A cardioviral 2C-ATP complex structure reveals the essential role of a conserved arginine in regulation of cardioviral 2C activity.

2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions. IMPORTANCE: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure-function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein.

Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

Management of encephalomyocarditis virus infection in Italian pig farms: a case report.

BACKGROUND: Encephalomyocarditis virus (EMCV) has been isolated from many animals, frequently as the cause of fatal myocarditis, but pigs are the most susceptible domestic specie. The virus was isolated in swine farms since 1958 in Panama and Europe from cases of sudden death in young pigs, and the main origin of outbreaks has been assumed to be local rodent populations. There is no treatment for the disease. CASE PRESENTATION: The clinical case describes an outbreak of encephalomyocarditis virus in a farrowing (farm A) and a weaning (farm B) site, with mortality that reached 24.2% in suckling piglets and 7.7% in weaners. The farms were located in an endemic Italian area, and the outbreak was characterised by high mortality with sudden death and clinical signs due to heart failure (trembling, dispnea and fever). The rodents control program was the key action in managing the outbreak. However, in the weaning site, the lack of rodent program in some unexplored areas of the barn (false ceiling) was responsible of a longer time of resolution of the outbreak. An unusual support treatment approach from human medicine suggestion was also applied using acetylsalicylic acid for its antiphlogistic and antithrombotic effects. CONCLUSIONS: To control the rodent population in a pig farm is often difficult and requires a deep knowledge also of the rodents habits. Considering the lack of treatment for the disease and the absence of available vaccines in several Countries, acetylsalicylic acid might be of interest for further studies as an important support for pigs' recovery.

Encephalomyocarditis Virus Abrogates the Interferon Beta Signaling Pathway via Its Structural Protein VP2.

Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-ß pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

Ablation of SigH+ pDCs in B6.Nba2 mice prevents lupus-like disease development only if started before disease is fully established.

Systemic lupus erythematosus is characterized by hyper-activation of the immune system, multi-organ inflammation, and end-organ damage. Type I interferons (IFN-I) have been strongly implicated a role in disease etiology as has the main IFN-I-producing cell subset, the plasmacytoid dendritic cell (pDC). The B6.Nba2 mouse model develops a lupus-like disease characterized by elevated IFN-I levels and pDC pathogenicity. We have previously shown that pDC ablation prior to disease development in B6.Nba2 mice effectively prevents disease; however, it remains unclear if a similar protection can be seen if pDC ablation is initiated during later disease stages. This is important as Systemic lupus erythematosus patients are rarely diagnosed until disease is well-established and thus preventative treatment is unlikely to take place. Here we show that ablation of pDCs in the B6.Nba2 mouse model must be initiated early in order to effectively block disease development and that sustained reduction in pDC numbers is necessary for sustained effects. Finally, targeting of pDCs have been hypothesized to affect immunity towards infectious agents, in particular virus and intracellular bacteria. We show here that pDC ablation in B6.Nba2 mice does not affect the anti-viral response to encephalomyocarditic virus or a model T-dependent antigen. In summary, pDC ablation does not affect general immunity, but needs to happen early and be sustained to prevent lupus-like disease development in B6.Nba2 mice.

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.

Caveolin-1 is involved in encephalomyocarditis virus replication in BHK-21 cells.

BACKGROUND: Encephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells. METHODS: The function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details. RESULTS: The results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection. CONCLUSIONS: Caveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.

Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells.

We are pursuing cancer immunotherapy with a neuro-attenuated recombinant poliovirus, PVSRIPO. PVSRIPO is the live attenuated type 1 (Sabin) poliovirus vaccine carrying a heterologous internal ribosomal entry site (IRES) of human rhinovirus type 2 (HRV2). Intratumoral infusion of PVSRIPO is showing promise in the therapy of recurrent WHO grade IV malignant glioma (glioblastoma), a notoriously treatment-refractory cancer with dismal prognosis. PVSRIPO exhibits profound cytotoxicity in infected neoplastic cells expressing the poliovirus receptor CD155. In addition, it elicits intriguing persistent translation and replication, giving rise to sustained type I interferon (IFN)-dominant proinflammatory stimulation of antigen-presenting cells. A key determinant of the inflammatory footprint generated by neoplastic cell infection and its role in shaping the adaptive response after PVSRIPO tumor infection is the virus's inherent relationship to the host's innate antiviral response. In this report, we define subversion of innate host immunity by PVSRIPO, enabling productive viral translation and cytopathogenicity with extremely low multiplicities of infection in the presence of an active innate antiviral IFN response.IMPORTANCE Engaging innate antiviral responses is considered key for instigating tumor-antigen-specific antitumor immunity with cancer immunotherapy approaches. However, they are a double-edged sword for attempts to enlist viruses in such approaches. In addition to their role in the transition from innate to adaptive immunity, innate antiviral IFN responses may intercept the viral life cycle in cancerous cells, prevent viral cytopathogenicity, and restrict viral spread. This has been shown to reduce overall antitumor efficacy of several proposed oncolytic virus prototypes, presumably by limiting direct cell killing and the ensuing inflammatory profile within the infected tumor. In this report, we outline how an unusual recalcitrance of polioviruses toward innate antiviral responses permits viral cytotoxicity and propagation in neoplastic cells, combined with engaging active innate antiviral IFN responses.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice.

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters. We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I-/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I-/- and wild-type mice. ATLANTIS was found in both wild-type and Reg I-/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I-/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I-/- mice did not differ from that of wild-type mice. These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 µL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.

Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKKε-IRF3 complex.

TRAF family member-associated NF-κB activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)-(IκB) kinase ε (IKKε) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN production in HEK293T cells. Mechanistically, EMCV 3C protease (EMCV 3C) cleaved TANK and disrupted the TANK-TBK1-IKKε-IRF3 complex, which resulted in the reduction in IRF3 phosphorylation and type I IFN production. Taken together, our findings demonstrate that EMCV adopts a novel strategy to evade host innate immune responses through cleavage of TANK.

E6AP/UBE3A catalyzes encephalomyocarditis virus 3C protease polyubiquitylation and promotes its concentration reduction in virus-infected cells.

The encephalomyocarditis virus (EMCV) 3C protease (3Cpro) is one of a small number of viral proteins whose concentration is known to be regulated by the cellular ubiquitin-proteasome system. Here we report that the ubiquitin-conjugating enzyme UbcH7/UBE2L3 and the ubiquitin-protein ligase E6AP/UBE3A are components of a previously unknown EMCV 3Cpro-polyubiquitylating pathway. Following the identification of UbcH7/UBE2L3 as a participant in 3Cpro ubiquitylation, we purified a UbcH7-dependent 3Cpro-ubiquitylating activity from mouse cells, which we identified as E6AP. In vitro reconstitution assays demonstrated that E6AP catalyzes the synthesis of 3Cpro-attached Lys48-linked ubiquitin chains, known to be recognized by the 26S proteasome. We found that the 3Cpro accumulates to higher levels in EMCV-infected E6AP knockdown cells than in control cells, indicating a role for E6AP in in vivo 3Cpro concentration regulation. We also discovered that ARIH1 functions with UbcH7 to catalyze EMCV 3Cpro monoubiquitylation, but this activity does not influence the in vivo 3Cpro concentration.

Transcriptional profiling of host cell responses to encephalomyocarditis virus (EMCV).

BACKGROUD: Encephalomyocarditis virus (EMCV) has been discovered on pig farms worldwide and can cause myocarditis in piglets and reproductive failure in sows. However, little is known about the host transcriptional responses to infection and host-pathogen interactions. METHODS: In this study, transcription profiling was performed by Illumina RNA-Sequencing (RNA-seq) to identify EMCV induced differentially expressed genes in BHK-21 cells at serial time points (12, 24, and 30 h post infection (hpi)), using mock infected cells as control. RESULTS: We identified 237, 241, and 207 differentially expressed genes (DEGs) respectively, majority of which were up-regulated. A large number of DEGs clustered into host defense, cellular signaling and metabolism categories. Moreover, short time series expression analysis revealed that 12 hpi was an important time point for expression change, indicating host virus resistance. CONCLUSIONS: This RNA-seq analysis provides the first data for understanding the network of virus host interactions under EMCV infection in vitro, and for identifying host components which involved in the virus infection course.

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.

Activation of type I interferon signaling in the parotid and exorbital lachrymal glands during the acute phase of encephalomyocarditis (EMC) virus infection in mice.

The present study was carried out to clarify the mechanisms of EMC virus-induced sialodacryoadenitis in mice during the acute phase infection focusing on the activation of type I interferon (IFN) signaling in the parotid and exorbital lachrymal glands. In the parotid gland, a few apoptotic acinar cells were detected at 2days post inoculation (DPI). The ratio of apoptotic acinar cells increased at 3 and 4DPI. On the other hand, in the exorbital lachrymal gland, apoptosis of acinar cells and infiltration of inflammatory cells mainly composed of mononuclear cells started at 3DPI, and prominent acinar cell damage developed at 4DPI. Viral RNA was detected at 3 and 4DPI in both glands and the expression level was higher in the exorbital lachrymal gland than in the parotid gland. The up-regulation of IFN-stimulated genes (ISGs), such as Irf7, Pkr and Oas, was quickly induced at 2DPI in the parotid gland, and this probably contributed to suppress viral replication and to eliminate affected cells by apoptosis. In the exorbital lachrymal gland, the expression levels of ISGs mRNAs were not elevated at 2DPI, suggesting no induction of an effective anti-viral response such as apoptosis at this time point. In the exorbital lachrymal gland, the mRNA expression of IFN beta and IFN alpha (type I IFNs) was weak- to strong-positive at 1DPI, and became negative at 2DPI. The weak- to strong-positive expression of IFNs at 1DPI is likely related to the abrupt viral replication and pathological changes in the exorbital lachrymal gland through activating the negative feedback regulation that depressed the IFN signaling cascade at 2DPI. In conclusion, the present study showed the changes in factors involved in the activation of type I IFN signaling cascade in the parotid and exorbital lachrymal glands and their differences between the two glands during the acute phase of EMC virus infection in mice.

A retrospective analysis of mortality in captive pygmy hippopotamus (Choeropsis liberiensis) from 1912 to 2014.

The pygmy hippopotamus (Choeropsis liberiensis) is an IUCN Red List Endangered species (CITES Appendix II) that has been housed in zoological collections since 1912. As wild populations continue to decline throughout the species' range, successful ex situ breeding and management, including an understanding of morbidity and mortality, are of utmost importance. This study is the first comprehensive review of mortality data from the captive population since 1982 and significantly expands on previous analyses. We solicited necropsy reports from 129/187 zoological institutions worldwide that currently or previously held pygmy hippos and received data for 404 animals (177 ♂, 220 ♀, 7 undermined sex), representing 43% of pygmy hippos that have died in captivity. Mortality in neonates was primarily due to perinatal causes (51.8%-stillbirth, failure to thrive, weakness, poor suckling reflex, maternal neglect) or parent-inflicted trauma (28%). Common causes of mortality in adult and geriatric animals included cardiovascular disease (16%), degenerative musculoskeletal conditions (10%), obstructive gastrointestinal disease (9%), and renal insufficiency (13%), sometimes associated with advanced polycystic kidney disease (PKD). Although not the direct cause of mortality, a number of adult and geriatric pygmy hippos were also overweight to obese. Infectious causes of mortality in included leptospirosis and encephalomyocarditis virus, the latter usually presenting as acute death due to cardiovascular demise. This comprehensive overview presents a useful guide for recommendations in preventative veterinary care and for improved husbandry and management of pygmy hippos in captivity. Zoo Biol. 35:556-569, 2016. © 2016 Wiley Periodicals, Inc.

Porcine encephalomyocarditis virus strain BD2 isolated from northern China is highly virulent for BALB/c mice.

Encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. The BD2 strain was isolated from the suspected piglets with EMCV in China. In order to establish an experimental animal model of EMCV, eight-weeks-old male BALB/c mice were intraperitonealy inoculated with 0.1 ml of 4×10(5) TCID50 suspension of the EMCV. Infected mice demonstrated hind limb paralysis, and movement disorder. The mortality rate of the infected group was 100% during the one-week observation period. The viral load in the brain of challenged mice gradually increased, with a peak level being 6.53 log CCID50/0.1 ml 5 days post infection. The pathological injury in infected mice was presented as neuronal necrosis. Brown positive staining could be detected in the cytoplasm of cerebral neurons. These results indicate that the porcine EMCV isolated from China could replicate in brain tissues and induce acute encephalitis in BALB/c mice.

The characteristics of synonymous codon usage in the initial and terminal translation regions of encephalomyocarditis virus.

The synonymous codon usage patterns in the initial and terminal translation regions (ITR, TTR) of the whole coding sequence of encephalomyocarditis virus (EMCV) were analyzed in relation to those in its natural hosts using the sequences accessible in databases. In general, some low-usage host codons were found over-represented in the ITR and TTR of the virus, while some high-usage host codons were found under-represented in the two viral regions. These relationships are thought to participate in the regulation of the speed of translation of viral proteins and in the suppression of ribosomal traffic jams, both aiming at the increase of virus yields.

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.

The complex interplay between encephalomyocarditis virus and the host defence system.

A variety of animals can be infected by encephalomyocarditis virus (EMCV). EMCV is the established causative agent of myocarditis and encephalitis in some animals. EMCV causes high fatality in suckling and weaning piglets, making pigs the most susceptible domestic animal species. Importantly, EMCV has zoonotic potential to infect the human population. The ability of the pathogen to avoid and undermine the initial defence mechanism of the host contributes to its virulence and pathogenicity. A large body of literature highlights the intricate strategies employed by EMCV to escape the innate immune machinery to suit its "pathogenic needs." Here, we also provide examples on how EMCV interacts with certain host proteins to dampen the infection process. Hence, this concise review aims to summarize these findings in a compendium of decades of research on this exciting yet underappreciated topic.