Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity.

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.

The challenge of using experimental infectivity data in risk assessment for Ebola virus: why ecology may be important.

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.

Emergence of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) in medium-scale swine farms in southeastern Cambodia.

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.

Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

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.

Knowledge, attitudes and practices of Cambodian swine producers in relation to porcine reproductive and respiratory syndrome (PRRS).

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.

The emergence and evolution of swine viral diseases: to what extent have husbandry systems and global trade contributed to their distribution and diversity?

Over the last 20 years, pig production has been characterised by a rapid increase in the volume of pig meat produced, greater intensification of the pig-rearing process and much greater international movement of products. There have also been many novel viral diseases that challenge the industry. Are these two developments linked and, if so, how? To understand how changes in the industry may influence the evolution of viruses, it is important to understand something of evolutionary theory. For RNA viruses, the concept of 'quasispecies' has moved solidly from theory to fact. Such viruses do not exist as a single entity, but as a 'cloud' of viruses, whose degree of diversity is influenced by a number of factors. Chief among these are the size and rate of the replicating population, along with the availability and diversity of susceptible hosts. A feature of RNA viruses is a high level of mutation, due to lack of capability to correct errors on the part of the host cell. Both in vivo and in vitro, RNA viruses have been shown to accumulate and fix these mutations, leading to bottleneck events and fitness loss, the phenomenon known as'Muller's ratchet'. Likewise, the opposite effect, fitness gain, can be achieved in an environment providing for high levels of replication and the generation of large populations of virus. This has been shown to be possible in vitro by high-volume passage. It is possible that the regular introduction of diverse viruses within large-scale pig production provides an in vivo equivalent that could drive quasispecies populations to increased fitness, and may explain why emergent viruses, either new to science or with new synergies and presentation, seem to be appearing more commonly.

Classical swine fever virus infection protects aortic endothelial cells from pIpC-mediated apoptosis.

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.

Dynamic differential regulation of innate immune transcripts during the infection of alveolar macrophages by the porcine reproductive and respiratory syndrome virus.

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.

Developing standards: the role of the OIE expert.

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.

Characterisation of a type 2 bovine viral diarrhoea virus isolated from cattle in the UK.

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.

Detection and genetic typing of type 2 porcine circoviruses in archived pig tissues from 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.

Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses.

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.

A review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus.

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.

Genetic typing of bovine pestiviruses from England and Wales.

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.

The detection of bovine viral diarrhoea virus in bulk milk samples by the use of a single-tube RT-PCR.

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.

Prevalence of antibodies to bovine virus diarrhoea virus and other viruses in bulk tank milk in England and Wales.

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.