Efficacy of sample collection without virus transport medium in suspected enteroviral infections for molecular diagnosis.

Clinical swabs with suspected viral infection are usually transported in virus transport medium (VMT). During epidemics/pandemics, tampons without VTM would be more suitable for saving space and cost. This study was conducted to verify the applicability of throat swabs without VTM in the diagnosis/screening of enteroviral infections by polymerase chain reaction (PCR) in a volunteer study group. Three different swab types were used in 40 volunteers: swabs with two different tips (cotton- or synthetic-tipped) without VTM and standard synthetic tips with VTM. The swabs were processed immediately or after 12 days of storage at either -80°C or +4°C. The molecular analysis included viral RNA extraction, and combination of reverse transcriptase PCR and nested PCR. Enteroviral RNA was detected in 15% (6/40) of the studied volunteers. When processed immediately, the results for all three swab types were compatible. Swabs without VTM may be used for collection of clinical samples in the diagnosis of suspected enteroviral infections or as potential screening tools for enteroviruses (Tab. 2, Ref. 15). Keywords: enterovirus infection, swab, transport medium, PCR, molecular diagnostics.

Should Enteroviruses Be Monitored in Natural Recreational Waters?

Enteroviruses (EVs) infections occur worldwide. Although, infections by these viruses are often asymptomatic and go unnoticed, they can be shed in stool for several weeks. The EVs are associated with sporadic outbreaks and a wide range of clinical symptoms, occasionally accompanied with fatal consequences. Presently in the Slovak Republic (SR) recreational waters are tested only for bacterial indicators. Our aim was to monitor EVs in recreational waters. Water samples were collected during the years 2012-2014 from different recreational natural lakes in Central and West regions of SR. The samples were concentrated by centrifugation using the two-phase separation method recommended by the World Health Organization (WHO) used for EVs surveillance in the treated sewage waste water. Each of the two phases collected from the samples was analysed by polymerase chain reaction for detection of EVs and primary sequencing was done. Our study demonstrated presence of EVs in three localities consecutively for three years, indicating a probability of constant local source of faecal contamination. This is the first monitoring report on the occurrence of EVs in the natural recreational waters in SR.

Pathophysiology of the pancreas after oral infection of genetically diverse mice with coxsackievirus B4-E2.

Coxsackievirus B4 strain E2 (CVB4-E2) and its association with type 1 diabetes (T1D) have been studied in experimental in vitro and in vivo murine models. CVB4-E2, known to be pancreotropic and diabetogenic in nature, is associated with acute pancreatitis in mice but shows differences in the induction of glycemia after intraperitoneal (i.p.) infection. Therefore, the aim of this work was to study the outcome of oral infection with CVB4-E2 in five mouse strains with different genetic backgrounds: two outbred (Swiss albino, CD1), two inbred (SJL, NOD) and one transgenic (NOD.SCID). Survival rates, fasting blood glucose, histopathology, viral titres and persistence were studied in selected organs and stool samples. Viral protein (VP1), proinflammatory cytokines, and interferon alpha (IFN-α) were analyzed by immunohistochemistry. We observed mortality only in infected NOD and NOD.SCID mice, with differing survival rates implying initial innate protection in the NOD.SCID mice and low virus clearance with replicating virus titres in the studied organs and stool up to day 40 post infection (p.i.). Independent of the mouse strain hyperglycemia, proinflammatory cytokines and histopathological changes were absent in the endocrine pancreas of infected mice. Only the pancreata of the dead NOD.SCID mice showed inflammation even in presence of IFN-α. Host-dependent viral RNA persistence was observed in all outbred mice. In conclusion, oral infection with CVB4-E2, despite the known affinity of this strain towards the pancreatic tissue and the presence of replicating virus, conferred total protection to the endocrine pancreas in all mice and failed to induce the proinflammatory cytokines studied by us.

History of the Wastewater Assessment of Polio and Non-Polio Enteroviruses in the Slovak Republic in 1963-2019.

We describe the genesis of poliovirus (PV) and non-polio enterovirus (NPEV) surveillance program of sewage wastewaters from its inception to the present in the Slovak Republic (SR). Sampling procedures and evolution of the methodology used in the SR for the detection of PVs and NPEVs are presented chronologically. For statistical data processing, we divided our dataset into two periods, the first period from 1963 to 1998 (35 years), and the second period from 1999 to 2019 (21 years). Generalized additive models were used to assess temporal trends in the probability of occurrence of major EV serotypes during both periods. Canonical correspondence analysis on relative abundance data was used to test temporal changes in the composition of virus assemblages over the second period. The probability of occurrence of major viruses PV, coxsackieviruses (CVA, CVB), and Echoviruses (E)) significantly changed over time. We found that 1015 isolated PVs were of vaccine origin, called "Sabin-like" (isolates PV1, PV2, PV3). The composition of EV assemblages changed significantly during the second period. We conclude that during the whole period, CVB5, CVB4, and E3 were prominent NPEVS in the SR.

Surveillance Program of Clinical Samples for Polio and Non-Polio Enteroviruses in the Slovak Republic during the 1958-2020 Period.

Enteroviruses (EVs) are associated with a wide spectrum of diseases involving various organs. Our aim was to give a historical overview of the genesis of clinical sample processing for EVs in the Slovak Republic (SR) during the 1958-2020 period, within the framework of the World Health Organization (WHO) polio program. Further, analyses were made of the data obtained from the archives of processed clinical sample surveillance using statistical methods. We used generalized additive models (GAM) with binomial distribution and logit link functions and an autoregressive moving average (ARMA) to analyze the data obtained during this 63-year period. Our results show trends in the composition of EV strains circulating in the population. Furthermore, statistically significant increasing trends of the non-polio enteroviruses (NPEVs) were observed over the studied time, represented by echoviruses (E) and coxsackieviruses A and B (CVA and CVB), with a cyclical pattern of occurrence. The most prevalent serotype over this period was CVB5, which became significantly more prevalent after 2000. While PVs, CVB1, and CVB3 were present in the second half of the studied period, CVA10, CVA16, E3, E25, and E30 appeared more frequently.

A single coxsackievirus B2 capsid residue controls cytolysis and apoptosis in rhabdomyosarcoma cells.

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.

Primary Site of Coxsackievirus B Replication in the Small Intestines: No Proof of Peyer's Patches Involvement.

BACKGROUND: Enterovirus (EV) infections are associated with a broad range of diseases. Since the first experimental infection of primates with poliovirus (PV), tonsils and the Peyer's patches (PPs) have been believed to be the primary replication sites of EVs. Our aim was to localize different viral markers in the small intestines (SI) of coxsackievirus B (CVB) orally and intraperitoneally (i.p.) infected mice. METHODS: Transverse sections of SIs of both infected and control male outbred mice were collected at different intervals post-infection (p.i) and analyzed for presence of interferon-alpha (IFN-α) and viral protein VP1 by immunohistochemistry and in situ hybridization (ISH). Fluorescent marker, eGFP, was identified in cryosections of mice infected with eGFP-CVB3. RESULTS: In the infected SIs, we observed enlarged germinating centers (GCs) in the PPs; IFN-α was detected in the PPs and mucosal layer of the SIs. However, VP1, viral RNA and the eGFP were absent in the GCs of PPs at all stages of infection irrespective of the virus strains used. CONCLUSIONS: Virus was present in the epithelial cells but not in GCs of the PPs of the murine SIs. Our results do not support the hypothesis of EV replication in the PP especially in the GCs.

Coxsackievirus B4 sewage-isolate induces pancreatitis after oral infection of mice.

Numerous serotypes which belong to the genus Enterovirus (EV) show variability in their virulence and clinical manifestations. They are also known to undergo changes caused by mutations and recombination during their circulation in the environment and the population. Various EV serotypes are prevalent in groundwater, wastewater and surface waters. Our previous studies showed that oral infection induces pancreatitis depending on specific conditions, such as gravidity, in an outbred murine model. Our aim in the present study was to further explore the pancreatic histopathology in an outbred mouse model following oral infection with clinical isolates from a patient who had aseptic meningitis and an isolate from a treated-sewage sample recovered from the residential area of the patient. The isolates were identified as coxsackievirus B4 (CVB4) in tissue culture. The CVB4 sewage-isolate induced pancreatitis after oral infection. In contrast, pancreatitis was absent following infection with the clinical isolates. Comparison of polyprotein sequences showed that the treated-sewage strains differed from the patient's isolates by 9 and 11 amino acids. We conclude that the isolates of clinical and environmental origin differed in their pathogenic properties and showed genetic variation.

Pancreas of coxsackievirus-infected dams and their challenged pups: A complex issue.

Enteroviral infections are frequent, often asymptomatic in humans and during gravidity. The present study is an extension of our previous investigations where we had shown pancreatitis in challenged pups of CVB4-E2-infected dams. Present investigation describes the effect of gestational infection with this virus on the pancreas of both dams and their challenged pups. Gravid CD1 outbred mice were orally infected with CVB4-E2 virus at different gestation times. Pups were challenged orally with the same virus after 25 days of birth. Organs were collected at selected intervals postinfection (p.i.), and replicating virus and viral-RNA copies were analyzed. Additional readouts included histopathology and immunohistochemical (IHC) analysis for localization and identification of Ly6G+ cells (neutrophils), CD11b+ cells (macrophages), and viral protein in pancreatic tissue sections of the infected dams and their challenged pups. Our results show the presence of replicating virus in the pancreas of infected dams and their challenged pups, with inflammation leading to chronic necrotizing pancreatitis and atrophy of pancreatic acini of the dams and their offspring. IHC analysis of the infiltrating cells showed pronounced Ly6G+ neutrophils in dams only, whereas CD11b+ macrophages were present in tissues of both, the pups and the dams. Time of infection during gravidity as well as the p.i. intervals when mice were sacrificed influenced the pancreatic pathophysiology in both groups. We conclude that coxsackievirus infection during pregnancy is a risk factor for chronic affliction of the exocrine tissue and could affect endocrine pancreas in the mother and child.

Assessment of a swab collection method without virus transport medium for PCR diagnosis of coxsackievirus infections.

Classically, detection of human enterovirus (EV) infections is based on virus isolation in tissue culture, proper sample collection and handling that optimizes virus viability. Samples are collected in virus transport medium (VTM) to ensure virus stability. High sensitivity and rapid results have made polymerase chain reaction (PCR) analysis increasingly popular for routine diagnosis. The PCR method enables simple sample collection and storage for EV diagnostics, which may eventually allow self-sampling at home. Our aim was to test a modification of the conventional clinical swab sample collection method for molecular diagnosis of EV infection. We compared swabs (cotton or synthetic) without VTM and the classical standard synthetic swabs with VTM. Effects of storage temperature (+4 °C or -80 °C) and duration were studied. EV-RNA could be detected by reverse transcriptase and nested PCR in both swab types without VTM. Differences depended on the storage duration and temperature. Optimum conditions were immediate processing or storage at -80 °C. Storage without VTM at +4 °C for longer periods is not advisable. We conclude that swabs without VTM can be considered for clinical EV-diagnostics based on PCR, and ultimately for epidemiological sample collection.

Biological activity of sophorolipids and their possible use as antiviral agents.

Sophorolipids are surface active glycolipids consisting of a hydrophilic sophorose unit and a hydrophobic portion composed of a fatty acid tail. Crude sophorolipid sample contains both acidic and lactonic forms of sophorolipid with different degrees of acetylation and varying lengths of the fatty acid chains depending on the substrates used in the production process. Carboxylic end in the acidic form of the fatty acid is free, whereas in the lactonic form, it is internally esterified. Sophorolipids show different physicochemical properties with wide range of applications for each structural compound. Lactonic form of sophorolipids shows surface tension reducing ability and biological activity, whereas the acidic form possesses better foam forming ability and higher solubility. Presence of acetyl groups gives hydrophilic nature to the sophorolipids which promotes its antiviral and cytokine-stimulating properties. The aim of this review is to explore and suggest the plausibility of sophorolipids as therapeutic and prophylactic agents for the treatment of viral diseases.

Type I diabetes mellitus: genetic factors and presumptive enteroviral etiology or protection.

We review type 1 diabetes and host genetic components, as well as epigenetics and viruses associated with type 1 diabetes, with added emphasis on the enteroviruses, which are often associated with triggering the disease. Genus Enterovirus is classified into twelve species of which seven (Enterovirus A, Enterovirus B, Enterovirus C, and Enterovirus D and Rhinovirus A, Rhinovirus B, and Rhinovirus C) are human pathogens. These viruses are transmitted mainly by the fecal-oral route; they may also spread via the nasopharyngeal route. Enterovirus infections are highly prevalent, but these infections are usually subclinical or cause a mild flu-like illness. However, infections caused by enteroviruses can sometimes be serious, with manifestations of meningoencephalitis, paralysis, myocarditis, and in neonates a fulminant sepsis-like syndrome. These viruses are often implicated in chronic (inflammatory) diseases as chronic myocarditis, chronic pancreatitis, and type 1 diabetes. In this review we discuss the currently suggested mechanisms involved in the viral induction of type 1 diabetes. We recapitulate current basic knowledge and definitions.

Preterm prelabor rupture of membranes (PPROM) is not associated with presence of viral genomes in the amniotic fluid.

BACKGROUND: The role of viral infections in preterm prelabor rupture of the membranes (PPROM) is not established. Studies on the presence of viral genomes in the amniotic fluid (AF) collected in pregnancies complicated by PPROM show contradictory outcomes. OBJECTIVES: To investigate AF samples of PPROM pregnancies for the presence of viral genomes. STUDY DESIGN: AF samples from patients with PPROM were collected during a 4-year (2008-2012) observational study. 174 women were included with selection criteria of singleton pregnancy, PPROM, and maternal age of 18 years and above. PCR was used for detection of human cytomegalovirus (HCMV), herpes simplex virus (HSV), parvovirus B19, human adenoviruses (HAdV), enteroviruses (EV) and human parechovirus (HPeV). The selection of these viral targets was based on literature regarding screening of AF for presence of viral genomes. RESULTS: Only a single sample was positive out of the 174 tested AFs, HCMV DNA was detected. CONCLUSIONS: PPROM is not associated with active viral infections.

Outcome of challenge with Coxsackievirus B4 in young mice after maternal infection with the same virus during gestation.

Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested.

Coxsackie B virus infection of mice: inoculation by the oral route protects the pancreas from damage, but not from infection.

The pathogenesis of coxsackie B virus (CVB) infections is generally studied in mice by intraperitoneal (i.p.) injection, whereas the gastrointestinal tract is the natural porte d'entrée in humans. The present study was undertaken to compare systematically the influence of infection route on morbidity and pathology. Swiss Albino mice were infected with CVB3 (Nancy) at different doses (5 x 10(3), 5 x 10(5), 5 x 10(7), 5 x 10(9) TCID50), given either i.p. or orally. Virus could be isolated from several organs (heart, spleen and pancreas), indicating systemic infection, irrespective of the infection route. Virus titres were 1-2 logs higher after i.p. infection, but kinetics were largely independent of infection route. Organs became negative for virus isolation after 21 days, with the exception of spleen tissue, which remained positive for up to 49 days. Thereafter, virus was detected only by immunohistochemistry and PCR up to 98 days post-infection (oral route). Histopathology showed mild inflammation and necrosis in heart tissue of all mice during the acute phase, with repair at later stages. Strikingly, pancreatic lesions were confined to the exocrine pancreas and observed only after i.p. infection. Under all experimental conditions, the pancreatic islets were spared. In contrast, immunohistochemistry showed the presence of viral VP1, protein 3A and alpha interferon (IFN-alpha) in exocrine as well as endocrine pancreas of all mice, irrespective of route and dose of infection. It is concluded that infection via the oral route protects the pancreas from damage, but not from infection, a process in which IFN-alpha is not the only factor involved.

Coxsackievirus B3 infection and type 1 diabetes development in NOD mice: insulitis determines susceptibility of pancreatic islets to virus infection.

Group B coxsackieviruses (CVB) are believed to trigger some cases of human type 1 diabetes (T1D), although the mechanism by which this may occur has not been shown. We demonstrated previously that inoculation of young nonobese diabetic (NOD) mice with any of several different CVB strains reduced T1D incidence. We also observed no evidence of CVB replication within islets of young NOD mice, suggesting no role for CVB in T1D induction in the NOD mouse model. The failure to observe CVB replication within islets of young NOD mice has been proposed to be due to interferon expression by insulin-producing beta cells or lack of expression of the CVB receptor CAR. We found that CAR protein is detectable within islets of young and older NOD mice and that a CVB3 strain, which expresses murine IL-4, can replicate in islets. Mice inoculated with the IL-4 expressing CVB3 chimeric strain were better protected from T1D onset than were mock-infected control mice despite intraislet viral replication. Having demonstrated that CVB can replicate in healthy islets of young NOD mice when the intraislet environment is suitably altered, we asked whether islets in old prediabetic mice were resistant to CVB infection. Unlike young mice in which insulitis is not yet apparent, older NOD mice demonstrate severe insulitis in all islets. Inoculating older prediabetic mice with different pathogenic CVB strains caused accelerated T1D onset relative to control mice, a phenomenon that was preceded by detection of virus within islets. Together, the results suggest a model for resolving conflicting data regarding the role of CVB in human T1D etiology.