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.

Tamoxifen Application Is Associated with Transiently Increased Loss of Hippocampal Neurons following Virus Infection.

Tamoxifen is frequently used in murine knockout systems with CreER/LoxP. Besides possible neuroprotective effects, tamoxifen is described as having a negative impact on adult neurogenesis. The present study investigated the effect of a high-dose tamoxifen application on Theiler's murine encephalomyelitis virus (TMEV)-induced hippocampal damage. Two weeks after TMEV infection, 42% of the untreated TMEV-infected mice were affected by marked inflammation with neuronal loss, whereas 58% exhibited minor inflammation without neuronal loss. Irrespective of the presence of neuronal loss, untreated mice lacked TMEV antigen expression within the hippocampus at 14 days post-infection (dpi). Interestingly, tamoxifen application 0, 2 and 4, or 5, 7 and 9 dpi decelerated virus elimination and markedly increased neuronal loss to 94%, associated with increased reactive astrogliosis at 14 dpi. T cell infiltration, microgliosis and expression of water channels were similar within the inflammatory lesions, regardless of tamoxifen application. Applied at 0, 2 and 4 dpi, tamoxifen had a negative impact on the number of doublecortin (DCX)-positive cells within the dentate gyrus (DG) at 14 dpi, without a long-lasting effect on neuronal loss at 147 dpi. Thus, tamoxifen application during a TMEV infection is associated with transiently increased neuronal loss in the hippocampus, increased reactive astrogliosis and decreased neurogenesis in the DG.

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.

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant (Loxodonta africana) occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats (Rattus rattus) captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.

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.

Role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and Japanese encephalitis virus infection.

Bats are potential natural hosts of Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV). Bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. Here, kidney epithelial cell lines derived from four bat species (Pteropus dasymallus, Rousettus leschenaultii, Rhinolophus ferrumequinum, and Miniopterus fuliginosus) and two non-bat species (Homo sapiens and Mesocricetus auratus) were infected with EMCV and JEV. The replication of EMCV and JEV was lower in the bat cell lines derived from R. leschenaultii, R. ferrumequinum, and M. fuliginosus with a higher expression level of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferon-beta (IFN-ß) than that in the non-bat cell lines and a bat cell line derived from P. dasymallus. The knockdown of TLR3, RIG-I, and MDA5 in Rhinolophus bat cell line using antisense RNA oligonucleotide led to decrease IFN-ß expression and increased viral replication. These results suggest that TLR3, RIG-I, and MDA5 are important for antiviral response against EMCV and JEV in Rhinolophus bats.

Viral Metagenomics Revealed a Novel Cardiovirus in Feces of Wild Rats.

BACKGROUND/AIMS: Cardiovirus is a genus of viruses belonging to the family Picornaviridae. Here, we used viral metagenomic techniques to detect the viral nucleic acid in the fecal samples from wild rats in Zhenjiang city in China. METHOD: Fecal samples were collected from 20 wild rats and pooled into four sample pools and then subjected to libraries construction which were then sequenced on Illumina MiSeq platform. The sequenced reads were analyzed using viral metagenomic analysis pipeline. RESULTS: A novel cardiovirus from feces of a wild rat was identified, named amzj-2018, of which the complete genome was acquired. Phylogenetic analysis based on the complete amino acid sequence of polyprotein revealed that amzj-2018 formed a separate branch located between clusters of Saffold virus and Rat Theilovirus 1 (RTV-1). Phylogenetic analysis based on different regions of the polyproteins, including P1, P2, P3, and P2+P3, respectively, showed discordant trees, where the tree based on P3 region indicated that amzj-2018 clustered separately between Theiler's murine encephalomyelitis virus and RTV-1. CONCLUSION: The complete genome of a cardiovirus was determined from the feces of wild rats which belonged to a novel type of cardiovirus based on phylogenetic analysis. Whether it is associated with disease needs further investigation.

Facets of Theiler's Murine Encephalomyelitis Virus-Induced Diseases: An Update.

Theiler's murine encephalomyelitis virus (TMEV), a naturally occurring, enteric pathogen of mice is a Cardiovirus of the Picornaviridae family. Low neurovirulent TMEV strains such as BeAn cause a severe demyelinating disease in susceptible SJL mice following intracerebral infection. Furthermore, TMEV infections of C57BL/6 mice cause acute polioencephalitis initiating a process of epileptogenesis that results in spontaneous recurrent epileptic seizures in approximately 50% of affected mice. Moreover, C3H mice develop cardiac lesions after an intraperitoneal high-dose application of TMEV. Consequently, TMEV-induced diseases are widely used as animal models for multiple sclerosis, epilepsy, and myocarditis. The present review summarizes morphological lesions and pathogenic mechanisms triggered by TMEV with a special focus on the development of hippocampal degeneration and seizures in C57BL/6 mice as well as demyelination in the spinal cord in SJL mice. Furthermore, a detailed description of innate and adaptive immune responses is given. TMEV studies provide novel insights into the complexity of organ- and mouse strain-specific immunopathology and help to identify factors critical for virus persistence.

Encephalomyocarditis virus is potentially derived from eastern bent-wing bats living in East Asian countries.

Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product.

Equine serum hepatitis (i.e., Theiler's disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares <50% protein identity with its phylogenetic relatives of the genus Copiparvovirus. Next, we experimentally infected 2 horses using a tetanus antitoxin contaminated with EqPV-H. Viremia developed, the horses seroconverted, and acute hepatitis developed that was confirmed by clinical, biochemical, and histopathologic testing. We also determined that EqPV-H is an endemic infection because, in a cohort of 100 clinically normal adult horses, 13 were viremic and 15 were seropositive. We identified a new virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products.

Complete genome sequences and phylogenetic analysis of encephalomyocarditis virus strains isolated from pigs and rats origin.

In order to evaluate the genetic variability of encephalomyocarditis virus (EMCV), the whole genomes of six EMCV field isolates originating from pigs and rats origin in different regions of central China, were phylogenetically and comparatively analyzed. Phylogenetic analysis of whole genome sequences, open reading frame (ORF), capsid coding region (CCR) and VP3/VP1 using neighbor-joining analysis revealed that these isolates belonged to lineage 1. Nucleotide sequences of six isolates showed more than 99% pairwise identity rates, and the sequences of isolates from pig and boar in the same region were completely identical with each other, without any genetic deletion or insertion. From comparative analysis of variability of each EMCV protein coding region, 3D and VP3 regions showed that the highest average identity rates, and was confirmed as highly conserved. In contrast, the protein coding regions 3A and 3B was confirmed to be highly variable region with the lowest average identity rate. Our data confirmed that the EMCV strains isolated from pigs and rats origin had high homology with each other, which implied rats may play an important role in EMCV transmission between domestic pigs and wild boars.

An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific real-time RT-PCR, indirect immunofluorescence assays and electron microscopy. In addition, the open reading frame sequence (ORF) of the EMCV C15 virus was determined. From sequence comparison and phylogenetic analysis among 24 reference EMCV strains, it appears that the EMCV C15 strain is closely genetically related to strain BEL2887A/91 (>99.0% nucleotide identity). In artificially challenged dogs, the heart and brain were important targets of EMCV C15. This study provides genetic and pathogenic characterization of the EMCV C15 strain isolated in Beijing and calls for sustained surveillance of EMCV infection in China to support better prevention and control of the disease.

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.

Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

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.

Isolation and molecular and phylogenetic analyses of encephalomyocarditis virus from wild boar in central China.

Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. In this study, an EMCV strain, designated JZ1202, was isolated from semi-captive wild boars that presented with acute myocarditis and sudden death in central China. It was identified by hemagglutination inhibition (HI) assay, reverse transcription polymerase chain reaction (RT-PCR) and genome sequencing. The subsequent results showed that the virus could produce a specific cytopathic effect on BHK cells and could cause clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that JZ1202 strain was 81.3%-99.9% identical with other isolates worldwide. Phylogenetic analysis of the whole genome, ORF, VP3/VP1 and 3D genes using neighbor-joining method revealed that JZ1202 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain JZ 1202 isolated from wild boar in central China was fatal to mice and provided new epidemiologic data on EMCV in China.