Prevalence of hepatitis E virus in slaughter-age pigs in Scotland.

The prevalence of anti-HEV isotype-specific antibodies and viraemia were investigated in serum samples collected from slaughter-age pigs (aged 22-24 weeks) from 23 farms in Scotland. Of 176 serum samples tested, 29·0% (n = 51) were anti-HEV IgG positive, 36·9% (n = 65) anti-HEV IgA positive and 29·0% (n = 51) anti-HEV IgM positive. Overall seroprevalence (anti-HEV IgG+ and/or IgA+ and/or IgM+) was 61·4% (n = 108). HEV RNA was detected in 72/162 serum samples (44·4%). Partial sequence of ORF2 (98 nt) was obtained from eight HEV RNA-positive samples and phylogenetic analysis confirmed that they were all of genotype 3. This is the first report on the prevalence of HEV in pigs in Scotland. Given the increasing incidence of locally acquired HEV infection in the UK, evidence that HEV is a foodborne zoonosis emphasizes the need for surveillance in pigs.

Costs of multidrug-resistant TB treatment in Finland and Estonia affected by the 2019 WHO guidelines.

BACKGROUND: Multidrug-resistant TB (MDR-TB) is a growing problem in the effort to end the global TB epidemic. In 2019, the WHO adopted a new standardised regiment for MDR-TB, consisting of only oral medications.METHODS: We estimated the impact of the new guidelines on the costs of TB treatment in Estonia and Finland. For both countries, the costs of the two most common new drug regimens were calculated, including drug costs, as well as care- and monitoring-related costs.RESULTS: In Turku, Finland, treatment costs with the old regimen were €178,714; this could either increase by 10% or decrease by 18%, depending on the duration of bedaquiline use (6 months vs. 20 months). In Estonia, treatment costs with the old regimen were €33,664, whereas the new regimens were associated with a 40% increase in overall costs.CONCLUSIONS: The 2019 WHO guidelines have led to significant changes in the costs of MDR-TB treatment in Finland and Estonia. These changes depend mostly on the drug regimen administered and on care-related practices, with important differences between countries and even within the same country due to local practices.

Excretion of hepatitis E virus by pigs of different ages and its presence in slurry stores in the United Kingdom.

Five faecal samples were collected from four different stages of production at each of 10 pig farms in the Yorkshire Humberside area of the UK, and samples of slurry were collected from nine of the farms. All the samples were tested for hepatitis E virus (HEV) RNA by a nested reverse transcriptase PCR. At least one sample from the pigs on each of the farms tested positive for hev; its prevalence in the 10 herds varied from 5 per cent to 35 per cent and its mean prevalence was 21.5 per cent. The mean prevalence in pigs aged three to five weeks was 26.0 per cent, in pigs aged 10 to 12 weeks 44.0 per cent, in pigs aged 22 to 24 weeks 8.9 per cent, and in adult dry sows 6.0 per cent. Two of the nine slurry lagoons tested positive for HEV RNA. Phylogenetic analysis of the sequence data indicated that the strains of the virus were of genotype 3 and closely related to strains detected in other pigs and in human beings in the UK.

Incursion of Schmallenberg virus into Great Britain in 2011 and emergence of variant sequences in 2016.

Schmallenberg virus (SBV) is a vector-borne orthobunyavirus in the family Bunyaviridae, first identified in Germany before rapidly spreading throughout Europe. To investigate the events surrounding the incursion of this virus into Great Britain (GB) and its subsequent spread, archived sheep serum samples from an unrelated field survey in 2011 were analysed for the presence of SBV specific antibodies, to determine the earliest date of seroconversion. This serological study, along with analysis of the spatial spread of the sources of samples submitted for SBV analysis after January 2012, suggests that SBV entered GB on more than one occasion and in more than one location. Phylogenetic analysis of SBV sequences from 2012 ovine samples, from a variety of counties and dates, demonstrated a non-linear evolution of the virus, i.e. there was no distinct clustering between host species, geographical locations or during the outbreak. This also supports the notion of multiple viruses entering GB, rather than a single virus incursion. Premature termination signals were present in several non-structural putative protein sequences. One SBV sequence exhibited large deletions in the M segment of the genome. After the first outbreak in 2011-2012, interest in SBV in GB waned and continuous surveillance was not upheld. The re-emergence of SBV in 2016 has raised renewed concern and ended speculation that SBV might have been eradicated permanently from GB. When SBV sequences from 2012 were compared with those from the re-emergence in 2016-2017, a second distinct clade of SBV was identified that separates recent strains from those observed during the first outbreak.

Swine and circovirus.

In the 1990s Post-weaning, multi-systemic wasting syndrome (PMWS) emerged in N. America and Europe as a major disease problem with significant welfare and economic consequences for pig producers. The disease, characterised by wasting, respiratory, enteric and lymphoid system problems in pigs of 4-16 weeks of age, has since spread so that today it has a global distribution. PCV-2 is consistently associated with PMWS, is more abundant in association with PMWS and is considered by many to be the causative agent of the syndrome. However, several lines of evidence indicate that PCV-2 is necessary but not sufficient to cause the full range of clinical signs associated with PMWS, suggesting the involvement of an as yet unidentified factor or factors. The process of identifying unknown agents and their respective roles in the pathogenesis of complex syndromes now has an ever broadening spectrum of analytical techniques available. Immune phenotyping, cytokine responses, micro-array profiling, and proteomics are just some of the techniques available. This paper describes the philosophy and the application of these and classical techniques in an integrated, holistic manner to the problem of PMWS and circoviruses, by examination of samples collected from a prospective, clinical case-control study, and discusses some of the preliminary findings in relation to the efforts to understand the aetiopathogenesis of PMWS.

Re-emergence of a genetic outlier strain of equine arteritis virus: Impact on phylogeny.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids, which is notifiable in some countries including the Great Britain (GB) and to the OIE. Herein, we present the case of a persistently infected stallion and the phylogenetic tracing of the virus strain isolated. Discussing EAV occurrence and phylogenetic analysis we review features, which may aid to harmonise and enhance the classification of EAV.

Genome sequence analysis of 10 Dutch porcine circovirus type 2 (PCV-2) isolates from a PMWS case-control study.

The factors responsible for the emergence of post-weaning multisystemic wasting syndrome (PMWS) as an epidemic disease with significant impact upon the pig industry are not all known. Porcine circovirus type 2 (PCV-2) has been shown to be necessary but not sufficient for the expression of PMWS. Retrospective serological and molecular surveys have shown that PCV-2 was widespread and was maintained with only occasional reports of sporadic PMWS in the 30 year period prior to the recent emergence of the epidemic form of the syndrome. However, the recent spread of the disease in Europe and elsewhere has pointed to the transmission of a novel pathogen. One explanation to reconcile this paradox is that PWMS is caused by a unique PCV-2 variant that is being spread through pig populations. To test this hypothesis, complete genomes (1767 bp) of 10 Dutch PCV-2 isolates from 4 PMWS affected premises and 6 farms without PMWS were sequenced. Phylogenetic analysis showed that these sequences were grouped together although they differed on 77 nucleotide positions relative to each other (95.6-100% identity between the 10 isolates). None of these nucleotide changes identified impacted upon transcriptional elements or other important recognised features of the genome of the PCV-2. Amino acid changes were recorded on 4 positions in ORF1 and on 16 positions in ORF2 but, importantly, no consistent pattern was evident between PCV-2 isolates from affected and control pigs. These data provide further evidence to suggest that factor(s) in addition to PCV-2 are necessary in the development of PMWS.

Evidence for the presence of hepatitis E virus in pigs in the United Kingdom.

Samples of serum, tissue and faeces from two pig herds in England were examined for hepatitis E virus by reverse-transcriptase PCR (RT-PCR), and a virus strain from each herd was partially sequenced. Eleven of 42 faecal samples and 16 of 21 tissue samples from two pigs were positive for the virus by RT-PCR. Analysis of two unique but closely related nucleotide sequences obtained from the two herds showed that the viruses clustered in genotype III (6) with a human strain of the virus from an autochthonously acquired case of acute hepatitis in the UK. An ELISA based on recombinant open reading frame 2 (ORF-2) was used to detect antibodies to hepatitis E virus in 256 pig sera from the UK; 85.5 per cent of the samples were positive, compared with 58 per cent of similar samples from Swedish pigs and 23.5 per cent of samples from Dutch pigs.

Replication of hepatitis E virus in three-dimensional cell culture.

Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

Systematic assessment of the impact of adenovirus infection on a captive reintroduction project for red squirrels (Sciurus vulgaris).

PCR was used to amplify adenoviral DNA, and transmission electron microscopy (TEM) to detect adenovirus particles in tissue and intestinal content samples from red squirrels (Sciurus vulgaris) associated with a reintroduction study on Anglesey (North Wales), from other populations on the island and from stock held at the Welsh Mountain Zoo, 38 km to the east. Samples were collected during the routine surveillance postmortem examinations of all 60 red squirrels with carcases retrieved in a suitable condition between 2004 and 2010, including 29 captive and 31 free-living animals. Following significant clusters of mortality in captive red squirrels, adenovirus was identified retrospectively in faecal material from 12 of 13 (92 per cent) examined carcases from squirrels captive on Anglesey, and 14 of 16 (88 per cent) from the Welsh Mountain Zoo. Virus was identified in 13 of 31 (42 per cent) free-living wild animals, with evidence of both subclinical and clinically significant enteric adenoviral infections in wild squirrels. Without ancillary PCR and TEM testing, the extent of adenovirus infection in such populations would have been underestimated. Screening protocols that include examinations for adenovirus should, therefore, be part of the routine biosecurity measures protecting reintroduction or captive breeding programmes for red squirrels.

Porcine circovirus type 2 infection before and during an outbreak of postweaning multisystemic wasting syndrome on a pig farm in the UK.

The presence of porcine circovirus type 2 (PCV-2) and other pathogens before and during an outbreak of postweaning multisystemic wasting syndrome (PWMS) in pigs is evaluated in this study. At the time of the outbreak on a large commercial pig farm in the UK, serum samples and data were collected in two independent on-going research projects, one in weaned pigs and the other in sows. Serum samples of growing pigs and sows were PCV-2-antibody and PCR positive before and during the PMWS outbreak. Upon sequencing, PCV-2 isolates collected before the outbreak were identified as PCV-2a, and isolates collected during the outbreak were identified as PCV-2b, suggesting a shift of PCV-2 genotypes present on the farm. Pigs in the weaner study were from sows originating from different breeders and an association of sow origin and PCV-2 serostatus in offspring was found. Further, pigs had higher odds to be PCV-2 antigen positive if the sow was PCV-2 antibody positive around farrowing, the sow was of higher parity, and were less likely to test antigen positive if the sow was sourced from a particular breeder. The findings of this study highlight the potential role of the immune status of the sow on the occurrence of PMWS.

Development of a DNA microarray for simultaneous detection and genotyping of lyssaviruses.

The lyssavirus genus of the Rhabdoviridae family of viruses includes 7 genotypes and several non-assigned isolates. The source of lyssavirus infections is diverse with numerous reservoirs in a wide geographical area. In many parts of the world reservoir hosts can potentially be carrying one of several lyssavirus strains and possibly new divergent isolates await discovery. Accordingly, generic detection methods are required to be able to detect and discriminate all lyssaviruses and identify new divergent isolates. Here we have allied a sequence-independent amplification method to microarray to enable simultaneous detection and identification of all lyssavirus genotypes. To do so, lyssavirus RNA was converted to cDNA and amplified in a random PCR, labelled and hybridized to probes on the microarray chip before being statistically analysed. The probes were to a 405 bp region of the relatively conserved N gene. Here we demonstrate a microarray capable of detecting each of the seven lyssavirus genotypes. The random amplification of lyssavirus RNA and the numerous oligonucleotide probes on the microarray chip also offer the potential to detect novel lyssaviruses.

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.