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.

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.

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.

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.

Saffold Cardiovirus Infection in a 2-Year-Old Boy with Acute Pancreatitis.

Saffold cardiovirus (SAFV), first identified in a stool sample in 2007, is thought to be associated with respiratory disease and gastroenteritis. On the other hand, animal experiments suggested that the major viral load, following intraperitoneal inoculation of SAFV in mice, may be detected in the pancreas. However, until now, no cases of SAFV in patients with pancreatitis have been reported. This report presents a unique case in a patient who developed relapsing acute pancreatitis (AP) after hand, foot, and mouth disease, and was suspected to have SAFV-1 infection. A 2-year-old boy was admitted to the hospital because of severe abdominal pain. His serum amylase and lipase levels were elevated. Enhanced computed tomography showed pancreatic swelling and dilation of the main pancreatic duct, leading to a diagnosis of severe AP. The viral genome of SAFV-1 was detected by reverse transcription polymerase chain reaction from fecal samples. Furthermore, the serum neutralization titer for SAFV was elevated during AP, but decreased after 1 year. These findings strongly suggest the patient developed SAFV-1 infection concurrent with AP. Therefore, we propose that a cohort study is required to clarify the relationship between SAFV and AP.

Saffold virus infection associated with human myocarditis.

BACKGROUND: Saffold virus was described in 2007 as one of the first human viruses within the genus cardioviruses. Cardioviruses may cause severe infections of the myocardium in animals, and several studies have associated saffold virus with human disease. As a result, saffold virus has been isolated from different anatomical compartments, including the myocardium, but, until now, it has not been possible to demonstrate the accompanying histopathological signs of inflammation. OBJECTIVES: The aim of the study was to examine if saffold virus is capable of causing invasive infection in the human myocardium. STUDY DESIGN: Using real-time PCR, we retrospectively examined formalin-fixed paraffin embedded cardiac tissue specimens from 150 deceased individuals diagnosed with myocarditis at autopsy. The results were compared with histological findings. RESULTS AND CONCLUSIONS: Saffold virus was detected in the myocardium, lung tissue and blood of one child and was accompanied by histopathological inflammation in the heart and lungs, which was supportive of a viral infection. These findings suggest that cardioviruses may be associated with myocarditis in humans.

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.

Rapid detection of encephalomyocarditis virus by one-step reverse transcription loop-mediated isothermal amplification method.

The encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect EMCV RNA. The RT-LAMP assay was highly sensitive and able to detect 2.2 × 10(-5)ng of EMCV RNA, as no cross-reaction was observed with other viruses. The RT-LAMP assay was conducted in isothermal (62 °C) conditions within 50 min. The amplified products of EMCV could be detected as ladder-like bands using agarose gel electrophoresis. This is the first report to demonstrate the application of a one-step RT-LAMP assay for the detection of EMCV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in clinical diagnosis and field surveillance of EMCV.

A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets.

A real-time polymerase chain reaction with SYBR Green was developed for the detection and quantification of encephalomyocarditis virus (EMCV) in porcine tissues; the method uses two primers specific for the 3D gene. The detection limit of this assay was 22 gene copies/reaction, equivalent to 0.001 TCID(50)/ml. The assay was linear over a 10(7) dilution range of template concentrations and was specific for EMCV; it did not amplify other porcine pathogens (porcine circovirus 2, porcine reproductive and respiratory virus, classical swine fever virus, pseudorabies virus, or porcine teschovirus). This assay detected EMCV titers at least 10(4) smaller than the routine PCR assay. To increase our understand of EMCV pathogenesis, the new method was used to quantify levels of EMCV genome in various tissues of artificially challenged sows and piglets. The virus was found mainly in the heart, lung, spleen, kidney, and endometrium of sows, and mainly in the heart, spleen, lung, and testis of fetuses. The real-time PCR method described here should be useful for the study of EMCV infection and distribution in pigs.

Social stress alters the severity and onset of the chronic phase of Theiler's virus infection.

Social stress alters the acute phase of Theiler's virus infection (TMEV), a model of multiple sclerosis. Stress applied prior to infection had deleterious disease outcomes, while stress applied concurrent with infection was protective. The current study examined multiple behavioral (motor impairment, open field activity) and immunological measures (IL-6, antibodies to virus and myelin proteins) in both the acute and chronic phases of TMEV. It was found that stress applied prior to infection exacerbated disease outcomes, while concurrent application was protective in both disease phases.

Estimation of seroprevalence of encephalomyocarditis in Dutch sow herds using the virus neutralization test.

Encephalomyocarditis virus (EMCV) has been found on pig farms worldwide and can cause myocarditis in young pigs and reproduction disorders in sows. So far, clinical signs of EMCV have not been reported in the Netherlands. The aim of this study was to estimate the seroprevalence of EMCV infection in Dutch sow herds. A total of 277 Dutch sow herds were randomly selected, from which 3237 serum samples were collected. These samples were tested for EMCV antibodies using the virus neutralization test (VN test). The apparent prevalence of EMCV antibodies was 9.3% in the total sow population, and the apparent herd prevalence was 58.8%. An exact determination of the prevalence of EMCV infections in the Dutch sow population was not possible because the characteristics of the VN test under field circumstances were not known. However, Dutch sow herds seem to be infected with EMCV because the distribution of positive blood samples in the tested sow population was significantly different from that expected if random false-positive reactions had occurred.

Dynamics of MRI lesion development in an animal model of viral-induced acute progressive CNS demyelination.

Theiler's murine encephalitis virus (TMEV) infection in mice is an established model of CNS demyelinating diseases. The aim of the study was to determine the chronological pattern of lesion development in this model of monophasic fulminant demyelinating disease. We followed six highly susceptible interferon-gamma receptor knockout mice with serial in vivo brain magnetic resonance imaging (MRI) studies to determine changes in overall T2 lesion load and gadolinium enhancement. Altogether, 163 individual lesions were followed over 52 days. The number of lesions increased linearly with time. Four chronological patterns of lesion development were seen: (a) expanding lesions (48.5% of all lesions, 54.05% volume contribution); (b) expanding-retracting lesions (20.85% of all lesions, 15.03% volume contribution); (c) fluctuating lesions (16.6% of all lesions, 28.8% volume contribution); (d) stable lesions (14.05% of all lesions, 2.12% volume contribution). Gadolinium enhancement was not seen in the evolution of every lesion. Enhancement was both time- and lesion type-dependent. Early in the disease course (<43 days after infection), enhancement was almost always seen, later on (>43 days after infection) it was only seen in 8% of new lesions. All of fluctuating, 85.3% of expanding, 83.5% of expanding-retracting, and 56.5% of stable lesions were associated with gadolinium enhancement. We conclude that the MRI features of TMEV-induced demyelination in this model showed four unique chronological patterns, and inconsistent gadolinium enhancement. These novel findings may provide new insights into the pathogenesis of acute fulminant multiple sclerosis (MS).

Therapeutic action of cannabinoids in a murine model of multiple sclerosis.

Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theiler's murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212-2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as MS; in particular, the possible involvement of cannabinoid receptor CB2 would enable nonpsychoactive therapy suitable for long-term use.

Detection of encephalomyocarditis virus in clinical samples by immunomagnetic separation and one-step RT-PCR.

A method of immunomagnetic separation and one-step reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of Encephalomyocarditis virus (EMCV). EMCV was captured from sample on magnetic beads with homologous monoclonal antibody and then heat denatured. The heated beads were used directly in one-step RT-PCR reaction to amplify a 285-bp PCR fragment at the 3' end of the genomic region that encodes the viral polymerase. This method detected as little as 3.5 TCID(50) of EMCV from infected cell culture. It was shown with this method that the sensitivity of RT-PCR increased when applied for the detection of EMCV added to fecal extract. Using this protocol EMCV was detected from heart homogenate samples containing less than 100 TCID(50)/ml. The amplified product was sequenced to ensure specificity. The immunomagnetic-RT/PCR procedure described here should be useful for the rapid, specific and sensitive detection of EMCV in clinical samples. This technique is rapid, reliable and can be readily adapted to detect EMCV from other clinical samples.

Rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction.

Encephalomyocarditis virus (EMCV) is widespread and the economic losses caused by an EMCV outbreak in pig holdings and the similarity between a foot-and-mouth disease virus (FMDV) and an EMCV infection in young piglets stress the need for a rapid, specific and broad diagnostic assay. An alternative to the time-consuming seroneutralisation assay, currently used for the characterisation of EMCV, is described. An EMCV specific reverse transcription-polymerase chain reaction (RT-PCR), using primers located in a conserved region of the 3D gene of the viral genome, was developed and tested on 114 different EMCV isolates. The identity of the respective amplicons was confirmed by sequencing. The potential of this assay for future diagnostic purposes was demonstrated by applying the RT-PCR on tissue samples collected from an experimentally infected piglet.