The long-lasting enigma of polycytidine (polyC) tract.

Long polycytidine (polyC) tracts varying in length from 50 to 400 nucleotides were first described in the 5'-noncoding region (NCR) of genomes of picornaviruses belonging to the Cardio- and Aphthovirus genera over 50 years ago, but the molecular basis of their function is still unknown. Truncation or complete deletion of the polyC tracts in picornaviruses compromises virulence and pathogenicity but do not affect replicative fitness in vitro, suggesting a role as "viral security" RNA element. The evidence available suggests that the presence of a long polyC tract is required for replication in immune cells, which impacts viral distribution and targeting, and, consequently, pathogenic progression. Viral attenuation achieved by reduction of the polyC tract length has been successfully used for vaccine strategies. Further elucidation of the role of the polyC tract in viral replication cycle and its connection with replication in immune cells has the potential to expand the arsenal of tools in the fight against cancer in oncolytic virotherapy (OV). Here, we review the published data on the biological significance and mechanisms of action of the polyC tract in viral pathogenesis in Cardio- and Aphthoviruses.

Generation of a recombinant Saffold Virus expressing UnaG as a marker for the visualization of viral infection.

BACKGROUND: Saffold virus (SAFV), which belongs to the genus Cardiovirus of the family Picornaviridae, is associated with acute respiratory or gastrointestinal illnesses in children; it is also suspected to cause severe diseases, such as acute flaccid paralysis and aseptic meningitis. However, the understanding of the mechanism of its pathogenicity is still limited due to the many unknowns about its lifecycle; for example, the cellular receptor for its infection remains to be determined. A system to monitor SAFV infection in vitro and in vivo is required in order to accelerate research on SAFV. RESULTS: We generated a recombinant SAFV expressing green fluorescent protein (GFP) or UnaG, a novel fluorescent protein derived from Japanese eel. HeLa cells infected by either GFP or UnaG-expressing SAFV showed a bright green fluorescent signal, enabling convenient monitoring of SAFV infection. However, the expression of GFP but not UnaG was quickly lost during virus passaging due to the difference in genetic stability in the SAFV virus genome; the UnaG gene was stably maintained in the virus genome after at least five passages. CONCLUSIONS: SAFV infection of cultured cells can easily be monitored using UnaG-expressing SAFV, which is superior to GFP in terms of genetic stability in the virus genome. This virus could be a useful tool for SAFV research, such as comparing the susceptibility of various cells to SAFV infection and evaluating the effects of antivirals on SAFV infection in high-throughput screening.

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

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

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

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

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.

A novel cardiovirus in wild rats.

BACKGROUND: Cardioviruses cause severe illnesses in rodents and humans. In recent years, novel cardioviruses have been frequently found, which promoted further studies of the genetic diversity of cardioviruses. Using viral metagenomics, we genetically characterized a novel cardiovirus (named SX1) from wild rat feces. The genomic structure of SX1 shared similar features with those of the Theiler's murine encephalomyelitis viruses, including a leader protein, four structural proteins and seven non-structural proteins. Phylogenetic analysis based on both structural proteins and non-structural proteins coding regions showed that SX1 was formed into a separate branch, being located between the branches of Theiler's murine encephalomyelitis viruses and Thera viruses. Variable resides presented in the Ser/Thr rich domain of L protein, VP1 loops, and VP2 puffs distinguished SX1 from Theiler's murine encephalomyelitis viruses, suggesting the different antigenicity and pathogenicity of SX1.

Genetic diversity of circulating Saffold viruses in Pakistan and Afghanistan.

Human cardioviruses or Saffold viruses (SAFVs) of the family Picornaviridae are newly emerging viruses whose genetic and phenotypic diversity are poorly understood. We report here the full genome sequence of 11 SAFV genotypes from Pakistan and Afghanistan, along with a re-evaluation of their genetic diversity and recombination. We detected 88 SAFV from stool samples of 943 acute flaccid paralysis cases using reverse transcriptase-PCR targeting the 5' untranslated region (UTR). Further characterization based on complete VP1 analysis revealed 71 SAFVs belonging to 11 genotypes, including three previously unidentified genotypes. SAFV showed high genetic diversity and recombination based on phylogenetic, pairwise distance distributions and recombination mapping analyses performed herein. Phylogenies based on non-structural and UTRs were highly incongruent indicating frequent recombination events among SAFVs. We improved the SAFV genotyping classification criteria by determining new VP1 thresholds based on the principles used for the classification of enteroviruses. For genotype assignment, we propose a threshold of 23 and 10 % divergence for VP1 nucleotide and amino acid sequences, respectively. Other members of the species Theilovirus, such as Thera virus and Theiler's murine encephalomyelitis virus, are difficult to classify in the same species as SAFV, because they are genetically distinct from SAFV, with 41-56 % aa pairwise distances. The new genetic information obtained in this study will improve our understanding of the evolution and classification of SAFV.

Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome.

The genus Cardiovirus (family Picornaviridae) currently comprises the species Encephalomyocarditis virus (EMCV) and Theilovirus. Cardioviruses have a positive-sense, single-stranded RNA genome that encodes a large polyprotein (L-1ABCD-2ABC-3ABCD) that is cleaved to produce approximately 12 mature proteins. We report on a conserved ORF that overlaps the 2B-encoding sequence of EMCV in the +2 reading frame. The ORF is translated as a 128-129 amino acid transframe fusion (2B*) with the N-terminal 11-12 amino acids of 2B, via ribosomal frameshifting at a conserved GGUUUUY motif. Mutations that knock out expression of 2B* result in a small-plaque phenotype. Curiously, although theilovirus sequences lack a long ORF in the +2 frame at this genomic location, they maintain a conserved GGUUUUU motif just downstream of the 2A-2B junction, and a highly localized peak in conservation at polyprotein-frame synonymous sites suggests that theiloviruses also utilize frameshifting here, albeit into a very short +2-frame ORF. Unlike previous cases of programmed -1 frameshifting, here frameshifting is modulated by virus infection, thus suggesting a novel regulatory role for frameshifting in these viruses.

Saffold virus, a novel human Cardiovirus with unknown pathogenicity.

Although cardioviruses have been thought to mainly infect rodents, a novel human cardiovirus, designated Saffold virus (SAFV), was identified in 2007. SAFV is grouped with Theiler-like rat virus and Theiler's murine encephalomyelitis virus (TMEV) in the species Theilovirus of the genus Cardiovirus of the family Picornaviridae. Eight genotypes of SAFV have now been identified. SAFV has been isolated from nasal and stool specimens from infants presenting with respiratory and gastrointestinal symptoms as well as from children with nonpolio acute flaccid paralysis; however, the relationship of SAFV to this symptomatology remains unclear. Of note, the virus has also been isolated from the cerebrospinal fluid specimens of patients with aseptic meningitis. This finding is of interest since TMEV is known to cause a multiple sclerosis-like syndrome in mice. The involvement of SAFV in various diseases (e.g., respiratory illness, gastrointestinal illness, neurological diseases, and type I diabetes) is presently under investigation. In order to clarify the pathogenicity of SAFV, additional epidemiological studies are required. Furthermore, identification of the SAFV cellular receptor will help establish an animal model for SAFV infection and help clarify the pathogenesis of SAFV-related diseases. In addition, investigation of the tissue-specific expression of the receptor may facilitate development of a novel picornavirus vector, which could be a useful tool in gene therapy for humans. The study of viral factors involved in viral pathogenicity using a reverse genetics technique will also be important.

Serious invasive Saffold virus infections in children, 2009.

The first human virus in the genus Cardiovirus was described in 2007 and named Saffold virus (SAFV). Cardioviruses can cause severe infections of the myocardium and central nervous system in animals, but SAFV has not yet been convincingly associated with disease in humans. To study a possible association between SAFV and infections in the human central nervous system, we designed a real-time PCR for SAFV and tested cerebrospinal fluid (CSF) samples from children <4 years of age. SAFV was detected in 2 children: in the CSF and a fecal sample from 1 child with monosymptomatic ataxia caused by cerebellitis; and in the CSF, blood, and myocardium of another child who died suddenly with no history of illness. Virus from each child was sequenced and shown to be SAFV type 2. These findings demonstrate that SAFV can cause serious invasive infection in children.

Adaptation of Saffold virus 2 for high-titer growth in mammalian cells.

Saffold viruses (SAFV) are a recently discovered group of human Cardioviruses closely related to Theiler's murine encephalomyelitis viruses (TMEV). Unlike TMEV and encephalomyocarditis virus, each of which is monotypic, SAFV are genetically diverse and include at least eight genotypes. To date, only Saffold virus 3 (SAFV-3) has been grown efficiently in mammalian cells in vitro. Here, we report the successful adaptation of SAFV-2 for efficient growth in HeLa cells after 13 passages in the alpha/beta interferon-deficient human glial cell line U118 MG. Nine amino acid changes were found in the adapted virus, with single mutations in VP2, VP3, and 2B, while 6 mutations arose in VP1. Most capsid mutations were in surface loops. Analysis of SAFV-2 revealed virus growth and cytopathic effect only in human cell lines, with large plaques forming in HeLa cells, with minimal cell association, and without using sialic acid to enter cells. Despite the limited growth of SAFV-2 in rodent cells in vitro, BALB/c mice inoculated with SAFV-2 showed antibody titers of >1:10(6), and fluorescence-activated cell sorting (FACS) analysis revealed only minimal cross-reactivity with SFV-3. Intracerebral inoculation of 6-week-old FVB/n mice produced paralysis and acute neuropathological changes, including meningeal infiltrates, encephalitis, particularly of the limbic system, and spinal cord white matter inflammation.

Insights from structural studies of the cardiovirus 2A protein.

Cardioviruses are single-stranded RNA viruses of the family Picornaviridae. In addition to being the first example of internal ribosome entry site (IRES) utilization, cardioviruses also employ a series of alternative translation strategies, such as Stop-Go translation and programmed ribosome frameshifting. Here, we focus on cardiovirus 2A protein, which is not only a primary virulence factor, but also exerts crucial regulatory functions during translation, including activation of viral ribosome frameshifting and inhibition of host cap-dependent translation. Only recently, biochemical and structural studies have allowed us to close the gaps in our knowledge of how cardiovirus 2A is able to act in diverse translation-related processes as a novel RNA-binding protein. This review will summarize these findings, which ultimately may lead to the discovery of other RNA-mediated gene expression strategies across a broad range of RNA viruses.

A novel cardiovirus species identified in feces of wild Himalayan marmots.

Recently a growing number of novel cardioviruses have been frequently discovered, which boosts interest in the search for the genetic diversity of cardioviruses. However, wild-marmot cardioviruses have been rarely reported. Here, a novel cardiovirus (tentatively named HHMCDV) was identified in fecal samples from wild Himalayan marmots in Qinghai Tibetan Plateau, China, by viral metagenomics analysis. 3 out of 99 fecal samples from Himalayan marmots were positive for HHMCDV, with the viral loads ranging from 2.7 × 105 to 1.3 × 107 gene copies/g. The complete genomic sequence of HHMCDV was 8108 nucleotides in length, with the typical cardiovirus genome organization and motifs. Coincidentally, while the data was analyzing, one marmot cardiovirus HT7 partial sequence was available in the Genbank, showing 95.1%, 95.6% and 96.0% amino acid (aa) identity in P1, P2 and P3, respectively. However, sequence analysis revealed that HHMCDV and HT7 are more closely related to species Cardiovirus F strain with 65.7%, 61.9-65.6%, 58.9-59.7%, 71.1-71.7%, 69.1-69.4% and 71.4-72.2% aa identity in polyprotein, P1, P2, P3, 2C and 3CD proteins, respectively. Phylogenetic analysis of P1, P2, P3 and 3CD aa sequences indicated that HHMCDV and HT7 clustered tightly and formed a distinct cluster in the Cardiovirus genus. Based on these data, we propose that HHMCDV and HT7 should be two different members of a potential novel species within the genus Cardiovirus. Further studies are needed to investigate the epidemiology and potential pathogenicity of the virus in Himalayan marmots.

Human cardioviruses, meningitis, and sudden infant death syndrome in children.

Cardioviruses cause myocarditis and encephalomyelitis in rodents; human cardioviruses have not been ascribed to any disease. We screened 6,854 cerebrospinal fluid and 10 myocardium specimens from children and adults. A genotype 2 cardiovirus was detected from a child who died of sudden infant death syndrome, and 2 untypeable cardioviruses were detected from 2 children with meningitis.

Cultivation and serological characterization of a human Theiler's-like cardiovirus associated with diarrheal disease.

Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.

Saffold virus, a human Theiler's-like cardiovirus, is ubiquitous and causes infection early in life.

The family Picornaviridae contains well-known human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and parechovirus). In addition, this family contains a number of viruses that infect animals, including members of the genus Cardiovirus such as Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelits virus (TMEV). The latter are important murine pathogens that cause myocarditis, type 1 diabetes and chronic inflammation in the brains, mimicking multiple sclerosis. Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). The virus is genetically related to Theiler's virus and classified as a new species in the genus Cardiovirus, which until the discovery of SAFV did not contain human viruses. By analogy with the rodent cardioviruses, SAFV may be a relevant new human pathogen. Thus far, SAFVs have sporadically been detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance remained unclear. Here we describe the first cultivated SAFV type 3 (SAFV-3) isolate, its growth characteristics, full-length sequence, and epidemiology. Unlike the previously isolated SAFV-1 and -2 viruses, SAFV-3 showed efficient growth in several cell lines with a clear cytopathic effect. The latter allowed us to conduct a large-scale serological survey by a virus-neutralization assay. This survey showed that infection by SAFV-3 occurs early in life (>75% positive at 24 months) and that the seroprevalence reaches >90% in older children and adults. Neutralizing antibodies were found in serum samples collected in several countries in Europe, Africa, and Asia. In conclusion, this study describes the first cultivated SAFV-3 isolate, its full-length sequence, and epidemiology. SAFV-3 is a highly common and widespread human virus causing infection in early childhood. This finding has important implications for understanding the impact of these ubiquitous viruses and their possible role in acute and/or chronic disease.

The preparation of an infectious full-length cDNA clone of Saffold virus.

The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

Persistent Detection of Cosavirus and Saffold Cardiovirus in Riachuelo River, Argentina.

Cosaviruses (CoSV) and Saffold cardiovirus (SAFV) are novel members of the Picornaviridae family. The Matanza-Riachuelo river basin covers a total area of 2200 km2 with approximately 60 km long. Its last section is called Riachuelo River. The aim of this study was to describe the circulation of both picornaviruses and their relationship with the environmental situation of the Riachuelo River using 274 samples collected from 2005 to 2015. CoSV and SAFV were investigated in samples available by two periods: 2005-2006 and 2014-2015 (103 and 101, respectively). Physicochemical and bacteriological parameters confirmed very high levels of human fecal contamination during the 11 years evaluated. CoSV was detected in 85.7% (66/77) and 65.4% (17/26) of the samples collected in 2005-2006 and 2014-2015 periods, respectively. Species A and D were identified, the first one being widely predominant: 74.1% (20/27) and 75.0% (3/4) in both periods. SAFV virus was detected in 47.1% (32/68) and 52.6% (10/19) in periods 2005-2006 and 2014-2015, respectively. SAFV-6 was the most identified genotype in the entire study, while SAFV-3 was predominant in 2005-2006. The contribution of genotypes 1, 2, 4 and 8 was minor. The high prevalence of CoSV and SAFV suggests that both viruses have been circulating in Argentina at least since 2005. Our results show that a watercourse with high rates of human fecal contamination can become a persistent source of new viruses which capacity to produce human diseases is unknown.

Saffold cardioviruses of 3 lineages in children with respiratory tract infections, Beijing, China.

To clarify the potential for respiratory transmission of Saffold cardiovirus (SAFV) and characterize the pathogen, we analyzed respiratory specimens from 1,558 pediatric patients in Beijing. We detected SAFV in 7 (0.5%) patients and identified lineages 1-3. However, because 3 patients had co-infections, we could not definitively say SAFV caused disease.

Genomic features and evolutionary constraints in Saffold-like cardioviruses.

This study identified the complete genomic sequence of four type 2 and type 3 human Saffold-like cardioviruses (SLCVs) isolated in Germany and Brazil. The secondary structures of the SLCV internal ribosome entry sites (IRESs) were deduced based on RNA base-pairing conservation and co-variation, using an established Theiler's murine encephalomyelitis virus (TMEV) IRES structure as a reference. The SLCV IRES was highly similar to that of TMEV, but motifs critical in TMEV for binding of the polypyrimidine tract-binding protein (PTB) were disrupted. In TMEV, corresponding alterations have been associated with reduced neurovirulence in mice. In the non-structural genome region, there was evidence of multiple intertypic recombination events between different SLCV types. Between viruses of the same type, recombination also occurred in the capsid-encoding genome region. There were apparently no recombination events between mouse TMEV and human SLCV. In another genus of the family Picornaviridae, Enterovirus, natural recombination occurs strictly within species and can serve as an additional criterion for delimiting species. Accordingly, the results of this study suggest that SLCV and TMEV may represent distinct species within the genus Cardiovirus.