Evaluation of Contamination Risks with Coxsackievirus B4 E2 in Swiss Albino Mice Stools.

Coxsackie B4 (CV-B4), is a major cause of viral myocarditis, dilated cardiomyopathy, and pancreatitis. Like other human enteroviruses, CV-B4 is ubiquitous, excreted in the stool, transmitted by fecal-oral route, and persists in the environment. In the context of studies on CV-B4 infection, it is important to investigate how this virus can be eliminated and to show the possibility of contamination risk with a CV-B4 E2 infected Swiss albino mice. Swiss albino female mice were inoculated with CV-B4 E2 strain and divided in two groups: the first was intraperitoneally (I.P.) infected; the second was orally infected. In order to study the CV-B4 E2 infection in mice, total RNA was extracted from thymus, spleen, pancreas, and intestine, and viral genome was detected using semi-nested (RT-PCR). To further demonstrate infection or immunization of mice, Sera obtained from infected mice were assayed in vitro for their neutralizing antibody. To detect virus in stool of infected mice, stool samples were collected at different post-infection (p.i.) times. Neutralizing antibodies were detectable all along the follow-up period (Day 0, 1, 3, 7, 9, 17, 22, 30, 45, 60 p.i.) in I.P and oral infected mice. Our results showed that when mice were inoculated successively at day 0 and day 8, neutralizing activity was increased in I.P route more than in the oral route. Viral isolation in HEp-2 cells showed negative results. Stool viral analyses reveal a low detection of the CV-B4 E2 genome for all infected mice. In conclusion, our experiments demonstrated that there are no risks linked with the stool of CV-B4 E2 of Swiss albino mice. It would be interesting to characterize the inhibitors of the virus infectivity in these biological samples (stool) and investigate their targets and mechanisms of action.

Anti-coxsackievirus B4 (CV-B4) enhancing activity of serum associated with increased viral load and pathology in mice reinfected with CV-B4.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.

Immunoglobulin G-dependent enhancement of the infection with Coxsackievirus B4 in a murine system.

It was demonstrated that specific IgG can enhance the infection with CV-B4, in vitro, in the human system. This enhancement could be involved in the pathophysiology of CV-B4 induced diseases. To investigate further the role of enhancing IgG in the infection with CV-B4 E2 in vivo, animal models are needed. Therefore, it was decided to assess whether inoculation of CV-B4 E2 to mice results in the appearance of IgG able to enhance the infection with this virus. Swiss albino mice were inoculated with CV-B4 E2 intraperitoneally. Serum samples were obtained from tail vein blood collected from day 0 to day 80 p.i. IgG were isolated by Protein G affinity chromatography. Seroneutralisation assays were carried out. In total murine spleen cells cultures inoculated with CV-B4 E2 mixed with various dilutions of serum or IgG samples, the enhancing activity was assayed through i) the antiviral activity titer of supernatants ii) the detection of intracellular viral RNA by RT-PCR iii) the level of infectious particles in supernatants. In most serum samples (76/105), neutralizing and enhancing activities were detected peaking between days 14 and 30 p.i and were higher in sera from mice inoculated with 2.10(6) TCID50 units than with lower doses. The enhancing activity was due to the IgG-enriched fraction of serum from CV-B4 E2 infected animals but not from control animals. These data show that IgG from immune mice can enhance the infection of splenocytes with CV-B4 E2 in vitro and open the way to explore whether such an enhancing activity can play a role in vivo.