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.

Louping ill virus: an endemic tick-borne disease of Great Britain.

In Europe and Asia, Ixodid ticks transmit tick-borne encephalitis virus (TBEV), a flavivirus that causes severe encephalitis in humans but appears to show no virulence for livestock and wildlife. In the British Isles, where TBEV is absent, a closely related tick-borne flavivirus, named louping ill virus (LIV), is present. However, unlike TBEV, LIV causes a febrile illness in sheep, cattle, grouse and some other species, that can progress to fatal encephalitis. The disease is detected predominantly in animals from upland areas of the UK and Ireland. This distribution is closely associated with the presence of its arthropod vector, the hard tick Ixodes ricinus. The virus is a positive-strand RNA virus belonging to the genus Flavivirus, exhibiting a high degree of genetic homology to TBEV and other mammalian tick-borne viruses. In addition to causing acute encephalomyelitis in sheep, other mammals and some avian species, the virus is recognized as a zoonotic agent with occasional reports of seropositive individuals, particularly those whose occupation involves contact with sheep. Preventative vaccination in sheep is effective although there is no treatment for disease. Surveillance for LIV in Great Britain is limited despite an increased awareness of emerging arthropod-borne diseases and potential changes in distribution and epidemiology. This review provides an overview of LIV and highlights areas where further effort is needed to control this disease.

Development of a real-time PCR to detect Streptococcus equi subspecies equi.

REASONS FOR PERFORMING STUDY: Infection with Streptococcus equi subspecies equi (S. equi) is endemic in the UK. A proportion of horses serve as long-term carriers and act as a reservoir of infection. Detection of these persistently infected horses is difficult using standard culture techniques owing to a lack of sensitivity and overgrowth by contaminating bacteria. In addition, differentiation of this causative bacterium from the closely related S. equi zooepidemicus has made the development of reliable and accurate diagnostic tests difficult. OBJECTIVE: To develop and validate a sensitive and specific real-time PCR assay to detect S. equi and to compare the results with traditional culture techniques. STUDY DESIGN: Retrospective cross-sectional study. METHODS: The assay was validated using a panel of 92 samples from suspected clinical cases of strangles. These were cultured using microbial techniques and tested using the S. equi real-time PCR. The results of the 2 methods were compared, and the diagnostic sensitivity and specificity of the real-time PCR were calculated. The real-time PCR was tested for cross-reactivity with horse commensal bacteria, and the efficiencies and limits of detection were established. RESULTS: The assay had a diagnostic sensitivity of 95% and specificity of 86%. No cross-reactivity was observed with any of the bacterial species tested, including S. equi zooepidemicus. The assay detected as few as 3 gene copies. CONCLUSION: The assay is fast, sensitive and specific and will detect S. equi DNA directly from a crude extract of clinical material on a swab. POTENTIAL RELEVANCE: This assay could aid in the rapid detection of subclinical shedders of S. equi, enabling quicker treatment and helping to limit the spread of strangles in equine populations.

Increased phylogenetic diversity of bovine viral diarrhoea virus type 1 isolates in England and Wales since 2001.

Currently, there are two recognised genotypes of Bovine viral diarrhoea virus (BVDV), type 1 and type 2. These genotypes are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2. Within this study, the genetic heterogeneity of BVDV-1 in England and Wales was investigated and compared to the situation in 1996/1997. Viral RNA was extracted from 316 blood samples collected between 2004 and 2009 that were previously identified as BVDV-1 positive. A region of the 5' untranslated region (UTR) was amplified by RT-PCR and the PCR products were sequenced. Phylogenetic analysis of the 5'UTR demonstrated the existence of five subtypes of BVDV-1 circulating in England and Wales, namely BVDV-1a (244 samples), BVDV-1b (50), BVDV-1e (3), BVDV-1f (1) and BVDV-1i (18). Phylogenetic analysis of the nucleotide sequence for the N(pro) region of the viral genome supported the classification obtained with the 5'UTR. Given the fact that only three subtypes were detected in 1999 this report supports the notion that the restocking of cattle from continental Europe, after the mass culling during the Foot-and-Mouth outbreak in 2001 and slaughter of cattle due to bovine tuberculosis infection, has increased the genetic diversity of BVDV-1 subtypes in England and Wales in the past 10 years.

Development and validation of RT-PCR tests for the detection and S1 genotyping of infectious bronchitis virus and other closely related gammacoronaviruses within clinical samples.

Two tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one-step, reverse transcription-polymerase chain reaction (RT-PCR) assay in which the amplification is monitored in real time using a TaqMan(®) probe. This real-time RT-PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls' eggs. A total of 323 field samples were tested; 176 samples were positive using the real-time RT-PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real-time RT-PCR results for a subset of samples. The test is suitable for swabs and post-mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high-throughput diagnostic laboratory.

Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy.

To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

Mucosal or systemic administration of rE2 glycoprotein antigen loaded PLGA microspheres.

We have evaluated the ability of recombinant E2 antigen, as a surfactant free formulation of poly (D,L-lactide-co-glycolide) (PLGA) microspheres, to elicit a systemic immune response after administration by mucosal routes (oral and nasal) in comparison to intramuscular route. The sequence encoding a truncated E2 glycoprotein of the classical swine fever virus (CSFV) was expressed in insect cells following infection with recombinant baculovirus, as a His-tagged recombinant antigen. The recombinant E2 glycoprotein (rE2) antigen was co-encapsulated with rabbit serum albumin (RSA) as a protein stabilizer. rE2/RSA loaded PLGA microspheres, with a mean diameter of 4 microm were obtained by a water in oil in water solvent extraction method (w/o/w). Rabbits were immunized with 10 microg of rE2 formulated in PLGA microspheres administrated by three different routes (oral, nasal and intramuscular). After 60 days, each rabbit in all three groups was challenge with 5 microg of rE2 glycoprotein solution by intradermal administration. Blood samples were collected weekly for 90 days and specific rE2 antigen antibodies measured. This work showed that rE2 antigen loaded microspheres was able to initiate an immune response. The intradermal challenge after nasal and oral administration had a clear boost effect on the systemic immune response. Moreover, the response after nasal administration was more intense and less variable than oral route. In conclusion, these data demonstrate a high potential of rE2 loaded PLGA microspheres for their use as a mucosal subunit vaccine.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue.

A LightCycler real-time PCR hybridization probe-based assay that detects a conserved region of the16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n=180) and aborted pig foetuses (n=24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n=7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n=30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents.

Bovine viral diarrhoea virus infection of alpacas (Vicugna pacos) in the UK.

Three alpacas (Vicugna pacos) aged two to 22 months with a history of illthrift and diarrhoea were examined postmortem, and tissues were collected for histology, including immunohistochemical labelling for pestivirus antigen, virus isolation and TaqMan reverse transcriptase-pcr assay. Blood samples from two clinical cases and the remaining herd members were tested for bovine viral diarrhoea virus (bvdv) antibody by serum neutralisation, antigen detection and pcr assay. The three affected alpacas were positive for bvdv by pcr of splenic tissue and/or heparinised blood. Non-cytopathic bvdv was isolated from several tissues and plasma of two of the alpacas. dna sequencing and phylogenetic analysis of the viral genome from the pcr product showed that the bvdv was of subgenotype 1b. Immunohistochemical examination of brain tissue was positive in two cases, consistent with a persistent infection. bvdv antibodies were detected in 16 of 25 clinically unaffected alpacas. There was no evidence of persistent infection in the in-contact animals. The source of the infection was not determined.

Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan PCR assays.

Three multiplex real-time TaqMan PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000Ptrade mark real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1h.

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.

Development of a real-time, differential RT-PCR TaqMan assay for lyssavirus genotypes 1, 5 and 6.

A number of RT-PCR methods have been reported for the detection of rabies and rabies-related viruses. Here, a single, closed tube, non-nested RT-PCR TaqMan assay to distinguish between Classical rabies virus and European bat lyssaviruses 1 and 2 in real time is described. The TaqMan assay is rapid, sensitive, specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. It can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. The efficiency and dynamic range of the PCR has been established using isolated viral RNA and cloned control template. Comparative performance of the TaqMan assay against other diagnostic methodologies for the detection of rabies virus has been determined and the assay validated against a panel of archival samples and virus of unknown genotype from both Germany and the Sudan. Despite sequence heterogeneity between the different genotypes in the N-gene, a universal forward and reverse primer set have been designed allowing simplification of previously described assays. Rapid genotyping of two recent EBLV2 cases in the U.K. in Daubenton's bats and a recently imported human case of rabies has been performed using this test.

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis.

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.

Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6.

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus.

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.