 days of gestation. The impact of this vaccination on maternal immunity and on the PRRSV infection pattern in piglets during their first weeks of life was evaluated. Upon vaccination with the farm-specific inactivated vaccine, a significant increase in farm-specific virus-neutralizing antibodies was detected in all sows. Virus-neutralizing antibodies were also transferred to the piglets via colostrum and were detectable in the serum of these animals until 5 weeks after parturition. In contrast, not all sows vaccinated with the commercial attenuated vaccine showed an increase in farm-specific virus-neutralizing antibodies and the piglets of this group generally had lower virus-neutralizing antibody titers. Interestingly, the number of viremic animals (i.e. animals that have infectious virus in their bloodstream) was significantly lower among piglets of both vaccinated groups than among piglets of mock-vaccinated sows and this at least until 9 weeks after parturition. The results of this study indicate that inactivated farm-specific PRRSV vaccines and commercial attenuated vaccines can be useful tools to boost PRRSV-specific (humoral) immunity in sows and reduce viremia in weaned piglets.

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus.

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Despite great efforts of pig holders, veterinarians, researchers and vaccine developers, the virus still causes major production losses. It is clear that efficient and correct monitoring and rational development of vaccines are crucial in the combat against this pathogen. PRRSV-specific monoclonal antibodies (mAbs) are essential tools for both diagnostic and research purposes. This study describes the production of PRRSV GP3-, GP5- and N-specific hybridomas and an extensive characterization of the mAbs. The N-specific mAbs generated in this study appear to be useful tools for diagnostics, as they were found to react with genetically very different PRRSV isolates and may serve to discriminate between European and American type PRRSV isolates. These mAbs also allowed detection of the PRRSV N protein in both formalin-fixed, paraffin-embedded tissue sections and frozen tissue sections of PRRSV-infected lungs, further illustrating their diagnostic value. Different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested shows virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the "major neutralizing epitope" suggested for American type PRRSV. The current findings argue against an important role of the identified antigenic regions in direct antibody-mediated neutralization of European type PRRSV in vivo. However, it is also clear that findings concerning a specific PRRSV epitope cannot always be generalized, as the antigenic determinants and their biological properties may differ radically between different virus isolates.

GP4-specific neutralizing antibodies might be a driving force in PRRSV evolution.

The structural envelope glycoprotein GP4 of European porcine reproductive and respiratory syndrome virus (PRRSV) strains contains a highly variable neutralizing epitope that is susceptible to neutralizing antibody-mediated selective pressure in vitro. In this study, it was analyzed what happens with this neutralizing epitope during infection in vivo in the presence of neutralizing antibodies. A neutralizing antibody-mediated selective pressure was created in 30 pigs by vaccination prior to inoculation with infectious Lelystad virus (LV). Nine viable neutralizing antibody-escape variants were isolated from 9 of these pigs and their neutralizing antibody-escape mutant-identity was confirmed by the acquired resistance to neutralization by autologous neutralizing sera. Six out of 9 neutralizing antibody-escape variants contained aa substitutions in the GP4 neutralizing epitope and had become resistant to neutralization by a monoclonal antibody (mAb) against this epitope. In addition, in all 6 corresponding pigs, antibodies against this epitope were detected early in infection. In contrast to these 6 virus variants, the 3 other antibody-escape variants did not contain aa substitutions in the GP4 neutralizing epitope and were still sensitive to neutralization by the GP4-specific mAb. These antibody-escape variants were isolated from pigs that did not contain antibodies against this epitope early in infection. All these findings together strongly indicate that aa substitutions in the GP4 neutralizing epitope can abrogate antibody recognition, and that neutralizing antibodies might be responsible for the selection of neutralizing antibody-resistant variants with aa substitutions in the neutralizing epitope on GP4. In conclusion, this study indicates that neutralizing antibodies in pigs might be a driving force in the rapid evolution of the neutralizing epitope on GP4 of European PRRSV strains.

A variable region in GP4 of European-type porcine reproductive and respiratory syndrome virus induces neutralizing antibodies against homologous but not heterologous virus strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce severe reproductive failure in sows, and is involved in the porcine respiratory disease complex. The glycoprotein GP4 of the European prototype PRRSV strain Lelystad virus (LV) contains a linear neutralizing epitope that is located in a highly variable region. The current study aimed to evaluate the antibody response against this and other epitopes on GP4 to infection of pigs with European-type PRRSV. It was shown that three virus strains, differing in the region that corresponds to the neutralizing epitope on GP4 of LV, strongly induce antibodies against this area. Antibodies against the epitopes of the different virus strains were purified from polyclonal swine sera, and used in virus-neutralization tests on primary alveolar macrophages. This revealed that antibodies against the variable region in GP4 of different virus strains are able to neutralize infection with homologous but not heterologous virus strains.

Development of an experimental inactivated PRRSV vaccine that induces virus-neutralizing antibodies.

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce reproductive disorders and is involved in the porcine respiratory disease complex, causing tremendous economic losses to the swine industry. Inactivated PRRSV vaccines are preferred over attenuated vaccines because of their safety and flexibility towards emerging virus strains, but the efficacy of current inactivated PRRSV vaccines is questionable. In this study, experimental inactivated PRRSV vaccines were developed, based on two formerly optimized inactivation procedures: UV irradiation and treatment with binary ethylenimine (BEI). In a first experiment, it was shown that vaccination with UV- or BEI-inactivated virus in combination with Incomplete Freund's Adjuvant induced virus-specific antibodies and strongly primed the virus-neutralizing (VN) antibody response. Subsequently, the influence of adjuvants on the immunogenicity of neutralizing epitopes on the inactivated virus was investigated. It was shown that vaccination with BEI-inactivated virus in combination with a commercial oil-in-water adjuvant induced high titers (3.4 log(2)) of VN antibodies in 6/6 pigs, instead of only priming the neutralizing antibody response. After challenge, neutralizing antibody titers in these vaccinated animals rose to a mean value of 5.5 log(2), and the duration of the viremia was reduced to an average of 1 week. This study shows that, by the use of an optimized inactivation procedure and a suitable adjuvant, inactivated PRRSV vaccines can be developed that induce VN antibodies and offer partial protection upon challenge.