Murine experimental models for studying the pathogenesis of coxsackieviruses.

Understanding the pathogenesis of communicable diseases often involves animal models. Mouse models are studied by researchers to achieve a better understanding of the relationship between the biological, physiochemical, and antigenic properties of the infectious agent, as well as the histopathological, immunological, and functional changes in the living system and the target organs, in short, the pathophysiological processes that the communicable agents bring about. The long-term objectives of the in vivo studies are important from a medical point of view, as they represent faithful (reliable and similar) human responses, which enhance the development of diagnostics, treatment, and measures for preventing the spread of the disease. Our work is devoted to the murine models used for understanding the pathogenesis of coxsackieviruses. We describe different mouse models used for studying the diseases caused by coxsackieviruses and the immune responses in different mouse models.  We then shortly elucidate experiences from our laboratory related to the oral route of infection, and compare the similarities and differences we found in this model. Keywords: pathogenesis; coxsackievirus; murine models.

Human enterovirus surveillance in the Slovak Republic from 2001 to 2011.

We report the outcome of an 11-year programme monitoring sewage water and acute flaccid paralysis (AFP) cases as part of the World Health Organization (WHO) strategy for polio eradication in the Slovak Republic (SR). Polioviruses (PV) and non-polio enteroviruses (NPEV), prior to and after the change in polio vaccination strategy, were detected. Sewage treatment plant samples from 48 localities spread over the Western, Central and Eastern regions and clinical material from AFP cases were examined. The WHO standard procedures were followed with regard to virus isolation and identification. There were 538 commonly detected human enteroviruses (HEVs) including 213 (40%) coxsackie B viruses (CBV), 200 (37%) echoviruses and 113 (21%) 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 fell to zero. CBV5, CBV2 and echovirus 3 were the NPEVs endemic during the study period.

[Use of PCR in diagnosis of enteroviral infections]. / Pouzitie metódy PCR v diagnostike enterovírusových infekcií.

Enteroviruses belonging to the family Picornaviridae are important human pathogens. Although most cases of infection caused by these viruses are asymptomatic, a wide range of clinical syndromes is observed in manifest cases. Conventional laboratory diagnostic methods based on virus isolation and identification, or on the detection of specific antiviral antibodies, are costly and time consuming. Therefore, they are of little benefit to treatment. We have implemented a commercially available PCR-based test for the detection of enteroviral infections. In 2008, we analyzed biological specimens from 125 patients with suspected enteroviral disease, most often involving the nervous system. The presence of enterovirus was detected in 39 patients. The results were compared with those obtained by the conventional methods. PCR appeared to be a valuable method for rapid, accurate and reliable diagnosis of enteroviral infections which is of major benefit to patient management.

[Circulation of coxsackieviruses in the Slovak population in 1985-2007]. / Cirkulácia coxsackievírusov v slovenskej populáicii v období rokov 1985 az 2007.

Enteroviral infections are of the most common infections in the human population. Circulation of these viruses is high in population and they cause a wide range of clinical syndromes in human. The aim of this study was to compare the coxsackievirus circulation intensity in the population of the Slovak Republic during the period of last 23 years and to identify changes also in relation to modification in polio vaccination scheme. Marker indicating suffered infection was the presence of virus specific antibodies, assessed by means of the virus-neutralizing test. High frequency of infections by all studied coxsackievirus serotypes was confirmed in this study. Consecutive decrease in rate of anti-coxsackievirus antibodies was identified. This may be a consequence of hygiene standard improvement. After modification of immunization scheme (2004/2005) the decrease has been stopped, a mild increase of seropositivity to all studied serotypes was observed, respectively.

Coxsackievirus infection of mice. I. Viral kinetics and histopathological changes in mice experimentally infected with coxsackieviruses B3 and B4 by oral route.

We followed the viral kinetics and histopathological changes in different organs of immunocompetent mice infected orally with coxsackieviruses B3 (CVB3) Nancy strain and B4 (CVB4) JVB strain separately. The viruses used were not adapted to mouse organs. In the acute phase of infection, the viral kinetics indicated virus replication in the heart, spleen, thymus, pancreas, and small and large intestines. This was accompanied by histopathological changes, mild infiltration of mononuclear cells and fibrosis in the heart. The necrotic changes with mononuclear infiltration and fibrosis in the myocard was observed on days 56 and 71 p.i. in the CVB4-infected animals only. In the mice infected with CVB3 and CVB4 a prolonged presence of infectious virus was shown in the spleen and small intestine; in the latter viral antigen was localized in smooth muscles of the muscular wall immunohistochemically. This is the first report on prolonged replication of coxsackieviruses (CV) in the spleen and small intestine in orally infected mice.

Coxsackievirus infection of mice. II. Viral kinetics and histopathological changes in mice experimentally infected with coxsackievirus B3 by intraperitoneal route.

The study was focused on kinetics of Coxsackievirus B3 serotype (CVB3) in different organs of Swiss albino mice following intraperitoneal (i.p.) infection. The results indicated that the virus replicated in the heart, spleen, thymus, pancreas, small and large intestines in the acute stage of the infection. Infectious virus was present in the spleen till day 35 post infection (p.i.). Histopathology of the hearts showed mild foci of infiltration of mononuclear cells in the acute stage of infection and massive inflammation of exocrine pancreas on day 5 p.i. These results, when compared to those of our previous study (Bopegamage et al., 2003), suggest that the pathogenesis of the disease may be influenced by the route of virus administration into the host.

Coxsackie viral infection and orofacial cleft.

The incidence of orofacial cleft (OC) in newborns was compared with the occurrence of virus-neutralizing antibodies to coxsackie viruses in the serum of newborns and their mothers. No significant difference was found when comparing the seropositivity rates between the group of patients and the control group of healthy newborns. If the patients were divided according to the place of residence however, marked differences occurred between the regions. The lowest incidence of both--coxsackie infection and OC was determined in the region of Bratislava and the highest in the region of Zilina. The explanation of these findings recquires a more detailed analysis of genetic background, social and hygienic status, style of life and other factors, known to influence the development of OC as multi-etiological developmental disorder. (Tab. 2, Fig. 4, Ref. 12.).

Detection of coxsackie B virus infection using a rapid screening method.

A Western blot method (WB) was adapted for rapid screening of antibodies against coxsackie virus B1-B6 in sera from patients with newly diagnosed insulin-dependent diabetes mellitus, myocarditis or febrile syndrome of suspected coxsackie viral aetiology. The use of a mixture of all 6 coxsackie virus B serotypes as the common antigen permitted a very rapid and inexpensive detection of antibody-positive sera for preliminary diagnosis and further detailed assay. Comparison of the results with those obtained in parallel run virus-neutralization tests showed a higher sensitivity and comparable specificity of WB.

Tumour necrosis factor alfa and glucose levels in sera of mice infected with coxsackie B4 and A7 viruses.

Cytokines have been suggested to play an important role in the pathogenesis of type I diabetes mellitus through their direct and indirect effects on the pancreatic islet cells. We studied the time course of tumour necrosis factor alpha (TNF-alpha) and glucose levels in the sera of mice infected with coxsackie B4 and A7 viruses. Two correlating peaks of TNF-alpha and glucose were found. These results suggest the involvement of TNF-alpha in the damage of the insulin producing cells and thus an immunity-related inflammatory process.

[Western blotting in the serologic diagnosis of enterovirus infections. I. Detection of coxsackie virus antibodies in reference sera and sera of experimentally infected mice]. / Pouzitie metódy Western blotting na sérologickú diagnostiku enterovírusových infekcií. I. Detekcia anti-coxsackievírusových protilátok v referencných diagnostických sérach a v sérach experimentálne infikovaných mysí.

The introduction of rapid serological diagnosis of enterovirus infections is faced with the problem of inter-type cross-reactions. Therefore the method of choice is still the virus-neutralization test on cell cultures and on suckling mice resp., i.e. methods which are expensive and time consuming and demanding on laboratory equipment. The detection of antibodies against the Coxsackie virus by means of Western blotting revealed a specific reaction of commercial hyperimmune diagnostic rabbit as well as of sera obtained after oral infection of mice. The application of the method in the diagnosis of human enterovirus infections will be the subject of the subsequent part of this study.

[Western blotting in the serologic diagnosis of enterovirus infections. II. Detection of coxsackievirus antibodies in the sera of patients with suspected enterovirus infection and in paired specimens of sera and saliva]. / Pouzitie metódy Western blotting na sérologickú diagnostiku enterovírusových infekcií. II. Detekcia anti-coxsackievírusových protilátok v sérach pacientov so suspektnou enterovírusovou infekciou a v párových vzorkách sér a slín.

Detection of anti-Coxsackie virus antibodies in sera with suspect enterovirus infection by the Western blotting (WB) method revealed complete agreement of results with virus neutralization tests (VNT) in those patients where in the VNT no antibodies against any of the tested Coxsackie viruses were detected. Comparison of the results of WB and VNT in other patients did not rule out an inter-type cross-reaction. In paired specimens of sera and saliva comparable results were obtained by WB, VNT and the complement fixation method. The rapid and relatively simple character of the WB method makes this method suitable for more detailed elaboration and introduction into routine diagnosis of enterovirus infections.

In vitro infection of mouse pancreatic islet cells with coxsackie viruses.

We have demonstrated the ability of 4 standard coxsackie viruses (B4, B5, A7, and A9) and one fresh isolate (A7) from a newly diabetic child with homologous serological response, to infect in vitro grown mouse pancreatic islet cells. Up to the 9th day after infection the multiplication of viruses in the cells was proved using virus titration and immunofluorescence test. Isolated pancreatic cells proved to be a suitable model for detailed studies of experimental infection of pancreatic cells with coxsackie viruses.