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 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.

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.

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.

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.

Isolation, molecular and phylogenetic analysis of encephalomyocarditis virus strain GS01 in China.

Encephalomyocarditis virus (EMCV) is a small non-enveloped, single-stranded RNA virus. It can infect many host species and cause acute myocarditis and respiratory failure in piglets, reproductive failure in pregnant sows. Diseases caused by EMCV currently affect the swine industry worldwide. In this study, an EMCV strain was isolated from an aborted fetus in western China. It was identified by 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-21 cells and could cause severe clinical symptoms and pathological changes in mice. Complete genome sequencing and multiple sequence alignment indicated that the GS01 strain was 79.9-99.9% identical with other isolates worldwide. Phylogenetic analysis showed that EMCV isolates fell into five clusters: lineage 1, 2, 3, 4, and 5 based on the nucleotide sequences of the entire ORF and VP3/VP1 junction, as well as 3D gene. GS01 isolate was grouped into lineage 1. The results of this study confirmed that an EMCV strain GS01 isolated from an aborted pig fetus in western China was fatal to mice and provided new epidemiologic data on EMCV in China.

[Isolation, identification and full-length genome sequence analysis of encephalomyocarditis virus from local aardvarks].

Encephalomyocarditis virus (EMCV) is a natural epidemic zoonotic pathogen. However, no reports have been published regarding the isolation, identification and full-length genome of EMCV from a local aardvark population. In present study, an EMCV isolate HNXX13 was isolated from aardvarks named Huainan-pig in Henan Province. The systematic identification, full-length genome sequencing and molecular characteristic analysis of the isolate HNXX13 were conducted. The result showed that the isolate was spherical with a diameter of 24-30 nm, neither heat- nor acid-resistant, sensitive to trypsin, insensitive to chloroform, not protected by bivalent cationic, and the specific fluorescence was observed in the cytoplasm of BHK-21 cells infected with the isolate by using indirect fluorescence assay. The full-length genome of EMCV HNXX13 generated a 7 725bp sequence (GenBank: F771002), with 81.0%-99.9% nucleotide identity to reference strains from different animals, and 99.5% with a Chinese reference strain isolated earlier from a commercial pig herd. The phylogenetic tree based on the full-length genome and ORF sequences identified that all EMCV strains were divided into three groups G1, G2 and G3, and strain HNXX13 belonging to the G1 group with other Chinese reference strains. The result also identified that this EMCV infection could cause severe clinical signs in a local aardvark population, and enriches the molecular epidemiological data of EMCV in China. Regional differences exist in EMCV genome and transmission is limited within a certain area. However, the cross-infection and transmission of EMCV between aardvark and mice appears most likely. Mutations have occurred in some amino acids of EMCV strain HNXX13 during the transmission in local aardvark herd and these mutations might make the virus easier to infect the aardvark.

Isolation, molecular characterization, and phylogenetic analysis of encephalomyocarditis virus from South China tigers in China.

Although encephalomyocarditis virus (EMCV) can infect many host species and cause myocarditis and sudden death in many species, little is known about EMCV infection in tigers. A virus was isolated from organs of dead South China tigers with sudden death in southern China. The production of cytopathic effect on BHK cells, and the results of PCR, electron microscopy (EM), and whole genome sequencing indicated that the pathogen was EMCV, the strain was named FJ13. Other pathogenic agents were excluded as possible pathogenic agents. Phylogenetic analyses of the whole genome, ORF (open reading frame) and CCR (capsid coding region) using the neighbour-joining method revealed that EMCV isolates cluster into two groups (group 1 and 2) with two sub-clusters within group 1 (group 1a and 1b), and FJ13 belongs to group 1a. Animal experiment showed that the isolated strain FJ13 could cause clinical symptoms and pathological changes. The results of this study indicated that FJ13 caused myocarditis of tigers and provided new epidemiologic data on EMCV in China.

A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

BACKGROUND: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens. METHODS: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted. RESULTS: Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range. CONCLUSIONS: The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

Isolation and molecular characterization of a second serotype of the encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) and Theilovirus are the two species of the Cardiovirus genus. Whereas theiloviruses comprise several sero-/genotypes, all known EMCV isolates are serologically very similar and are thought to belong to one serotype, named EMCV-1. Here, a novel EMCV type is described. Strain RD 1338 (D28/05) was isolated from a wood mouse (Apodemus sylvaticus) in Germany and can be distinguished from EMCV-1 by serological and molecular means. Failure of EMCV-1 specific hyperimmune sera to neutralize RD 1338 suggests a distinct serotype. The viral genome was de novo sequenced using next-generation Illumina/Solexa technologies. Considerable differences of the BC-loop/loop I/loop II sequences of VP1, the VP2 puff and the VP3 knob provide a structural basis for deviant serological properties. Sequence alignments reveal amino acid identities of 75 percent for the P1 region and 84 percent for the P2 and P3 regions when comparing RD 1338 to EMCV-1 strains and some 60 percent and less than 50 percent amino acid identities, respectively, for comparisons with theilovirus strains. Phylogenetic analyses of the P1, 2C and 3CD gene regions support the establishment of an EMCV-2 serotype. In contrast to the theilovirus sero-/genotypes that show a narrow host range, EMCV-1 infects a wide variety of hosts. The host range of EMCV-2 remains to be determined.

Encephalomyocarditis virus may use different pathways to initiate infection of primary human cardiomyocytes.

Encephalomyocarditis virus (EMCV) can infect a wide range of vertebrate species including swine and non-human primates, but few data are available for humans. We therefore wanted to gain further insight into the mechanisms involved in EMCV infection of human cells. For this purpose, we analyzed the permissiveness of primary human cardiomyocytes towards two strains of EMCV; a pig myocardial strain (B279/95) and a rat strain (1086C). In this study, we show that both strains productively infect primary human cardiomyocytes and induce complete cytolysis. Binding and infection inhibition experiments indicated that attachment and infection are independent of sialic acid and heparan sulfate for B279/95 and dependent for 1086C. Sequence comparison between the two strains and three-dimensional analysis of the capsid revealed that six of the seven variable residues are surface-exposed, suggesting a role for these amino acids in binding. Moreover, analysis of variants isolated from the 1086C strain revealed the importance of lysine 231 of VP1 in the attachment of EMCV to cell-surface sialic acid residues. Together, these results show a potential for EMCV strains to use at least two different binding possibilities to initiate infection and provide new insights into the mechanisms involved in primary human cell recognition by EMCV.

Encephalomyocarditis virus mortality in semi-wild bonobos (Pan panicus).

BACKGROUND: Fatal myocarditis from encephalomyocarditis virus (EMCV) infection has previously been identified in sporadic and epidemic forms in many species of captive non-human primates probably including one bonobo (Pan paniscus). METHODS: We investigated the deaths of two bonobos that were suspicious of EMCV using a combination of histopathology, immunohistochemistry and, for one of the two bonobos, reverse transcription PCR. RESULTS: Histopathological examination of heart tissue from the two bonobos showed changes characteristic of EMCV. Immunohistochemical studies confirmed the presence of EMCV antigen in heart tissue of both and in kidney and intestine of one of the bonobos. EMCV RNA was also isolated from the serum of the bonobo tested. CONCLUSION: Together, these findings confirm that EMCV was responsible for deaths of the two bonobos. Strict separation of bonobos in particular and captive primates in general from potential sources of EMCV contamination should be maintained to prevent mortality caused by EMCV.

[Sequencing and analysis of the complete genome of encephalomyocarditis virus strain GXLC isolated from swine].

The complete genome of encephalomyocarditis virus (EMCV)strain GXLC isolated from swine was sequenced and analyzed. Five overlapped gene fragments covering the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified by the 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE method, respectively. The genome sequences of strain GXLC were obtained by assembling the sequences of RT-PCR-generated cDNA fragments. The length of the complete genome was 7 725 nucleotides (nt). The homology comparison and phylogenetic analysis of the nucleotide and deduced amino acid sequences between strain GXLC and other EMCV strains available in GenBank were performed. The results showed that the complete genome identity between GXLC strain and the strains from China, i.e. GX0601, GX0602, BJC3 and HB1 and the strains from other countries, i.e. CBNU, K3, K11, TEL-2887A, EMCV-R and PV21 was over 99%. The phylogenetic trees based on the complete genome, the structural protein or the non-structural protein gene sequences revealed that the tree topology was similar. All the EMCV strains could be divided into two groups: group I and group II, and group I could be subdivided into subgroup Ia and subgroup Ib. The strains from swine belonged to subgroup Ia or Ib, and the strains from mice belonged to subgroup Ia, while the strains from Sus scro fa belonged to group II. Strain GXLC, together with other EMCV isolates from China, belonged to subgroup Ia.

Immune responses and expression of the virus-like particle antigen of the porcine encephalomyocarditis virus.

Virus-like particles (VLPs) are particles that consist of viral capsid proteins and are structurally similar to authentic virus. To express VLPs of the porcine encephalomyocarditis virus (EMCV) and investigate their efficacy and immuno response in vivo, a plasmid (P12A3C-pCI) containing the P12A and 3C genes of the EMCV-K3 viral strain was constructed. The VLPs of EMCV-K3 were successfully assembled in 293FT cells on 3 days after transfection with P12A3C-pCI and were identified as particles of about 30-40 nm using transmission electron microscopy (TEM). In an in vivo experiment, the murine cytokines induced by VLPs of naked DNA vaccine showed that the Th1 indicators IL-2, TNF-alpha and GM-CSF, and the Th2 indicators IL-4 and IL-10 were increased. The immunization of mice with the P12A3C-pCI plasmid induced high levels of neutralizing antibody from 128- to 256-fold and led to a significant protection ratio (90%) after challenge with EMCV-K3 (wild-type strain). These VLPs may represent a novel vaccine strategy for the control of EMCV infection on pig farms.

Human febrile illness caused by encephalomyocarditis virus infection, Peru.

Etiologic studies of acute febrile disease were conducted in sites across South America, including Cusco and Iquitos, Peru. Patients' clinical signs and symptoms were recorded, and acute- and convalescent-phase serum samples were obtained for serologic examination and virus isolation in Vero E6 and C6/36 cells. Virus isolated in Vero E6 cells was identified as encephalomyocarditis virus (EMCV) by electron microscopy and by subsequent molecular diagnostic testing of samples from 2 febrile patients with nausea, headache, and dyspnea. The virus was recovered from acute-phase serum samples from both case-patients and identified with cardiovirus-specific reverse transcription-PCR and sequencing. Serum samples from case-patient 1 showed cardiovirus antibody by immunoglobulin M ELISA (acute phase <8, convalescent phase >1,024) and by neutralization assay (acute phase <10, convalescent phase >1,280). Serum samples from case-patient 2 did not contain antibodies detectable by either assay. Detection of virus in serum strongly supports a role for EMCV in human infection and febrile illness.

Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China.

Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5-99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3-84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.

Destruction of human retinoblastoma after treatment by the E variant of encephalomyocarditis virus.

The oncolytic effects of encephalomyocarditis (EMC) virus were examined in human retinoblastoma cell (Y79) cultures which were infected with 10(4 )tissue culture infectious doses (TCIDs) of the E variant of EMC (EMC-E) virus. The TCIDs were used to titer the maximum effect of EMC virus on L-929 cells. In-vitro studies showed 90% cytopathic effect (CPE) at 24 h and 100% CPE at 52 h. The CPE was used to observe pathologic effects of the cells. In-vivo studies employing human retinoblastoma grown as a tumor in nude mice, revealed degeneration of 80% of the tumor cells at 3 days and total destruction at 4 days following inoculation with the EMC-E virus. The virus is highly neurotropic in mice, but is usually not pathogenic in man. These studies suggest a possible new direction in the treatment of retinoblastoma and other malignant tumors using the viral technology.

A wild-type porcine encephalomyocarditis virus containing a short poly(C) tract is pathogenic to mice, pigs, and cynomolgus macaques.

Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 C's) at the 5' nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7- to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tract-containing virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with >/==" BORDER="0">4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n = 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC(5)UC(8)) was comparable to that of strain 2887A/91 (C(10)UCUC(3)UC(10)), another recent porcine isolate.

A comparative study of the pathogenic properties and transmissibility of a Greek and a Belgian encephalomyocarditis virus (EMCV) for piglets.

Thirteen susceptible piglets, aged 40 days, were divided into two groups and were experimentally infected either with a Greek (myocardial) or a Belgian (reproductive) encephalomyocarditis virus (EMCV) strain (total dose 5 x 10(6) TCID50, intramuscularly and intranasally). Six piglets were placed in the same rooms, 24 h later, as contact controls. The following criteria were studied: ante mortem: clinical signs, serum cardiac isoenzyme activities (CK-MB and LD-1), viraemia, nasal and faecal virus excretion and serological response. Post mortem (after death or euthanasia): gross lesions, virus isolation from tissues, RT-PCR, as well as histopathological and immunohistochemical findings. The Greek strain was more pathogenic, producing mortality, with high cardiac isoenzyme activities and pronounced macroscopic myocardium lesions. The Belgian strain was able to induce mild heart lesions, as detected only by cardiac isoenzyme activity and histopathologically. All contact pigs were infected, within the first 1-2 days of their introduction, that coincided with the period of viral excretion by the experimentally infected pigs (up to the 3rd day post infection). Disease was mild, with no mortality.

Phylogenetic analysis of European encephalomyocarditis viruses: comparison of two genomic regions.

The phylogenetic relationships of encephalomyocarditis (EMC) viruses isolated from pigs and rodents in Europe were determined by comparison of nucleotide sequences from two different regions of the virus genome, the VP3/VP1 gene junction (part of the capsid-coding region) and part of the 3D polymerase-coding region. Thirty-five European EMC viruses could be divided into two genetic groups, one which contained viruses from Greece isolated between 1986 and 1997 and from Belgium in 1991 and the other which contained viruses from Italy (1986-1996), Cyprus (1994-1995), France (1995) and Belgium (1995-1996).