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.

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.

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.

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.

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 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.

First Occurrence of Saffold Virus in Sewage and River Water Samples in Karaj, Iran.

Saffold virus as a newly discovered virus, which seems to be related to acute gastroenteritis as with other enteric viruses and to human airway diseases in children belongs to Cardiovirus genus in picornaviridae family with 11 genotypes. Saffold virus initially was detected in America from infant stool sample. Saffold virus has also been detected in environmental water samples. Until now, two reports have demonstrated that sewage water sources are contaminated with Saffold viruses. Molecular detection of Saffold virus mostly depended on reverse transcription PCR methods and RT-qPCR, which had targeted 5'UTR region of the viral genome. The present study aims to evaluate the molecular detection and quantity of Saffold virus in sewage water and river water specimens by RT-qPCR assay in Karaj, Iran. Fifty samples collected from environmental waters containing treated and untreated sewage water and river water samples were included in this study. After viral RNA extraction, the Real-time PCR was developed to amplify the 5'UTR sequence of Saffold virus genome and viral load was assessed. Out of the 50 samples tested (consisting 28 river water samples and 22 sewage water samples), the Saffold virus genomic RNA was identified in 10/28 (35.7%) of river water samples and in 4/12 (33.3%) of treated and 4/10 (40%) of untreated sewage samples. The maximum viral load was 6.8 × 106 copies/l in untreated sewage water sample in December, and the lower viral load was 1.2 × 106 copies/l related to treated sewage water taken in October. Our results for the first time indicate that Saffold virus has apparently been circulating among Iranian peoples. Also, the viral prevalence of Saffold virus in each of the three sets of tested samples was within moderate to high in range.

Bacterial Stabilization of a Panel of Picornaviruses.

Several viruses encounter various bacterial species within the host and in the environment. Despite these close encounters, the effects of bacteria on picornaviruses are not completely understood. Previous work determined that poliovirus (PV), an enteric virus, has enhanced virion stability when exposed to bacteria or bacterial surface polysaccharides such as lipopolysaccharide. Virion stabilization by bacteria may be important for interhost transmission, since a mutant PV with reduced bacterial binding had a fecal-oral transmission defect in mice. Therefore, we investigated whether bacteria broadly enhance stability of picornaviruses from three different genera: Enterovirus (PV and coxsackievirus B3 [CVB3]), Kobuvirus (Aichi virus), and Cardiovirus (mengovirus). Furthermore, to delineate strain-specific effects, we examined two strains of CVB3 and a PV mutant with enhanced thermal stability. We determined that specific bacterial strains enhance thermal stability of PV and CVB3, while mengovirus and Aichi virus are stable at high temperatures in the absence of bacteria. Additionally, we determined that bacteria or lipopolysaccharide can stabilize PV, CVB3, Aichi virus, and mengovirus during exposure to bleach. These effects are likely mediated through direct interactions with bacteria, since viruses bound to bacteria in a pulldown assay. Overall, this work reveals shared and distinct effects of bacteria on a panel of picornaviruses.IMPORTANCE Recent studies have shown that bacteria promote infection and stabilization of poliovirus particles, but the breadth of these effects on other members of the Picornaviridae family is unknown. Here, we compared the effects of bacteria on four distinct members of the Picornaviridae family. We found that bacteria reduced inactivation of all of the viruses during bleach treatment, but not all viral strains were stabilized by bacteria during heat treatment. Overall, our data provide insight into how bacteria play differential roles in picornavirus stability.

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.

Recombination Located over 2A-2B Junction Ribosome Frameshifting Region of Saffold Cardiovirus.

Here we report the nearly full-length genome of a recombinant Saffold virus strain (SAFV-BR-193) isolated from a child with acute gastroenteritis. Evolutionary analysis performed using all available near-full length Saffold picornavirus genomes showed that the breakpoint found in the Brazilian strain (SAFV-BR-193) is indeed a recombination hotspot. Notably, this hotspot is located just one nucleotide after the ribosomal frameshift GGUUUUU motif in the SAFV genome. Empirical studies will be necessary to determine if this motif also affects the binding affinity of RNA-dependent RNA-polymerase (RdRp) and therefore increases the changes of RdRp swap between molecules during the synthesis of viral genomes.

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.

VPg unlinkase/TDP2 in cardiovirus infected cells: Re-localization and proteolytic cleavage.

Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5' end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for cardioviruses to regulate TDP2 activity during infection.

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.

The Language of Life.

Science is our best current approximation of the way things work. You cannot do science unless you believe there is a discernable truth inherent to the arrangement of our tangible world. The problem is, we in our given time never know where exactly the asymptote lies or how far we are from it. My curiosity about the natural world is innate, but fate has variously gifted me with outstanding personal opportunities to indulge that curiosity through the study of viruses. To a woman of the boomer generation, professional paths were not always open-door, and to a certain extent they still aren't. Whether such points should now be viewed as obstacles or stepping stones is a matter of perspective. RNA viruses, and the multiple, seminal mentors who taught me their secrets, have defined my career. Some of their stories are told here as they dovetail with mine. If there is any unity to this, it would be a pursuit of the language of life, or sequence analysis, as taught to us by natural selection. The intent here is not a legacy but an example. Science is a beautiful fate.

The Pathogenesis of Saffold Virus in AG129 Mice and the Effects of Its Truncated L Protein in the Central Nervous System.

Saffold Virus (SAFV) is a human cardiovirus that has been suggested to cause severe infection of the central nervous system (CNS). Compared to a similar virus, Theiler's murine encephalomyelitis virus (TMEV), SAFV has a truncated Leader (L) protein, a protein essential in the establishment of persistent CNS infections. In this study, we generated a chimeric SAFV by replacing the L protein of SAFV with that of TMEV. We then compared the replication in cell cultures and pathogenesis in a mouse model. We showed that both SAFV and chimeric SAFV are able to infect Vero and Neuro2a cells well, but only chimeric SAFV was able to infect RAW264.7. We then showed that mice lacking IFN-α/ß and IFN-γ receptors provide a good animal model for SAFV infection, and further identified the locality of the infection to the ventral horn of the spine and several locations in the brain. Lastly, we showed that neither SAFV nor chimeric SAFV causes persistence in this model. Overall, our results provide a strong basis on which the mechanisms underlying Saffold virus induced neuropathogenesis can be further studied and, hence, facilitating new information about its pathogenesis.

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.

Prevalence and genetic characteristics of Saffold cardiovirus in China from 2009 to 2012.

The epidemiology and clinical features of the Saffold cardiovirus (SAFV) remain ambiguous. The present study was designed to systematically and intensively investigate the epidemiological features of SAFV in pediatric patients in China. Three cohorts of pediatric patients were recruited from 2009 to 2012. Cohort 1 comprised patients with acute respiratory tract infections. Cohort 2 comprised patients with diarrhea. Cohort 3 comprised hand, foot, and mouth disease (HFMD) patients. A total of 115 patients (1.6%) among 6052 (17/1647, 12/2013, and 86/2392 in cohorts 1, 2, and 3, respectively) were SAFV-positive. The samples from 82 SAFV-positive patients were successfully sequenced, and four genotypes were identified: 8 SAFV-1, 41 SAFV-2, 29 SAFV-3, and 4 SAFV-6. A significantly higher detection rate was found in the HFMD patients than in other two cohorts (both P <0.001). A higher frequency of severe clinical outcome and nervous system manifestation were also observed in the SAFV-positive HFMD patients. Additionally, 6 (3.5%) cerebrospinal fluid and 7 (2.2%) serum samples from the HFMD-associated encephalitis patients were SAFV-positive. Based on the VP1 sequences, all four genotypes displayed distinct geographical clustering. SAFV infection might be associated with a wide clinical spectrum and contribute to HFMD.