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The present study investigates the pathogenicity of two recently isolated strains of Suid herpesvirus 1 (SuHV1), the Greek strain Hercules and the Chinese strain HeN1, in unvaccinated pigs and in pigs vaccinated with a Bartha-K61 strain. In an experiment performed in negative pressure kiosks (isolators), 45-day old seronegative pigs previously oronasally /intramuscularly vaccinated with the Bartha-K61 vaccine strain, along with unvaccinated controls, were challenged either with the Hercules strain or the HeN1 strain of SuHV1. All animals were observed daily for clinical signs and body temperature and nasal swabs, faeces, blood and bodyweight were collected up to a maximum period of 20 days post-challenge (dpc). The results showed that, in the unvaccinated pigs, HeN1 strain was more virulent than the Hercules strain, with increased mortality, shorter time to death and higher group clinical score (p < 0.05). However, after vaccination with the Bartha-K61 vaccine, there was a drastic reduction in morbidity, mortality, bodyweight loss and virus excretion to almost a similar extent in both strains (p < 0.05). No significant differences were seen among the pigs of the two vaccinated groups compared to unvaccinated unchallenged controls, except a slight elevation in body temperature and in clinical score in the HeN1 vaccinees at 2 and 3 dpc, while bodyweight gain was similar to that of the negative control pigs. Our study showed that despite differences in virulence, the standard vaccination scheme with the Bartha-K61 strain could equally protect nursery pigs against both the European and Chinese strains., (© 2022. The Author(s).)

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT 50 ) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Singanallur, van Vuren, McAuley, Bruce, Kuiper, Gwini, Riddell, Goldie, Drew, Blasdell, Tachedjian, Mangalaganesh, Chahal, Caly, Druce, Juno, Kent, Wheatley and Vasan.)

As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivatives that can withstand 100 °C for 90 min and 37 °C for four weeks and help eliminate cold-chain requirements. We show that mice immunised with these vaccine formulations elicit high titres of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an average 14.4-fold reduction in neutralisation against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold reduction for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clinical trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modelling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerisation domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 10 4 TCID 50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3-7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies., (© 2022. The Author(s).)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Vaccines against SARS-CoV-2 are likely to be critical in the management of the ongoing pandemic. A number of candidates are in Phase III human clinical trials, including ChAdOx1 nCoV-19 (AZD1222), a replication-deficient chimpanzee adenovirus-vectored vaccine candidate. In preclinical trials, the efficacy of ChAdOx1 nCoV-19 against SARS-CoV-2 challenge was evaluated in a ferret model of infection. Groups of ferrets received either prime-only or prime-boost administration of ChAdOx1 nCoV-19 via the intramuscular or intranasal route. All ChAdOx1 nCoV-19 administration combinations resulted in significant reductions in viral loads in nasal-wash and oral swab samples. No vaccine-associated adverse events were observed associated with the ChAdOx1 nCoV-19 candidate, with the data from this study suggesting it could be an effective and safe vaccine against COVID-19. Our study also indicates the potential for intranasal administration as a way to further improve the efficacy of this leading vaccine candidate.

The 'D614G' mutation (Aspartate-to-Glycine change at position 614) of the SARS-CoV-2 spike protein has been speculated to adversely affect the efficacy of most vaccines and countermeasures that target this glycoprotein, necessitating frequent vaccine matching. Virus neutralisation assays were performed using sera from ferrets which received two doses of the INO-4800 COVID-19 vaccine, and Australian virus isolates (VIC01, SA01 and VIC31) which either possess or lack this mutation but are otherwise comparable. Through this approach, supported by biomolecular modelling of this mutation and the commonly-associated P314L mutation in the RNA-dependent RNA polymerase, we have shown that there is no experimental evidence to support this speculation. We additionally demonstrate that the putative elastase cleavage site introduced by the D614G mutation is unlikely to be accessible to proteases., Competing Interests: Competing interestsT.R.F.S. and K.E.B. are the co-inventors of INO-4800 and employees of Inovio Pharmaceuticals. Other authors declare no competing interests., (© Crown 2020.)

Background: The rate at which COVID-19 has spread throughout the globe has been alarming. While the role of fomite transmission is not yet fully understood, precise data on the environmental stability of SARS-CoV-2 is required to determine the risks of fomite transmission from contaminated surfaces., Methods: This study measured the survival rates of infectious SARS-CoV-2, suspended in a standard ASTM E2197 matrix, on several common surface types. All experiments were carried out in the dark, to negate any effects of UV light. Inoculated surfaces were incubated at 20 °C, 30 °C and 40 °C and sampled at various time points., Results: Survival rates of SARS-CoV-2 were determined at different temperatures and D-values, Z-values and half-life were calculated. We obtained half lives of between 1.7 and 2.7 days at 20 °C, reducing to a few hours when temperature was elevated to 40 °C. With initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. Conversely, infectious virus survived less than 24 h at 40 °C on some surfaces., Conclusion: These findings demonstrate SARS-CoV-2 can remain infectious for significantly longer time periods than generally considered possible. These results could be used to inform improved risk mitigation procedures to prevent the fomite spread of COVID-19.

Pre-clinical responses to fast-moving infectious disease outbreaks heavily depend on choosing the best isolates for animal models that inform diagnostics, vaccines and treatments. Current approaches are driven by practical considerations (e.g. first available virus isolate) rather than a detailed analysis of the characteristics of the virus strain chosen, which can lead to animal models that are not representative of the circulating or emerging clusters. Here, we suggest a combination of epidemiological, experimental and bioinformatic considerations when choosing virus strains for animal model generation. We discuss the currently chosen SARS-CoV-2 strains for international coronavirus disease (COVID-19) models in the context of their phylogeny as well as in a novel alignment-free bioinformatic approach. Unlike phylogenetic trees, which focus on individual shared mutations, this new approach assesses genome-wide co-developing functionalities and hence offers a more fluid view of the 'cloud of variances' that RNA viruses are prone to accumulate. This joint approach concludes that while the current animal models cover the existing viral strains adequately, there is substantial evolutionary activity that is likely not considered by the current models. Based on insights from the non-discrete alignment-free approach and experimental observations, we suggest isolates for future animal models., (© 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Dual use is defined as the application of materials, knowledge or technologies for military or terrorist purposes, as well as for good. In biological science, it is considered to be a growing threat as the genetics of pathogenicity traits and toxins are becoming on one hand elucidated in a detail that was not anticipated 20years ago and on the other hand technological advances in genetic engineering and synthetic biology are continually enabling easier access to these technologies. On a theoretical and policy level, much has happened over the past decade, but translating these policies and concepts to operational level awareness and robust processes requires more attention. Where the research is conducted, scientists have to make ethical judgements and account for their data sharing and publication policies. How can we ensure the requirement for dual use review is taken on board, but is not skewing research detrimentally and imposing a disproportionate burden?, (Copyright © 2017. Published by Elsevier Ltd.)

Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5-week-old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock-infected and used as controls. The animals were euthanized at 3, 7 and 35 days post-infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes-macrophages. BE-infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV-infected cells in the tissues., (© 2014 Crown copyright. This article is published with the permission of the Controller of HMSO/Queen‘s Printer for Scotland and Animal and Plant Health Agency.)

In 2009, the UK's OIE Reference Laboratory for rabies, based at the APHA in Weybridge, was awarded a project to twin with the Changchun Veterinary Research Institute in the People's Republic of China to help the institute develop the skills and methods necessary to become an OIE Reference Laboratory itself. Here, Tony Fooks, Trevor Drew and Changchun Tu describe the OIE's twinning project and the success that has since been realised in China., (British Veterinary Association.)

Analysis of published data shows that experimental passaging of Zaire ebolavirus (EBOV) in guinea pigs changes the risk of infection per plaque-forming unit (PFU), increasing infectivity to some species while decreasing infectivity to others. Thus, a PFU of monkey-adapted EBOV is 10(7) -fold more lethal to mice than a PFU adapted to guinea pigs. The first conclusion is that the infectivity of EBOV to humans may depend on the identity of the donor species itself and, on the basis of limited epidemiological data, the question is raised as to whether bat-adapted EBOV is less infectious to humans than nonhuman primate (NHP)-adapted EBOV. Wildlife species such as bats, duikers and NHPs are naturally infected by EBOV through different species giving rise to EBOV with different wildlife species-passage histories (heritages). Based on the ecology of these wildlife species, three broad 'types' of EBOV-infected bushmeat are postulated reflecting differences in the number of passages within a given species, and hence the degree of adaptation of the EBOV present. The second conclusion is that the prior species-transmission chain may affect the infectivity to humans per PFU for EBOV from individuals of the same species. This is supported by the finding that the related Marburg marburgvirus requires ten passages in mice to fully adapt. It is even possible that the evolutionary trajectory of EBOV could vary in individuals of the same species giving rise to variants which are more or less virulent to humans and that the probability of a given trajectory is related to the heritage. Overall the ecology of the donor species (e.g. dog or bushmeat species) at the level of the individual animal itself may determine the risk of infection per PFU to humans reflecting the heritage of the virus and may contribute to the sporadic nature of EBOV outbreaks., (© 2015 Crown copyright. © 2015 Society for Applied Microbiology.)

Porcine reproductive and respiratory syndrome viruses (PRRSV) show high genetic differences both among and within genotypes. Recently, several highly pathogenic PRRSV (HP-PRRSV) strains have been described. This study compares and characterizes the production of cytokines by pulmonary macrophages in pigs experimentally infected with four different PRRSV-1 strains: two low-virulent strains, Lelystad (LV) and a British field strain (215-06); a HP strain (SU1-bel) from Belarus and the attenuated vaccine strain DV (Porcilis(®) PRRS). Animals were clinically monitored and post-mortem examinations were performed at 3, 7 and 35 days post-infection (dpi). Lung samples were processed for histopathological and immunohistochemical studies by using specific antibodies against PRRSV, IL1-α, IL-6, TNF-α, IL-10 and IFN-γ. SU1-bel infected animals presented the highest mean scores for clinical observations, gross and microscopic lesions as well as for PRRSV expression compared with the other infected groups (p≤0.027). These animals displayed the highest expression of IL1-α at 7dpi, together with the highest score for lung pathology, whereas LV, 215-06 and DV inoculated animals only showed a transient enhancement in some of these cytokines. SU1-bel-infected pigs showed a positive correlation between the amount of PRRSV antigen and IL-1α expression (r=0.645, p<0.001). The highest expression of IL-10 was detected in 215-06-infected animals (p≤0.004), with a positive correlation with the numbers of virus-infected cells (r=0.375, p≤0.013). In conclusion, the HP-PRRSV SU1-bel strain replicated more efficiently in the lung of infected animals and induced a higher expression of IL-1α than the other PRRSV-1-infected groups, which may have played a key role in the onset of the clinical signs and interstitial pneumonia., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1 log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13 °C, 14 °C, 12 °C and 10 °C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215-06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.

Since 2006, reports from China and Viet Nam have alerted of an emergent highly pathogenic variant of porcine reproductive and respiratory syndrome virus (HP-PRRSV) in that region. The frequent occurrence of outbreaks in these countries puts Cambodian pig farms at high risk of infection, but no study had been conducted to investigate the presence of HP-PRRS in Cambodian farms. We investigated the presence of HP-PRRS in medium-scale (semi-commercial) swine farms in the Cambodian southeastern region. Specifically, one province bordering Viet Nam (Takeo) was selected due to the concentration of most semi-commercial farms in that province. A cross-sectional study was carried out, between July and September 2010 to assess whether the prevalence of infection in these farms was indicative of recent spread of PPRSV and to identify risk factors for infection. The number of farms to be sampled was established using methods for Lot Quality Assurance Surveys (LQAS), in order to achieve a pre-established ability to discriminate between two different prevalence settings. The target population comprised all semi-commercial farms in Takeo province from which a random sample of 35 farms was selected. Selected farms were visited and questionnaires administered to gather information on farm characteristics and husbandry practices. Blood samples from individual pigs were collected in each of the study farms and tested for PRRSV, along with a number of other swine respiratory pathogens in order to investigate potential interactions. Our results showed that the virus was already present in Takeo semi-commercial pig population (LQAS herd prevalence ≥85%) at the time of sampling. The presence of sows in the farm and farm density were significantly associated (P<0.05) with the introduction and the presence of PRRS - but this was an unadjusted association as small sample size precluded multivariate analysis. Spatiotemporal description of the supposed pattern of infection revealed that the 1st farms infected were closely located to major national and provincial roads, connecting the Cambodian capital Phnom Penh to Viet Nam., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) was first detected in Cambodia in 2010. The disease was responsible for high morbidity and high mortality in adult pigs and the outbreak had a costly impact on those farmers affected. The aim of this study was to generate a better understanding of Cambodian swine producers' behaviour, in relation to PRRS and its control, in areas that have previously been affected by the disease. A survey of the knowledge, attitude and practices (KAPs) of pig owners with regard to PRRS was conducted in semi-commercial and backyard farms in Takeo province in southeast Cambodia. The survey was designed to assess knowledge of PRRS disease and its transmission, farmers' attitudes and practices related to preventive and control measures, knowledge on vaccination and perception towards local veterinary authority activities. Descriptive statistics were used to summarise qualitative data, while multivariate regression analyses were used to assess the association between selected outcomes and a number of hypothetical predictors. When presented with clinical signs typical of PRRS, most farmers identified an infectious disease as the most likely explanation for the listed clinical conditions. Farmers were also confident in recognising direct contact between pigs as one of the main ways of disease transmission; however, other viral transmission patterns typical of PRRS were mostly unknown or ignored. In general, male farmers and farmers with a higher level of education were more likely to have a better knowledge of transmission routes between pigs. In terms of attitude towards control measures, vaccination and disinfection were perceived as the most effective control practices. Farmers with a better knowledge of vaccine protocols were more likely to find vaccination effective. Village animal health workers (VAHWs) were generally in contact more with backyard farmers, while semi-commercial farmers were more prone to treat pigs themselves, raising the issue of easy and uncontrolled access to medication and vaccination. In general, farmers had a positive attitude towards local veterinarians, and lack of contact between farmers and the veterinary authority was associated more with logistic constraints than with farmers' mistrust towards the authority., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.

Porcine reproductive and respiratory syndrome (PRRS) continues to be a significant problem for European pig producers, contributing to porcine respiratory disease complex, neonatal piglet mortality, infertility and occasional abortion storms. PRRS virus (PRRSV), a member of the arterivirus family with two defined major genotypes, has been shown to be quite genetically diverse. In the present study, genetic analysis of multiple gene regions of over 100 viruses isolated in Britain between 2003 and 2007 revealed that the diversity of British strains is now far greater than during the early 1990s. All isolates belong to genotype 1 (European). While some recent isolates are still very similar to early isolates, a wide range of more diverse viruses is now also circulating. Interestingly, some isolates were found to be very similar to a modified-live vaccine strain, and it is suggested that use of the vaccine has affected the evolution pattern of PRRS virus strains in Britain. Evidence of deletions in one viral gene, ORF3, and of genome recombination was also seen. A molecular clock model using the ORF7 sequences estimates the rate of substitution as 3.8 × 10(-3) per site per year, thereby dating the most recent common ancestor of all British viruses to 1991, coincident with the first outbreak of disease. Our findings therefore have implications for both the diagnostic and prophylactic methods currently being used, which are discussed., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers. To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990 s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously. For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues. As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Little is known how the virus subverts the innate immune response to initiate its replication in alveolar macrophages. Large-scale transcriptional responses of macrophages with different levels of susceptibility to PRRSV infection were compared over 30 h of infection. This study demonstrates a rapid and intense host transcriptional remodelling during the early phase of the replication of the virus which correlates with transient repression of type-I interferon transcript as early as 8 h post-infection. These results support the suggestion from previous studies that host innate immune response inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.

Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins N(pro) and E(rns) both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis.

Emerging evidence places deubiquitylation at the core of a multitude of regulatory processes, ranging from cell growth to innate immune response and health, such as cancer, degenerative and infectious diseases. Little is known about deubiquitylation in pig and arterivirus infection. This report provides information on the biochemical and functional role of the porcine USP18 during innate immune response to the porcine respiratory and reproductive syndrome virus (PRRSV). We have shown that UBP gene is the ortholog of the murine USP18 (Ubp43) gene and the human ubiquitin specific protease 18 (USP18) gene and encodes a biochemically functional de-ubiquitin enzyme which inhibits signalling pathways that lead to IFN-stimulating response element (ISRE) promotor regulation. Furthermore we have demonstrated that overexpression of the porcine USP18 leads to reduced replication and/or growth of PRRSV. Our data contrast with the conclusion of numerous reports demonstrating that USP18-deficient mice are highly resistant to viral and bacterial infections and to oncogenic transformation by BCR-ABL, and highlight USP18 as a potential target gene that promotes reduced replication of PRRSV.

The phylogenetic diversity of classical swine fever virus (CSFV) in China has been extensively studied previously, with the report of the classification of Chinese CSFVs into four subgroups within two of the established genotypes, but the antigenic differences amongst Chinese CSF viruses still remain unknown. To address this issue, 21 CSFV field strains isolated in China between 1996 and 2006 were grown in cell culture and characterized in comparison with two Chinese reference strains: a virulent strain Shimen and a vaccine strain CSF lapinized virus (hog cholera lapinized virus in China, HCLV), by indirect immunofluorescence assay (IFA) with a panel of 28 monoclonal antibodies (mAbs) against four pestiviruses, CSFV, bovine viral diarrhoea virus-1 (BVDV-1), bovine viral diarrhoea virus-2 (BVDV-2) and border disease virus (BDV). All 23 CSFV strains reacted only with CSFV-specific mAbs, not with those raised against BVDV-1, BVDV-2 and BDV. Of the former mAbs, those directed against CSFV E2 protein recognized more isolates than those directed against E(rns) and NS2/3. Of nine CSFV E2-specific mAbs used, WH303 and WH302 reacted with all 23 strains, confirming their value in differentiating CSFV from other pestiviruses. Furthermore, different strains had different patterns of reactivity with CSFV-specific mAbs, and mAbs other than WH303 and WH302 did not recognize all strains. This study provides the first evidence for the existence of antigenic differences among Chinese CSFVs.

Much work has been done to investigate participants' ability to detect repeated targets, but the presentstudy examined the influence of recently attended stimuli on target masking. Participants performed a target identification task in which the item that masked the target was either a recently attended item or a novel item. When it was identical to a previously attended stimulus, the mask was rendered considerably less effective. We have termed this effect a repeated mask reduction (RMR). This simple manipulation resulted in a large, reliable effect on the efficacy of visual masking in a single-target identification paradigm. In Experiments 1, 2, and 3, we demonstrated the basic effect and noted that the RMR increased as task difficulty increased. Experiments 4 and 5 suggested that attention to the first instance of the mask was crucial to this effect by showing that the magnitude of the RMR was unaffected by repetition of nonmask distractors and that the magnitude of the effect was reduced when less attention was paid to the first instance of the mask.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, is the etiologic agent of an infectious disease of that name, characterized by respiratory disorders, abortion in pregnant sows and high mortality in piglets, resulting in significant economic losses in the pig industry worldwide. In order to identify whether genetic differences in PRRSV response may exist in pigs, alveolar macrophages were used to assess the progression of the type-I interferon (IFN) transcript response in porcine alveolar macrophages infected by PRRSV. Our results suggest that a dynamic differential regulation of the type-I IFN and chemokine transcripts may operate during the first hours of infection with and entry of the virus in alveolar macrophages, and provide a compelling mechanism for the establishment of PRRSV replication in susceptible cells.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Identifying the genetic components involved in host resistance/susceptibility would represent an important step forward in the design of disease control programs. In this study, alveolar macrophages derived from five commercial pig lines were used to study the innate immune response to PRRSV infection in vitro. Analysis by flow cytometry has demonstrated that bronchial alveolar lavage fluid (BALF) preparations were almost exclusively composed of alveolar macrophages and that the pigs tested were free from infection. Macrophages from the Landrace line showed significantly reduced virus replication and poor growth of PRRSV during 30 h of infection. By 72 h, PRRSV viral load was down to 2.5 log(10) TCID(50) compared with an average of 5 log(10) TCID(50) for the other breeds tested. These observations suggest that factors intrinsic to the Landrace breed may be responsible for this reduced or delayed response to PRRSV. Preliminary investigation suggests that the PRRSV coreceptor, sialoadhesin, may not be responsible for the Landrace macrophage phenotype as its abundance and localisation were comparable in all the breeds. Strikingly, we found that the reduced or delayed growth of PRRSV was temporally associated with high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-8 mRNA accumulation and substantial reduction of secretion of IL-8, suggesting a key contributory role for cytokine synthesis and secretion during the innate immune response to PRRSV infection.

The increasingly global nature of animal trade, along with greater focus on improving the production and trading opportunities for emerging nations has highlighted the need for a more standardised, harmonised approach to standardisation of diagnostics and prophylaxis. This has driven a need, on the part of both the competent authorities of member countries and international agencies, for OIE itself to develop its role as the repository and source of expertise in these fields. With this role has emerged a strong demand to develop standardisation and harmonisation of diagnostic processes, along with an ever-expanding role played by the OIE Reference Expert in networking and consultancy. This expansion comes at a price, both in terms of the burden on Reference Laboratories and the financial cost of the activities themselves. Accordingly, the role of the Reference Expert is pivotal, both in improving standards and in assuring the viability of the Reference Laboratory they represent. At a higher level, the need for strategic changes are highlighted, involving establishment of networks to share the burden and increase efficiency of delivery, along with greater interaction among international agencies and assistance in providing project-based financial support. Closer collaboration with the diagnostics and vaccine industries is also foreseen, which, if managed properly, will provide benefits to OIE Reference Laboratories through income to support their activities, to national laboratories thorough advanced, reliable and validated tests and, ultimately, to animal health itself, at the global level.

Visual working memory is a limited capacity system that temporarily maintains information about objects in the immediate visual environment. Psychophysical experiments have shown that most people are able to actively maintain 3 or 4 items in visual working memory at any point in time. To better understand how this process works and why our working memory capacity is so limited, a variety of neurophysiological approaches have been employed. In recent years, there has been a surge of interest in understanding how visual information is maintained in working memory at the neural level. Single-cell research with nonhuman primates has shown that neuronal firing during the retention period reflects the information that is currently held in working memory. In humans, event-related potentials (ERPs) have been used to examine the maintenance of information in working memory. An event-related potential component, known as the negative slow wave (NSW), has been used to measure the maintenance of information in working memory "online" during a given trial. More recently, another ERP component, the contralateral delay activity (CDA) has been shown to be a fairly specific correlate of the current contents of working memory. This component is sensitive to an individual's working memory capacity and may provide a window into the operations of this central cognitive construct.

We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (Npro) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from Npro-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.

Two genotypes of bovine viral diarrhoea virus (BVDV) are recognised. Type 2 was first recognised when virulent strains caused significant losses among cattle in North America. Subsequently, BVDV type 2 has been found in many other countries, but recent studies have shown that only type 1 BVDV is circulating in the UK herds (sheep and cattle) with type 1a predominating. During routine genotyping of UK BVDV isolates, a type 2 isolate was identified. Phylogenetic analysis of the 5'-untranslated region of the viral genome showed it to be a BVDV type 2a, most similar to a low virulent US strain of BVDV type 2. Antigenic typing with a panel of monoclonal antibodies verified this classification. This is the first confirmed isolation of BVDV type 2 found circulating in the UK.

Porcine circovirus 2 (PCV-2) is implicated as the causative agent of post-weaning multisystemic wasting syndrome (PMWS) and is also associated with porcine dermatitis and nephropathy syndrome (PDNS). The recent emergence of epidemic PMWS in the United Kingdom was predated by sporadic cases of PDNS dating back to the early 1980's. The aim of this study was to investigate whether PCV-2 DNA was present in archival tissues, and if so, to investigate the relatedness of these viruses with contemporary strains of PCV-2. DNA extracted from paraffin wax-embedded tissue blocks ( n = 68), was subjected to a TaqMan polymerase chain reaction (PCR) targeting a fragment of ORF1 of PCV-2. Positive results were obtained from 41% (9/22), 31% (4/13) and 32% (8/25) of submissions from the 1990's, 1980's and 1970's respectively. The presence of PCV-2 antigen in some of these tissues was confirmed by immunohistochemistry (IHC). A PCR targeting ORF2 was used to obtain sequence data for phylogenetic analysis. Sequences from 5 archival tissues were unique but showed high genetic identity to PCV-2 sequence obtained from a 2000 PDNS case. These data demonstrate that similar isolates of PCV-2 have been present in the UK pig population for more than 30 years.

Infection with virulent strains of classical swine fever virus (CSFV) results in an acute haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopenia and immunosuppression, whereas for less virulent isolates infection can become chronic. In view of the haemorrhagic pathology of the disease, the effects of the virus on vascular endothelial cells was studied by using relative quantitative PCR and ELISA. Following infection, there was an initial and short-lived increase in the transcript levels of the proinflammatory cytokines interleukins 1, 6 and 8 at 3 h followed by a second more sustained increase 24 h post-infection. Transcription levels for the coagulation factor, tissue factor and vascular endothelial cell growth factor involved in endothelial cell permeability were also increased. Increases in these factors correlated with activation of the transcription factor NF-kappaB. Interestingly, the virus produced a chronic infection of endothelial cells and infected cells were unable to produce type I interferon. Infected cells were also protected from apoptosis induced by synthetic ouble-stranded RNA. These results demonstrate that, in common with the related pestivirus bovine viral diarrhoea virus, CSFV can actively block anti-viral and apoptotic responses and this may contribute to virus persistence. They also point to a central role for infection of vascular endothelial cells during the pathogenesis of the disease, where a proinflammatory and procoagulant endothelium induced by the virus may disrupt the haemostatic balance and lead to the coagulation and thrombosis seen in acute disease.

Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan assay. The results of TaqMan and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan assay is a rapid, reliable method for the detection of EAV.

A closed-tube polymerase chain reaction (PCR) was developed to allow the rapid detection of African swine fever virus (ASFV) DNA. This assay targets the VP72 gene of ASFV and uses the 5'-nuclease assay (TaqMan) system to detect PCR amplicons, avoiding tube opening and potential cross-contamination of post-PCR products. An artificial mimic was engineered with the TaqMan probe site replaced by a larger irrelevant DNA fragment allowing discrimination from ASFV by using two-colour TaqMan probe reporters. When added to the samples, successful amplification of this mimic demonstrated the absence of substances inhibitory to PCR, thereby validating negative results. Assay sensitivity was confirmed by obtaining positive signals with a representative selection of ASFV isolates. Many of the clinical and post-mortem features of ASF resemble those of classical swine fever (CSF) and porcine dermatitis and nephropathy syndrome (PDNS). Therefore, fast and reliable detection of ASFV is essential not only for the implementation of control measures to prevent the spread of ASF, but also in the differential diagnosis from CSF and PDNS. This assay should prove to be a valuable tool in the laboratory diagnosis of ASF and will complement existing molecular methods to provide rapid differential diagnosis in cases of suspected swine fever., (Copyright 2002 Published by Elsevier Science B.V.)

We determined 22 partial porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences, representing pathogenic field strains mainly from Poland and Lithuania, and two currently available European-type live PRRSV vaccines. Also, the complete ORF7 of two Lithuanian and two Polish strains was sequenced. We found that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity. The most diverse present-day European-type PRRSV sequences were from Poland (2000) and Lithuania (2000), and exhibited only 72.2% nucleotide identity in the investigated ORF5 sequence. While all sequences determined in the present study were clearly of European type, inclusion of the new Lithuanian sequences in the genealogy resulted in a common ancestor for the European type virus significantly closer to the American-type PRRSV than previously seen. In addition, the length of the ORF7 of the Lithuanian strains was 378 nucleotides, and thus intermediate between the sizes of the prototypical EU-type (387 nucleotides) and US-type (372 nucleotides) ORF7 lengths. These findings for the Lithuanian PRRSV sequences provide support for the hypothesis that the EU and US genotypes of PRRSV evolved from a common ancestor. Also, this is the first report of ORF7 protein size polymorphism in field isolates of EU-type PRRSV.

Accounts of field disease and experimental studies involving porcine reproductive and respiratory syndrome (PRRS) are reviewed for evidence of immunomodulation or immunosuppression by the causative virus. The conclusion is that immunomodulation through infection of alveolar macrophages is likely to occur, but that it is transient and at a local level, in the lung. There is some evidence for more subtle effects via more disseminated replication or induction of apoptosis with some isolates, but more definitive studies are needed. There is some emerging evidence of interaction between PRRSV and different cells of the immune system, but its significance for the course of disease or pig health are unclear. Likewise, the current experimental evidence for any interaction of PRRSV with other pathogens is ambiguous and therefore no firm conclusions can yet be drawn. Strains of PRRSV do vary in pathogenicity, which may be related to their degree of ability to cause overt respiratory disease in the absence of other agents. Experimentally, varying degrees of interstitial pneumonia are a common histological finding. There is, as yet, no firm evidence of general immunosuppression--in fact, some contrary evidence exists in the form of observations of a transient enhancement of humoral response, possibly through polyclonal B cell activation. The basis of pathogenicity of PRRSV and of any interaction with other agents is still unknown and is likely to remain unclear. Virus interaction with the pig's immune system must be addressed before any assessment of virulence of any known or emergent strains of PRRSV can be made.

Using RNA purified directly from stored clinical specimens, a collection of 62 pestiviruses were typed by RT-PCR and sequencing within the 5'-untranslated region of the genome. All the specimens had been obtained in 1966/1967 from diary cattle in England and Wales. Eight further pestiviruses, grown in cell culture, were characterised in the same way. Seven of these viruses were representatives of a panel of British isolates, obtained from cattle ten years before. The eighth was the virus used in a British bovine viral diarrhoea (BVD) vaccine. Most of the viruses were genetically unique and were of BVDV type Ia. One recent isolate was BVDV type Ib, two others were intermediate between Ia and Ib. No BVDV type II or border disease virus (BDV) isolates were found. There was no overall association between geographical and phylogenetic clustering, suggesting long-distance virus dispersal, presumably via trading of infected cattle. The sequences of the recently obtained cattle viruses were very similar or, in one case, identical to the older isolates in the region studied. Their close similarity to some previously characterised pestiviruses from British sheep suggests that a common pool of BVDV Ia is shared by these two livestock species, although another pestivirus--BVDV--is confined to sheep. The British cattle viruses were mostly distinct from continental European isolates, but more similar to type Ia isolates from North American cattle.

A single step, single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) test was developed to detect the presence of bovine viral diarrhoea virus (BVDV) in somatic cells from bulk milk samples. The test was configured using commercial kit-form RNA extraction and RT-PCR procedures. The test was validated by examining bulk milk samples from approximately 80 herds with a history of BVDV and comparing results with those obtained from samples from a similar-sized control group. The test proved highly specific, giving a positive result in 20.5% of herds with a history of BVDV, with no control herds positive. Its sensitivity was likewise high, detecting, at its maximum, one persistently infected (PI) cow in a herd of 162 lactating animals. In 19 herds where follow-up blood tests were performed, the RT-PCR gave a positive result in all ten herds where at least one lactating PI animals was present. In control involving the detection of PI cattle, the test provides a rapid and inexpensive alternative to individual animal testing for those cows in milk at the time of sampling.

Bulk tank milk samples from 1070 dairy herds in England and Wales were tested by ELISA for antibodies to bovine virus diarrhoea virus (BVDV). A subset of 341 herds was tested by ELISA for antibodies to bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV). None of the herds had less than 40 dairy cows and none had been vaccinated against BVDV. The prevalence of BVDV antibody-positive herds in the national population was estimated at 95 per cent and approximately 65 per cent of the herds had a high level of bulk tank antibody suggestive of recent infection with BVDV. Dairy herds in East Anglia and the south-east of England had a significantly lower risk of being BVDV antibody-positive than herds in the rest of England and Wales. However, these regional differences tended to diminish with increasing herd size. Around 69 per cent of the herds were BHV-1 antibody-positive and all the herds were antibody positive to BRSV and BCV. Comparison with earlier serological surveys revealed that there had been little change in the prevalence and distribution of BVDV antibody-positive herds in England and Wales over the last 20 years, but that there had been an increase in the prevalence of BHV-1 antibody-positive herds.

Previous studies using monoclonal antibodies (mAbs) have revealed antigenic variation among UK isolates of porcine reproductive and respiratory syndrome viruses (PRRSV) and the use of in vitro translation products has shown that this variation lies in the protein encoded by open reading frame (ORF) 3. This protein has been shown to be present in purified virion preparations, suggesting that it is a structural protein. The original objective was to investigate the degree of variation of ORF3 among a number of UK isolates of different mAb reactivity and diverse chronology by sequencing and to correlate this with the mAb reactivity, in an attempt to define conserved and variable antigenic sites. A number of PRRSV isolates, from different outbreaks in the UK between 1991 and 1994, were propagated in pig alveolar macrophages and RNA extracted. The ORF3 and ORF7 regions of the individual viruses were amplified by the polymerase chain reaction (PCR) and their sequences were determined using internal sense and antisense primers. A number of differences among the sequences were noted within specific regions of the ORF3, with a hypervariable area detected at the carboxyterminal end, in the area of overlap with ORF4. With the most divergent isolate, 9.5% of the 84 translated amino acids encoded by the area of overlap were different from Lelystad isolate, translating the sequence in both reading frames. In view of consistent changes elsewhere in the ORF that suggest a common ancestry among the isolates studied, we conclude that this region may be subject to rapid change in comparison to other regions studied, and therefore may be an area subjected to immunoselective pressure.

The members of a European Community Concerted Action participated in a study to compare in-house and commercial tests for the serodiagnosis of porcine reproductive and respiratory syndrome (PRRS). These tests included the immunoperoxidase monolayer assay (IPMA) and enzyme-linked immunosorbent assay (ELISA). The trials involved assays on experimentally-produced reference sera, a blind trial of sera of known status, and a comparative study of negative and 'problem' field sera. The results showed a high level of agreement among IPMA tests used in the participating laboratories at the herd level, with only minor inconsistencies in testing early post-infection sera. A blocking ELISA, used in experimental work by one laboratory, was almost as sensitive as IPMA in assays of reference and validation trial sera. Commercial ELISAs were generally less sensitive than IPMA when used to assay ten-day post-infection sera. Discrepant results among certain field sera are likely to be due to antigenic variation among PRRS viruses in Europe. This may lead to increasing instances of misdiagnosis using tests which rely solely on test antigens derived from single PRRS virus isolates.