In What Ways Does Health Related Stigma Affect Sustainable Employment and Well-Being at Work? A Systematic Review.

PURPOSE: Studies are increasingly showing that health related stigma is a barrier to employment, but it is not known how. The aim of this systematic review is to identify, appraise and analyse studies that have directly or indirectly addressed ways in which stigma affects sustainable employment and well-being at work of people with disabilities. METHODS: Using a multiphase screening process, this review is based on a comprehensive literature search (2000-2019) carried out in six electronic databases: Embase, Web of Science, Medline Ovid, Cochrane CENTRAL, PsycINFO and Google Scholar. RESULTS: 7.263 publications were identified; 96 studies were found eligible to be included in the review. 72% of the studies were conducted in North America or Europe. Few studies directly assessed how stigma affects the employment of people with disabilities. Most studies highlighted that attitudes and behaviour of employers formed a barrier to employment, as well as anticipated stigma and self-stigma in people with health problems. However, the findings also showed that the attitudes and behaviour of co-workers, health care professionals, reintegration professionals, customers, and family and friends could act as a barrier to employment although these influences are under-researched. Although many similarities were seen in the relevant findings of studies about both physical and mental disabilities, several nuances were found. CONCLUSION: Stigma hampers sustainable employment and well-being in multiple ways. Whereas the number of publications on this topic is rapidly increasing, the roles of health care professionals, reintegration professionals, co-workers, customers, and family and friends particularly warrant more attention.

Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations.

Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress Clostridium difficile abundance, delay the onset of diet-induced obesity, and increase the relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout the evolution of the host-microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.

Persistent viral shedding of SARS-CoV-2 in faeces - a rapid review.

AIM: In addition to respiratory symptoms, COVID-19 can present with gastrointestinal complaints suggesting possible faeco-oral transmission. The primary aim of this review was to establish the incidence and timing of positive faecal samples for SARS-CoV-2 in patients with COVID-19. METHODS: A systematic literature review identified studies describing COVID-19 patients tested for faecal virus. Search terms for MEDLINE included 'clinical', 'faeces', 'gastrointestinal secretions', 'stool', 'COVID-19', 'SARS-CoV-2' and '2019-nCoV'. Additional searches were done in the American Journal of Gastroenterology, Gastroenterology, Gut, Lancet Gastroenterology and Hepatology, the World Health Organization Database, the Centre for Evidence-Based Medicine, New England Journal of Medicine, social media and the National Institute for Health and Care Excellence, bioRxiv and medRxiv preprints. Data were extracted concerning the type of test, number and timing of positive samples, incidence of positive faecal tests after negative nasopharyngeal swabs and evidence of viable faecal virus or faeco-oral transmission of the virus. RESULTS: Twenty-six relevant articles were identified. Combining study results demonstrated that 53.9% of those tested for faecal RNA were positive. The duration of faecal viral shedding ranged from 1 to 33 days after a negative nasopharyngeal swab with one result remaining positive 47 days after onset of symptoms. There is insufficient evidence to suggest that COVID-19 is transmitted via faecally shed virus. CONCLUSION: There is a high rate of positive polymerase chain reaction tests with persistence of SARS-CoV-2 in faecal samples of patients with COVID-19. Further research is needed to confirm if this virus is viable and the degree of transmission through the faeco-oral route. This may have important implications on isolation, recommended precautions and protective equipment for interventional procedures involving the gastrointestinal tract.

Uncertainty in and around biophysical modelling: insights from interdisciplinary research on agricultural digitalization.

Agricultural digitalization is providing growing amounts of real-time digital data. Biophysical simulation models can help interpret these data. However, these models are subject to complex uncertainties, which has prompted calls for interdisciplinary research to better understand and communicate modelling uncertainties and their impact on decision-making. This article develops two corresponding insights from an interdisciplinary project in a New Zealand agricultural research organization. First, we expand on a recent Royal Society Open Science journal article (van der Bles et al. 2019 Royal Society Open Science 6, 181870 (doi:10.1098/rsos.181870)) and suggest a threefold conceptual framework to describe direct, indirect and contextual uncertainties associated with biophysical models. Second, we reflect on the process of developing this framework to highlight challenges to successful collaboration and the importance of a deeper engagement with interdisciplinarity. This includes resolving often unequal disciplinary standings and the need for early collaborative problem framing. We propose that both insights are complementary and informative to researchers and practitioners in the field of modelling uncertainty as well as to those interested in interdisciplinary environmental research generally. The article concludes by outlining limitations of interdisciplinary research and a shift towards transdisciplinarity that also includes non-scientists. Such a shift is crucial to holistically address uncertainties associated with biophysical modelling and to realize the full potential of agricultural digitalization.

A novel hepatitis B virus subgenotype D10 circulating in Ethiopia.

Hepatitis B virus (HBV) is genetically highly divergent and classified in ten genotypes and forty subgenotypes in distinct ethno-geographic populations worldwide. Ethiopia is a country with high HBV prevalence; however, little is known about the genetic variability of HBV strains that circulate. Here, we characterize the complete genome of 29 HBV strains originating from five Ethiopian regions, by 454 deep sequencing and Sanger sequencing. Phylogenetically, ten strains were classified as genotype A1 and nineteen as genotype D. Fifteen genotype D strains, provisionally named subgenotype D10, showed a novel distinct cluster supported by high bootstrap value and >4% nucleotide divergence from other known subgenotypes. In addition, the novel D10 strains harboured nine unique amino acid signatures in the surface, polymerase and X genes. Seventy-two per cent of the genotype D strains had the precore premature stop codon G1896A. In addition, 63% genotype A and 33% genotype D strains had the basal core promoter mutations, A1762T/G1764A. Furthermore, four pre-S deletion variants and two recombinants were identified in this study. In conclusion, we identified a novel HBV subgenotype D10 circulating in Ethiopia, underlining the high genetic variability of HBV strains in Africa.

Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters.

The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 µM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction.

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus, but the exact modes of transmission of MERS-CoV to humans remain unclear. In February 2014, an assessment was made of the suitability and sensitivity of different types of sample for the detection of MERSCoV by real-time reverse-transcription polymerase chain reaction (RT-PCR) for three gene targets: UpE (upstream of the E gene), the N (nucleocapsid) gene and open reading frame (ORF) 1a. Fifty-three animals presented for slaughter were sampled. A high percentage of the sampled camels (79% [95% confidence interval 66.9-91.5%, standard error 0.0625]; 42 out of 53) were shown to be shedding MERS-CoV at the time of slaughter, yet all the animals were apparently healthy. Among the virus-positive animals, nasal swabs were most often positive (97.6%). Oral swabs were the second most frequently positive (35.7%), followed by rectal swabs (28.5%). In addition, the highest viral load, expressed as a cycle threshold (Ct) value of 11.27, was obtained from a nasal swab. These findings lead to the conclusion that nasal swabs are the candidate sample of choice for detecting MERS-CoV using RT-PCR technology in apparently healthy camels.

Des travaux de recherche approfondis sont encore nécessaires concernant le coronavirus responsable du syndrome respiratoire du Moyen-Orient (MERSCoV), un virus identifié récemment et qui provoque des troubles respiratoires sévères en particulier chez les individus atteints de pathologies multiples. Les études effectuées au Qatar et ailleurs ont démontré que les dromadaires font office de réservoirs du virus ; toutefois, les modalités précises de la transmission du MERS-CoV à l'être humain demeurent obscures. En février 2014, une équipe de chercheurs a évalué l'adéquation et la sensibilité de plusieurs types d'échantillons pour détecter le MERS-CoV en utilisant l'amplification en chaîne par polymérase couplée à une transcription inverse en temps réel (RT-PCR) spécifique pour trois cibles génétiques, à savoir la séquence UpE (en amont du gène E), le gène N (nucléocapside) et le cadre de lecture ORF1a. Pour ce faire, divers prélèvements ont été effectués sur 53 dromadaires destinés à l'abattage. Un fort pourcentage de ces dromadaires (79 % [intervalle de confiance à 95 % compris entre 66,9 et 91,5 %, erreur standard : 0,0625], soit 42 sur 53) excrétaient le MERSCoV au moment de l'abattage, mais aucun ne présentait le moindre signe clinique. Les échantillons dans lesquels le plus de cas positifs ont été détectés étaient les écouvillons nasaux (97,6 %). Venaient ensuite les écouvillons oraux, qui ont détecté 35,7 % de cas positifs, puis les écouvillons rectaux (28,5 % de cas positifs détectés). Par ailleurs, ce sont les écouvillons nasaux qui ont permis d'obtenir l'intensité la plus élevée de la réponse de la RT-PCR, exprimée en une valeur du seuil de cycles de 11,27. Ces résultats permettent de conclure que les écouvillons nasaux sont les échantillons à privilégier pour la détection du MERS-CoV par RTPCR chez les dromadaires asymptomatiques.

Es preciso investigar más a fondo el coronavirus del síndrome respiratorio de Oriente Medio (MERS-CoV), recién identificado, que provoca una grave enfermedad respiratoria, sobre todo en personas con afecciones concomitantes. Estudios realizados en Qatar y otros lugares han deparado pruebas de que los dromedarios son un reservorio del virus, pero aún no están del todo claros los modelos exactos de transmisión del MERS-CoV al ser humano. Los autores describen un análisis realizado en febrero de 2014 de la idoneidad y sensibilidad de distintos tipos de muestra para detectar el MERS-CoV mediante una reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (RTPCR) dirigida contra tres genes: el gen UpE (upstream of the E gene: en dirección 5' desde el gen E); el gen N (nucleocápside) y el marco de lectura abierto (ORF) 1a. Para ello se tomaron muestras de 53 animales enviados al sacrificio. Se comprobó que un elevado porcentaje de los dromedarios analizados (un 79% [intervalo de confianza al 95%: 66,9­91,5%; error estándar: 0,0625], esto es, 42 de 53) excretaban virus en el momento del sacrificio, pese a que todos los animales parecían estar sanos. Entre los ejemplares positivos para el MERS-CoV, las muestras que con más frecuencia arrojaban resultado positivo eran los frotis nasales (97,6%). Las segundas, por orden de frecuencia, eran los frotis bucales (35,7%), seguidos de los frotis rectales (28,5%). Además, la carga viral más alta, expresada por un valor de ciclo umbral (Ct) (o punto de cruce) de 11,27, se obtuvo a partir de un frotis nasal. Estos resultados llevan a la conclusión de que los frotis nasales son el tipo de muestra más adaptado para detectar el MERS-CoV en dromedarios aparentemente sanos mediante la técnica de RT-PCR.

Human bocavirus infection as a cause of severe acute respiratory tract infection in children.

In 2005 human bocavirus (HBoV) was discovered in respiratory tract samples of children. The role of HBoV as the single causative agent for respiratory tract infections remains unclear. Detection of HBoV in children with respiratory disease is frequently in combination with other viruses or bacteria. We set up an algorithm to study whether HBoV alone can cause severe acute respiratory tract infection (SARI) in children. The algorithm was developed to exclude cases with no other likely cause than HBoV for the need for admission to the paediatric intensive care unit (PICU) with SARI. We searched for other viruses by next-generation sequencing (NGS) in these cases and studied their HBoV viral loads. To benchmark our algorithm, the same was applied to respiratory syncytial virus (RSV)-positive patients. From our total group of 990 patients who tested positive for a respiratory virus by means of RT-PCR, HBoV and RSV were detected in 178 and 366 children admitted to our hospital. Forty-nine HBoV-positive patients and 72 RSV-positive patients were admitted to the PICU. We found seven single HBoV-infected cases with SARI admitted to PICU (7/49, 14%). They had no other detectable virus by NGS. They had much higher HBoV loads than other patients positive for HBoV. We identified 14 RSV-infected SARI patients with a single RSV infection (14/72, 19%). We conclude that our study provides strong support that HBoV can cause SARI in children in the absence of viral and bacterial co-infections.

Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

Identification and characterization of a novel adenovirus in the cloacal bursa of gulls.

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

The role of Pitx3 in survival of midbrain dopaminergic neurons.

Dopamine belongs to the most intensively studied neurotransmitters of the brain, because of its implications in psychiatric and neurological disorders. Although, clinical relevance of midbrain dopaminergic (mDA) neurons is well recognized and dopaminergic dysfunction may have a genetic component, the genetic cascades underlying developmental processes are still largely unknown. With the advances in molecular biology, mDA neurons and their involvement in psychiatric and neurological disorders are now subject of studies that aim to delineate the fundamental neurobiology of these neurons. These studies are concerned with developmental processes, cell-specific gene expression and regulation, molecular pharmacology, and genetic association of dopamine-related genes and mDA-associated disorders. Several transcription factors implicated in the post-mitotic mDA development, including Nurr1, Lmx1b, Pitx3, and En1/En2 have contributed to the understanding of how mDA neurons are generated in vivo. Furthermore, these studies provide insights into new strategies for future therapies of Parkinson's Disease (PD) using stem cells for engineering DA neurons in vitro. Here, we will discuss the role of Pitx3 in molecular mechanisms involved in the regional specification, neuronal specification and differentiation of mDA neurons.

Strategies to unravel molecular codes essential for the development of meso-diencephalic dopaminergic neurons.

Understanding the development of neuronal systems has become an important asset in the attempt to solve complex questions about neuropathology as found in Parkinson's disease, schizophrenia and other complex neuronal diseases. The development of anatomical and functional divergent structures in the brain is achieved by a combination of early anatomical patterning and highly coordinated neuronal migration and differentiation events. Fundamental to the existence of divergent structures in the brain is the early region-specific molecular programming. Neuronal progenitors located along the neural tube can still adapt many different identities. Their exact position in the developing brain, however, determines early molecular specification by region-specific signalling molecules. These signals determine time and region-specific expression of early regulatory genes, leading to neuronal differentiation. Here, we focus on a well-described neuronal group, the meso-diencephalic dopaminergic neurons, of which heterogeneity based on anatomical position could account for the difference in vulnerability of specific subgroups as observed in Parkinson's disease. The knowledge of their molecular coding helps us to understand how the meso-diencephalic dopaminergic system is built and could provide clues that unravel mechanisms associated with the neuropathology in complex diseases such as Parkinson's disease.

Signalling through phospholipase C beta 4 is not essential for midbrain dopaminergic neuron survival.

The most prominent progressive neurodegenerative movement disorder, Parkinson's disease, is attributed to selective loss of dopamine neurons in the substantia nigra pars compacta, resulting in severe deficiency of dopamine. The homeo-domain gene, Pit x 3, is essential for proper development of midbrain dopaminergic neurons in the substantia nigra pars compacta and might be involved in midbrain dopaminergic survival pathways. The mGluR1-signaling downstream-effector phospholipase C beta 4 was identified in a suppression subtractive hybridization screen comparing wild-type and Pit x 3-deficient Aphakia midbrain dopaminergic neurons. Expression pattern analysis revealed that phospholipase C beta 4 was expressed in midbrain dopaminergic neurons of the substantia nigra pars compacta and part of the ventral tegmental area, whereas expression of mGluR1alpha was predominantly observed in the more vulnerable midbrain dopaminergic neurons in the lateral substantia nigra pars compacta. However, clear expression of phospholipase C beta 4 in spared midbrain dopaminergic neurons of Aphakia mice located in the ventral tegmental area, indicated that induction and maintenance of phospholipase C beta 4 expression is Pit x 3-independent in these neurons. Furthermore, we report here a normal distribution of midbrain dopaminergic cell bodies and axonal projection to the striatum in phospholipase C beta 4-/- mice, indicating that signaling of phospholipase C beta 4 is not essential for the survival of midbrain dopaminergic neurons.

Public awareness of risk factors and screening for colorectal cancer in Europe.

Colorectal cancer (CRC) is the commonest site for malignancy in Europe. The Commissioner for Health wishes to promote screening for colorectal, breast and cervical cancer in Europe. The aim of this study was to assess public knowledge of CRC in Europe and likely take up of free screening. To this end 20710 members of the public from 21 European countries were interviewed by means of a regular survey amongst consumers (Omnibus survey) using 13 stem questions. Forty-eight per cent thought the population were at equal risk of CRC, only 57% were aware of age and 54% of family history as risk factors. Although 70% were aware of dietary factors, only 30% knew that lack of exercise might be a risk factor. Only 51% had knowledge of CRC screening but 75% were 'very', or 'quite interested, in taking up faecal occult blood (FOB) screening if offered free. Barriers to screening were lack of awareness of risk (31%), youth (22%) and an un-anaesthetic test (19%). There was a big cultural difference in willingness of the public to discuss bowel symptoms: there was a major barrier in Finland (91%), Britain (84%), Luxembourg (82%), Poland (81%) and Portugal (80%); less of a barrier in Spain (49%), Italy (44%) and Iceland (39%). In conclusion, the challenge of achieving high compliance for CRC screening must be a major objective amongst EU member states and non-aligned countries of Europe in the next decade, because it is known that the non-compliant group are those at greatest risk of death from CRC. This study has shown that awareness of CRC is low in Europe and that an educational programme will be essential to achieve high compliance for CRC screening as a means of reducing deaths from bowel cancer.

Potato tuber proteins efficiently inhibit human faecal proteolytic activity: implications for treatment of peri-anal dermatitis.

BACKGROUND: Frequent diarrhoea after intestinal resections and faecal incontinence in healthy infants may lead to perianal injury. A causative agent may be a high concentration of pancreatic proteases in faeces. The aim of the present study was to assess whether protease inhibitors are applicable for treating and preventing peri-anal dermatitis by inhibiting the initial cause of the inflammation, the faecal proteases. DESIGN: Proteolytic activity was estimated in faeces of subjects frequently suffering from peri-anal dermatitis: patients with intestinal resections and healthy infants. The development of perianal dermatitis was studied after the construction of a reservoir with ileoanal anastomosis. The inhibitory effect of crude and partly purified potato juice on proteolytic activity of faecal output from patients with intestinal resections and healthy infants was investigated in vitro and in vivo (skin tests). RESULTS: Faecal protease activity in faeces from patients with intestinal resections and healthy infants was found to be significantly higher than in healthy adults. After the construction of an ileum reservoir, 46 of 48 patients developed a protease-related peri-anal dermatitis. The partly purified protein fraction from potatoes inhibited the larger part of faecal proteases in vitro and completely prevented skin irritation by pancreatic proteases dissolved in sterilized faecal fluid, in a 24-h skin test, on the back of healthy human volunteers. CONCLUSIONS: Potato proteins contain protease inhibitors, which suppress almost the complete proteolytic activity in faeces. Topical application of potato protease inhibitors might be a novel approach in preventing protease-induced peri-anal dermatitis, and therapeutic studies are needed to confirm our results.

Phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events.

Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.

Discontinuous and non-discontinuous subgenomic RNA transcription in a nidovirus.

Arteri-, corona-, toro- and roniviruses are evolutionarily related positive-strand RNA viruses, united in the order Nidovirales. The best studied nidoviruses, the corona- and arteriviruses, employ a unique transcription mechanism, which involves discontinuous RNA synthesis, a process resembling similarity-assisted copy-choice RNA recombination. During infection, multiple subgenomic (sg) mRNAs are transcribed from a mirror set of sg negative-strand RNA templates. The sg mRNAs all possess a short 5' common leader sequence, derived from the 5' end of the genomic RNA. The joining of the non-contiguous 'leader' and 'body' sequences presumably occurs during minus-strand synthesis. To study whether toroviruses use a similar transcription mechanism, we characterized the 5' termini of the genome and the four sg mRNAs of Berne virus (BEV). We show that BEV mRNAs 3-5 lack a leader sequence. Surprisingly, however, RNA 2 does contain a leader, identical to the 5'-terminal 18 residues of the genome. Apparently, BEV combines discontinuous and non-discontinuous RNA synthesis to produce its sg mRNAs. Our findings have important implications for the understanding of the mechanism and evolution of nidovirus transcription.