12^th World Congress on Virology October 16-17, 2017 Baltimore, Maryland, USA

Renata Kissova, MSc., PhD is a Virologist currently heads the Virology Laboratory of the Regional Authority of Public Health in Banska Bystrica, Slovak Republic. Her work is focused mainly on the surveillance and diagnosis of enteroviruses and influenza viruses, especially virus isolations in cell cultures techniques. She received her MSc. Microbiology degree from the Safarik University in Kosice and the Slovak Medical University in Bratislava, Slovakia. She got her PhD. at the Faculty of Public Health of Slovak medical University in Bratislava, Slovakia. Since 2013 she has been teaching at the Slovak Medical University, Faculty of Health in Banska Bystrica

We present the monitoring programme of sewage water according to the World Health Organization (WHO) strategy for polio eradication in the Slovak Republic (SR). Polioviruses (PV) and non-polio enteroviruses (NPEV). \r\nWe cover the period from 2001 to 2016, which ranges from before and after the change in polio vaccination strategy, in the Slovak Republic. Samples from the sewage treatment plants of 48 localities from Western, Central and Eastern regions were tested. We have compared the circulation pattern of PVs and NPEVs in the SR, as a component of the PV surveillance program. WHO standard procedures were followed for virus isolations and identifications. 870 human enteroviruses (EVs) were detected: 357 (41%) coxsackie B viruses (CBV), 309 (36%) echoviruses, 76 (9%) NPEV not-typed and 114 (13%) Sabin-like PVs (PV1, 2, 3) including vaccine-derived poliovirus (VDPV) isolates. The percentage of PV isolates fell from 66% to 30% during 2001–2005 and thereafter to zero. CBV5, echoviruses 3 and 11 were the NPEVs endemic during the study period. In conclusion we have shown that the changes in the vaccination program of oral polio vaccine towards inactivated polio vaccine in the year 2005 changed the balance of circulating serotypes from Sabin-like PV and vaccine derived PV towards the NPEV. In September 2015, wild PV type 2 was officially declared eradicated. According to the WHO Global Action Plan to minimize poliovirus facility-associated risk after type-specific eradication of wild polioviruses, Slovak republic disposed all material containing PV2.\r\n

Brigita Benkőová, MSc, is a Virologist currently working at the Enterovirus Laboratory and the National Reference Centre for Identification of Enteroviruses at the Medical Faculty of the Slovak Medical University in Bratislava, Slovakia. She received her BSc. and MSc. Virology degree at Comenius University in Bratislava, Faculty of Natural Sciences. Her Bachelor thesis contains a literature review of neonatal enteroviral infections, their pathogenesis and clinical manifestation. In her MSc research thesis was focused on adaptive and innate immune response caused by coxsackievirus infection.\r\n\r\n

Coxsackieviruses (CV) are small positive-stranded RNA viruses, important human pathogens that cause both acute and chronic diseases. These viruses induce lytic infections; their cytopathic effect (CPE) includes morphological changes and destruction of the host cell monolayer in vitro. Their viral RNA can remain persistent for prolonged times in cells and organs post infection. Literature reports show moderate replication of enteroviruses in NIH 3T3 cells. Our aim was to study the replication of coxsackieviruses in NIH 3T3 clones with resistance to Puromycin alone or along with Blastomycin, in combination with presence of a truncated variant of the Dicer ribonuclease. Four genetically modified clones of NIH 3T3 cells were infected with CVB3 (Nancy strain). This virus was previously passaged in Vero cells with a titer of 106.75 TCID50. Cells were infected with a multiplicity of infection of 0.1. We checked the cells for presence of viral replication (CPE) and viral RNA by the reverse transcriptase polymerase chain reaction (PCR) and Nested-PCR. We also made an attempt to adapt the virus to the cells by blind passages in the NIH 3T3 cells. Morphological changes were absent in the infected cells as compared to the mock-infected control of the NIH 3T3 cell lines. After third and fourth passages of the virus in the same cell line, rounding of cells was observed but this effect did not increase on further passages of the virus. When checked for presence of viral RNA, the cultures were found to be positive irrelevant of the rounding effect. Considering the viral kinetics we hypothesize a slow viral replication in the first phase of infection in these cells which might allow a time frame for the induction of cytokines, resulting in further slowing down the virus replication and spread of infection between cells and ensure cell survival and leading to persistence of viral RNA.

Maria Borsanyiova, MSc., PhD., is a Virologist, she works at the Enterovirus Laboratory and the National Reference Centre for Identification of Enteroviruses at the Medical Faculty of the Slovak Medical University in Bratislava, Slovakia. She received PhD. in Microbiology degree from Faculty of Medicine, Comenius University, Bratislava, Slovak Republic. She is involved in research, diagnosis of enteroviruses and teaching. She has experience with mouse model infection

Statement of the Problem: Coxsackieviruses B (CVB) are associated with asymptomatic, acute and number of chronic inflammatory diseases. Based on the clinical manifestations nasal, throat or rectal swabs are collected for enteroviral diagnosis. Traditional method makes use of the clinical sampling swabs with virus transport medium (VTM) to ensure the stability of the viral genome. Molecular diagnostic tests may not require the VTM. The purpose of this study is to evaluate swabs consisting of different material: synthetic nylon swabs with plastic sticks, cotton swabs with wooden sticks without VTM and swabs with the VTM. Methodology & Theoretical Orientation: We have made a systematic comparison of different types of swabs. For evaluation the swabs were dipped in different serial dilutions of CVB3-Nancy strain, stored as follows in 3 sets: Set (1) processed immediately; Set (2) frozen at -80°C and had three groups a) processed after 12 days, b) 1 month and c) 2 months; Set (3) stored at + 4°C (and processed after 12 days). Viral RNA was isolated and detected in the processed suspension (made by vortexing the swabs in 500μl RNAse free water). Findings: The results show a possibility of using swabs without VTM for diagnostics and epidemiological purposes for identifying the enteroviral infections by PCR. Conclusion & Significance: All three types of swabs can be used. Presently the cotton tip swabs are not much in use in diagnostic and clinical laboratories, however their positivity was higher when compared to polyester tipped plastic swabs. Best option for swabs without VTM observed was freezing the swabs at -80°C, but viral RNA was also detectable after storage for 12 days up to 4°C. The use of swabs without transport medium will be suitable for diagnosis/screening of EV infections. Detection of viral RNA after storage depends on virus concentration.

Sona Sarmirova is a Virologist presently working at the Enterovirus Laboratory and the National Reference Center for Identification of Enteroviruses at the Medical Faculty of the Slovak Medical University in Bratislava. She has completed her BSc, MSc and PhD degree in Virology from Comenius University in Bratislava, Faculty of Natural Sciences. Her PhD research was focused on the study of the influence of a challenge infection in the offspring of experimentally infected mice during the gestation period.

Statement of the Problem: Enteroviruses are distributed worldwide. Very often infections caused by these viruses go unnoticed due to subclinical course. In a pregnant woman such infections remain frequently undiagnosed because of mild and non-specific manifestations. In neonates, however, coxsackie B virus (CVB) infections can cause a sepsis syndrome, sometimes followed by severe disease and death. Moreover, seroepidemiological studies relate CVB infections during pregnancy with increased risk for childhood-onset type 1 diabetes. The objective of this study was to follow-up the pathophysiological process after coxsackievirus infection in offspring born to dams infected with the same virus in the first, second and third week of gravidity. Methodology & Theoretical Orientation: The selected organ tissues (pancreas and heart) of pups orally infected with CVB4-E2 were analyzed for viral RNA, quantification of RNA copies and histopathological changes. Furthermore, analysis of multiple cytokines induced simultaneously in sera of these mice was performed. Findings: Independent of the copies of RNA which were observed in the heart and pancreas of the challenged offspring, we observed histopathological changes only in the pancreas of the challenged pups. After oral infection we observed inflammation of the islets of Langerhans, the endocrine tissue of the pancreas for the first time. The inflammation was present in the offspring of the mice infected in the third week of gravidity. Conclusion & Significance: We conclude that infection during gravidity enhances the pathophysiological process especially in the pancreas, and the time of infection during gravidity may also rule the intensity of the infection, which may be enhanced by the innate and adaptive immune responses.

Martin Sojka, MSc., PhD is Microbiologist and Virologist, currently working at the Enterovirus Laboratory and Institute of Microbiology at the Medical Faculty of the Slovak Medical University in Bratislava, Slovakia. His work in the field of Virology is focused on the pathogenesis of enteroviruses. He received his BSc, MSc and PhD in Microbiology and examina rigorosa in Virology from Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. He is involved in research, teaching and is guiding several BSc and MSc. students. He has participated several national and international projects.

High mutation rates of enteroviruses create a “cloud” of potentially beneficial mutations at the population level, which allow the viral quasispecies a greater probability to evolve and adapt to new environments. \r\nOur aim was to study the influence of intratypic virus strain variability on the course of coxsackieviral (CV) infection. \r\nCD1 mice were infected with CV: (i) clinical isolate from the cerebrospinal fluid of a patient – CVB4 AL, (ii) isolate from the stool of the same patient – CVB4 AS, (iii) environmental isolate – CVB4 COV. Mice were observed up to 45 days. Presence of replicating virus, viral RNA and localization of the virus was assessed. 5´NCR and VP1 regions of the viral genomes were sequenced. \r\nDifferences in weights were observed during the course of experiment. The differences between weights in CVB4 AL-infected and control mice were significant from day 7. The differences between CVB4 AS-, CVB4 COV- infected and control mice were statistically significant from the day 13. CVB4 AL had an affinity for the brain tissue and was detected in brain up to 45 days. CVB4 AS isolate was detected in pancreas and unlike other viruses; this virus was shedded in the stool up to day 45. CVB4 COV was detected in pancreas of mice up to day 45, this was the only isolate which induced acute pancreatitis in mice. The environmental virus isolate was localized in the acinar pancreatic tissue as well as in islets of Langerhans, whereas clinical isolates were detected only in islets of Langerhans. Sequence analysis of the isolates showed no differences between the CVB4 AL and CVB4 AS in VP1 region. In the 5´NCR one difference was found between the CVB4 AL/AS and CVB4 COV, three differences were found between the CVB4 AL/AS isolates and CVB4 COV in the VP1 region.\r\n