Prevalence and Molecular Analysis of Encephalomyocarditis Virus-2 in the Hazel Dormouse.

The hazel dormouse (Muscardinus avellanarius) population in the UK continues to decline due to habitat loss, despite reintroductions of captive-bred individuals being conducted nationally for over 30 years. Disease surveillance of captive-bred and wild dormice is performed to identify novel and existing disease threats which could impact populations. In this study, we firstly investigated cause of death in seven hazel dormice found dead in England, through next-generation sequencing identifying a virus closely related to a wood mouse encephalomyocarditis virus-2 (EMCV-2). Subsequently, lung tissue samples from 35 out of 44 hazel dormice tested positive for EMCV-2 RNA using a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Sanger sequencing methods developed in this study. Formalin-fixed tissues available for nine hazel dormice which tested positive for EMCV-2 RNA were examined microscopically. Three cases showed moderate interstitial pneumonia with minimal to mild lymphoplasmacytic myocarditis, but no evidence of encephalitis. However, the presence of possible alternative causes of death in these cases means that the lesions cannot be definitively attributed to EMCV-2. Here, we report the first detection of EMCV-2 in hazel dormice and conclude that EMCV-2 is likely to be endemic in the hazel dormouse population in England and may be associated with clinical disease.

A recent view about encephalomyocarditis virus circulating in compartmentalised animal population in Northern Italy.

Encephalomyocarditis virus (Picornaviridae, Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis and reproductive disorders in various mammals, especially in swine. Despite the disease occurred endemically in pig farms since 1997, the recent increase of death experimented in Northern Italy prompted to furtherly investigate the evolution of the virus and the actual spread of the infection. Italian EMC viruses, collected between 2013 and 2019, showed an overall antigenic stability. The in-house ELISA Monoclonal Antibodies based, able to reveal changes in seven different antigenic sites, showed only sporadic and occasional mutations in considered samples and the subsequent phylogenetic analysis confirmed antigenic panel's remarks. All the isolates could be classified within a unique lineage, which comprise other European strains and confirm that the viruses currently circulating in Italy developed from a unique common ancestor. Despite the demonstrated stability of virus, some putative newly emerged variants were detected through antigenic profile analysis and phylogenesis. Finally, the serosurvey proved that spread of EMCV is greater than the diffusion of fatal infections would suggest, due to subclinical circulation of EMCV. It demonstrated an increase in the proportion of seropositive farms, if compared with previous data with no remarkable differences between farms with and without clinical evidence of disease.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Saffold virus infection in elderly people with acute gastroenteritis in Sweden.

Viral gastroenteritis is a major source of morbidity and mortality, predominantly caused by so-called NOROAD viruses (norovirus, rotavirus, and adenovirus). In approximately onethird of all cases, however, the exact etiology is unknown. The in 2007 discovered human cardiovirus Saffold virus (SAFV) may prove to be a plausible candidate to explain this diagnostic gap. This virus, a member of the Picornaviridae family which is closely related to the murine viruses Theiler's murine encephalomyelitis virus and Theravirus, is a widespread pathogen and causes infection early in life. Screening of 238 fecal or vomitus samples obtained from NOROAD-negative, elderly patients with acute gastroenteritis at the University Hospital of Linköping showed that SAFV is present in low abundance (4.6%). Phylogenetic analysis of the VP1 gene revealed a Swedish isolate belonging to the highly common and in Europe widespread SAFV-3 genotype. This genotype is also related to previously reported Asian strains. This study describes the first molecular typing of a Swedish SAFV isolate and is the first report to document the circulation of SAFV among elderly people. The pathogenicity of SAFV is, as of yet, still under debate; further studies are necessary to determine its role in the development of disease.

Encephalomyocarditis virus infection in alpacas.

Encephalomyocarditis virus (EMCV) infection was detected by real-time reverse transcription PCR (qRT-PCR) in four adult alpacas (Vicugna pacos) from two properties on the Far North Coast of New South Wales (NSW) in April and May 2018 and in two adult alpacas from a third property on the Central Coast of NSW in October 2018. Viral RNA was detected in a range of samples, including blood, fresh body organs and mucosal swabs. EMCV was isolated from the blood and body organs of five of these alpacas. These animals displayed a range of clinical signs, including inappetence, colic, recumbency and death. Necropsy findings included multifocal to coalescing areas of myocardial pallor, pulmonary congestion and oedema, hepatic congestion and serosal effusion. Histopathological changes comprised acute, multifocal myocardial degeneration and necrosis, with mild, neutrophilic and lymphocytic inflammation (5/5 hearts) and mild, perivascular neutrophilic meningoencephalitis (1/3 brains). This is the first report of disease due to EMCV in alpacas under farm conditions, and it identifies EMCV infection as a differential diagnosis for acute disease and death in this camelid species. In addition to the samples traditionally preferred for EMCV isolation (fresh heart, brain and spleen), blood samples are also appropriate for EMCV detection by qRT-PCR assay.

Evidence of Saffold virus circulation in Italy provided through environmental surveillance.

Saffold virus (SAFV) is an emerging human cardiovirus associated with respiratory and gastrointestinal infection, and, more recently, to symptoms related to the endocrine, cardiovascular, and neurological systems. Information about SAFV circulation in Italy is scarce. In order to provide insights into the epidemiology of SAFV in Italy, 141 raw sewage samples collected throughout Italy were tested using broad-range nested RT-PCR primers targeting the 5'-NC region. Seven samples (5·0%) were confirmed as SAFV in samples collected in North, Centre and Southern Italy. Typing was attempted through amplification of the VP1 coding region, using both published and newly designed primers, and one sample was characterized as SAFV-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential.

Clinical Characteristics of Saffold Virus Infection in Children.

BACKGROUND: Saffold virus (SAFV) is a novel human cardiovirus that was identified in 2007. Recently, SAFV has been isolated from nasal and stool specimens of infants presenting with respiratory and gastrointestinal symptoms and from cerebrospinal fluid (CSF) specimens of children with central nervous system infection. However, little is known regarding clinical characteristics of SAFV in children. METHODS: We reviewed 5412 specimens from the database of the infectious agents surveillance system in Niigata prefecture, Japan, between January 2006 and December 2013, and identified SAFV-infected patients. Subsequently, we retrospectively reviewed their medical records and evaluated their clinical characteristics. RESULTS: We identified 9 SAFV-infected patients (median age: 5 years; range: 2-16 years). Seven patients were diagnosed with pharyngitis, one with meningitis and one with fever of unknown origin. Dominant symptoms were high fever, appetite loss and headache. The median duration of the fevers was 2 days in patients with pharyngitis; however, the patient with meningitis remained febrile for 5 days. All blood tests available in this case series revealed leukocytosis with a predominance of neutrophils. CSF profiles showed mild lymphocytic pleocytosis. All patients recovered fully without complications. CONCLUSIONS: A few clinical characteristics of SAFV infection were clarified, including high fever of short duration in patients with pharyngitis, and neutrophil-dominant leukocytosis. The clinical course and CSF profiles of a case of meningitis were similar to those of other aseptic meningitis. SAFV needs to be included in the differential diagnosis of pharyngitis or meningitis when commonly identified viruses are not identified in such patients.

Detection of Saffold viruses from children with acute respiratory infections in Yamagata, Japan, between 2008 and 2015.

Although Saffold virus (SAFV) was reported as a novel human cardiovirus in 2007, no causative association between SAFV and clinical disease has been proven and the longitudinal epidemiology of SAFVs is not available. To establish the relationship between SAFVs and acute respiratory infections (ARIs) and to clarify the longitudinal epidemiology of SAFVs, 7258 nasopharyngeal specimens were collected from children with ARIs in Yamagata, Japan between 2008 and 2015. The specimens were inoculated on a microplate including six cell lines as part of routine surveillance, and molecular screening was performed for SAFVs using a reverse transcription (RT)-PCR method. Throughout the study period, 95 (1.3%) SAFV genotype 2 (SAFV2), and 28 (0.4%) SAFV3 were detected, mainly between September and November. There were two outbreaks of SAFV2 in 2009 and 2013, and one outbreak of SAFV3 in 2012 and the positive rates during these outbreaks were 12.1% (53/439), 11% (35/319), and 4.4% (20/453), respectively. Sixty-three SAFV2 and 28 SAFV3 strains were detected as a single virus from children with ARIs such as pharyngitis, herpangina, and tonsillitis. These results suggested that SAFV2 and SAFV3 are possible causative agents of ARIs among children and their infections occur mainly in the autumn season in Japan.

Isolation and genetic characterization of encephalomyocarditis virus 1 from a deceased captive hamadryas baboon.

In 2007, numerous hamadryas baboons (Papio hamadryas) died suddenly in an aviary of a primate institute in Sochi, Russia, in the absence of prior clinical signs. Necropsies were suggestive of encephalomyocarditis virus infection, but RT-PCR assays with commonly used primers were negative. Here we report the histopathological results obtained during necropsies and the isolation and genomic characterization of a divergent strain of encephalomyocarditis virus 1 (EMCV-1) from heart tissue of one of the succumbed hamadryas baboons. Phylogenetic analysis indicates that the isolated virus belongs to the newly proposed EMCV-1 lineage G, which clusters alongside lineage C ("Mengo virus"). This study is the first report describing a lineage G strain of EMCV-1 as the etiological agent of a lethal disease outbreak among captive nonhuman primates in Europe.

Saffold virus, an emerging human cardiovirus.

Saffold virus (SAFV) is an emerging human cardiovirus that has been shown to be ubiquitous. Initial studies of SAFV focused on respiratory and gastrointestinal infection; however, it has also recently been associated with diverse clinical symptoms including the endocrine, cardiovascular, and neurological systems. Given the systemic nature of SAFV, and its high prevalence, understanding its pathogenicity and clinical impact is of utmost importance. This comprehensive review highlights and discusses recent developments in epidemiology, human pathogenicity, animal, and molecular studies related to SAFV. It also provides detailed insights into the neuropathogenicity of SAFV. We argue that human studies have been confounded by coinfections and therefore require support from robust molecular and animal research. Thereby, we aim to provide foresight into further research to better understand this emerging virus.

Encephalomyocarditis virus infection in Macaca sylvanus and Hystrix cristata from an Italian rescue centre for wild and exotic animals.

BACKGROUND: The Encephalomyocarditis virus (EMCV) is a small, non enveloped, positive sense single-stranded RNA virus in the genus Cardiovirus, family Picornaviridae, with two known serotypes. It is spread worldwide and infects a huge range of vertebrate hosts with zoonotic potential for humans. The pig is the mammal most likely to be impacted on with the disease, but EMCV occurrence has also been reported in non-human primates and in a variety of domestic, captive and wild animals. Until now, human cases have been very rare and the risk appears to be almost negligible in spite of human susceptibility to the infection. CASE PRESENTATION: Between September and November 2012 a fatal Encephalomyocarditis virus outbreak involving four Barbary macaques and 24 crested porcupines occurred at a rescue centre for wild and exotic animals in Central Italy. In this open-field zoo park located near Grosseto, Tuscany about 1000 animals belonging to different species, including various non-human primates were hosted at that time. Sudden deaths were generally observed without any evident symptoms or only with mild nonspecific clinical signs. The major gross change was characterised by grey-white necrotic foci in the myocardium and the same EMCV strain was isolated both in macaques and crested porcupines. Phylogenetic analysis has confirmed that only one EMCV strain is circulating in Italy, capable of infecting different animal species. CONCLUSIONS: This report confirms the susceptibility of non-human primates to the EMCV infection and describes the disease in porcupine, a common wild Italian and African species. No human cases were observed, but given the zoonotic potential of EMCV these findings are of importance in the context of animal-human interface.

The impact of compartmentalised housing on direct encephalomyocarditis virus (EMCV) transmission among pigs; insight from a model.

Although generally considered a rodent virus, pigs sometimes were suggested a potential reservoir host for encephalomyocarditis virus (EMCV), implying pig-to-pig transmission can cause major outbreaks in a pig population (basic reproduction ratio, R0>1). An earlier experimental study on EMCV transmission among pigs was inconclusive in this respect (R0≈1.24; CI 0.4-4.4). In this study we used a simulation model to extrapolate the experimental results to commercial, compartmentalised pig housings and tested to what extend contacts between pigs in different pens needed to be reduced in order to prevent major outbreaks in a compartment following a single introduction. The final size of simulated outbreaks was measured and the probability to observe outbreaks that affected at least 50 or 80% of the pens was calculated. Simulation scenarios compare one homogeneously mixing compartment (no fence) to epidemiological theory and an increasing effect of fencing on the pig-to-pig transmission between pigs in neighbouring pens. For any R0<1.24 the probability to observe outbreaks affecting more than 50% of the pens remained below 10% if compartmentalisation was introduced leaving per capita transmission rate unchanged. If fences also reduced contact transmission the probability to observe major outbreaks was below 50% for any R0<2.7. Only for R0>4, major outbreaks occurred with more than 50% chance even if only minimal contact between adjacent pens was allowed. In conclusion the results suggested that in a compartmentalised pig housing one single EMCV introduction is unlikely to cause a major outbreak by direct pig-to-pig transmission alone. Other mechanisms e.g. multiple introductions from a rodent reservoir may be required for large outbreaks to occur.

Detection of antibodies against Theiler's murine encephalomyelitis virus GDVII strain in experimental guinea pigs.

A disease affecting guinea pigs called 'guinea pig lameness' characterized by clinical signs of depression, lameness of limbs, flaccid paralysis, weight loss and death within a few weeks was first described by Römer in 1911. After a research group in our facility kept laboratory guinea pigs from two different origins together in one room, lameness was observed in two animals. Further investigations revealed a serological immune response against Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) in these animals. Histopathology of the lumbar spinal cord of these animals showed mononuclear cell infiltration and necrotic neurons in the anterior horn. Therefore, all guinea pigs from this contaminated animal unit, from other units in our facility, as well as from different European institutions and breeding centres were screened for antibodies directed against GDVII. Our investigations showed that approximately 80% of all guinea pigs from the contaminated animal unit were seropositive for GDVII, whereas animals from other separate units were completely negative. In addition, 43% of tested sera from the different European institutions and breeding centres contained antibodies against GDVII. The present data confirm that an unknown viral infection causes an immune response in experimental guinea pigs leading to seroconversion against GDVII and that guinea pigs from a commercial breeder are the source of the infection.

National serosurvey of encephalomyocarditis virus in healthy people and pigs in China.

Encephalomyocarditis virus (EMCV) is a zoonotic pathogen that has a wide spectrum of host range. The virus has been discovered on swine farms worldwide and can cause acute fatal myocarditis in piglets and reproductive disorders in sows. Although EMCV infection has been documented in farmed pigs in China, seroprevalence in humans has not been reported. In this study, we conducted nationwide serological surveys for EMCV in humans and farmed pigs in China in 2013, by the use of a double antigen sandwich ELISA method. A total of 3305 serum samples from healthy people were obtained from seven geographical regions in China, of which 1010 samples (30.56%) were positive for EMCV antibodies. The overall seroprevalence for EMCV in the age groups of 0-20, 21-40, 41-60 and >60 years were 13.5%, 30.25%, 36.83% and 38.71% respectively, showing a tendency of increasing with age (P = 0.000). A total of 3470 serum samples from farmed pigs were collected and tested for antibodies to EMCV. A high seroprevalence of 77% was recorded, and significant regional differences were observed. It was concluded that people and pigs in China were commonly infected by EMCV. In addition, in order to characterize changes of seroprevalence during natural EMCV infection in pigs, 240 serial serum samples were collected from 30 pigs (at 0, 15, 30, 60, 75, 90, 120, and 150 days of age) in a farrow-to-finish farm in China. The data showed that there were two EMCV antibody peaks: the first peak appeared at day 30, followed by a decrease in EMCV antibody titer, and the second occurred after day 75. Thus, the most susceptible period of pigs for EMCV infection was between day 30 and day 75 of age.

Molecular epidemiological analysis of Saffold cardiovirus genotype 3 from upper respiratory infection patients in Taiwan.

BACKGROUND: Saffold cardiovirus (SAFV) belongs to the Cardiovirus genus of Picornaviridae family, and may be a relevant new human pathogen; Thus far, eleven genotypes have been identified. The SAFV type 3 (SAFV-3) is thought to be the major genotype and is detected relatively frequently in children with acute gastroenteritis and respiratory illness. The epidemiology and pathogenicity of SAFV-3 remain unclear. OBJECTIVES: To investigate the genomic and epidemiologic profiles of SAFV-3 infection in Taiwan. STUDY DESIGN: Virus was detected in respiratory samples from children suffering for URI. SAFV-3 isolates were detected by isolation on cell culture and IF assay. The molecular typing was performed by RT-PCR and was sequenced to compare with reference strains available in the NCBI GeneBank. Serum samples were collected from 2005 to 2013 in Taiwan for seroprevalence investigation. RESULTS: A total of 226 specimens collected from children with URIs, 22 (9.73%) were positive for SAFV-3. The majority of SAFV-3 infections were found in children less than 6 years of age (14 of 22, 63.6%). Genetic analysis of VP1 coding region of Taiwanese isolates shown an 83.2-97.7% difference from other available SAFV-3 sequences in NCBI GenBank. Phylogenetic analysis revealed there is three genetic groups of SAFV-3 co-circulated in Taiwan during the study period. In addition, seroprevalence investigation results indicated that SAFV-3 infection occurs early in life and 43.7-77.8% of children aged between 6 months to 9 years old, had neutralizing antibodies against SAFV-3. CONCLUSION: SAFV-3 may have circulated in Taiwan for some time and it appears to be one of the etiological agents responsible for URIs in children.

Serological prevalence of viral agents that induce reproductive failure in South Korean wild boar.

BACKGROUND: Viral agents associated with reproductive failure such as Aujeszky's disease virus (ADV), encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV) have also been identified in European wild boar. To screen for the presence of antibodies against ADV, EMCV, and PPV from wild boar (Sus scrofa) in South Korea, 481 serum samples were collected from wild boar hunted between December 2010 and May 2011. RESULTS: Of the 481 serum samples tested, 47 (9.8%) and 37 (7.7%) were seropositive for ADV and EMCV antibodies, respectively, based on a neutralization test (VNT), and 142 (29.5%) were seropositive for PPV antibodies based on a hemagglutination inhibition (HI) test. CONCLUSIONS: This was the first survey to identify the seroprevalence of the three major viruses associated with reproductive failure in the wild boar population of South Korea. Wild boar may act as a reservoir for many viruses that cause infectious diseases in domestic pigs. Thus, strict prevention and control measures, such as continuous wildlife disease surveillance and strategic methods of downsizing the population density, should be implemented to prevent disease transmission from wild boar to domestic pigs.

Prevalence and genetic characteristics of Saffold cardiovirus in China from 2009 to 2012.

The epidemiology and clinical features of the Saffold cardiovirus (SAFV) remain ambiguous. The present study was designed to systematically and intensively investigate the epidemiological features of SAFV in pediatric patients in China. Three cohorts of pediatric patients were recruited from 2009 to 2012. Cohort 1 comprised patients with acute respiratory tract infections. Cohort 2 comprised patients with diarrhea. Cohort 3 comprised hand, foot, and mouth disease (HFMD) patients. A total of 115 patients (1.6%) among 6052 (17/1647, 12/2013, and 86/2392 in cohorts 1, 2, and 3, respectively) were SAFV-positive. The samples from 82 SAFV-positive patients were successfully sequenced, and four genotypes were identified: 8 SAFV-1, 41 SAFV-2, 29 SAFV-3, and 4 SAFV-6. A significantly higher detection rate was found in the HFMD patients than in other two cohorts (both P <0.001). A higher frequency of severe clinical outcome and nervous system manifestation were also observed in the SAFV-positive HFMD patients. Additionally, 6 (3.5%) cerebrospinal fluid and 7 (2.2%) serum samples from the HFMD-associated encephalitis patients were SAFV-positive. Based on the VP1 sequences, all four genotypes displayed distinct geographical clustering. SAFV infection might be associated with a wide clinical spectrum and contribute to HFMD.

SAffold viruses in pediatric patients with diarrhea in Thailand.

Saffold virus (SAFV) is a newly discovered human virus which is classified into the genus Cardiovirus of the family Picornaviridae. A total of 608 fecal specimens collected during January 2012 to December 2013 from children with diarrhea in Chiang Mai, Thailand were investigated for SAFV by RT-nested PCR and sequence analysis. Of these, nine out of 608 (1.5%) were positive for SAFVs and four genotypes were identified, SAFV1, SAFV2, SAFV3, and SAFV4. SAFV mono-infection was found in five cases (CMH-S038-12, CMH-S071-12, CMH-S102-12, CMH-N029-12, and CMH-S048-13), while co-infection with other viruses causing diarrhea was observed in four cases (CMH-S021-12, CMH-S115-12, CMH-N048-13 and CMH-N103-13). This study provides more information about the genetic background of SAFV circulating in pediatric patients with diarrhea in Thailand.

Isolation and molecular analysis of porcine encephalomyocarditis virus strain BD2 from northern China.

Encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, an EMCV strain (BD2) was isolated from the suspected piglets with EMCV in China. It was identified by an indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. The virus could reproduce on BHK-21 cells and reach a peak titer by 16 hpi with maximum titers of 10(8.22)TCID50/0.1 ml. Phylogenetic analyses of open reading frame and the VP3/VP1 genes using neighbor-joining method revealed that EMCV isolates fell into two clusters: groups I and II. The BD2 isolate belonged to group I, along with strains NJ08, HB1, BJC3, CBNU, K3, K11, BEL-2887A, GX0601, GXLC, pEC9, and PV21, whereas four strains (D variant, EMCV-B, EMCV-D, and PV2) belonged to group II.