Prolonged viral RNA detection in the central nervous system of one-week-old Swiss albino mice following coxsackievirus B4 and echovirus 9 infection.

OBJECTIVES: Type B coxsackieviruses (CV-B), together with echoviruses (E), are among the most common pathogens encountered in aseptic meningitis and meningoencephalitis. They frequently infect the central nervous system (CNS). The mechanisms of virus spreading in the CNS are poorly understood. In the present study, we investigated CV-B4 and E-9 spreading and neurotropism within intraperitoneally inoculated one-week-old Swiss albino mice. METHODS: Seminested RT-PCR and virus isolation were used to assay viral distribution. RESULTS: Viral RNA was present in various organs: brain, spinal cord, spleen and heart at various times post-infection (p.i.); ranging from 1 day p.i. up to 30, 60 and 90 days p.i, respectively, for CV-B4-JVB-, E-9 Barty- and CV-B4-E2-infected mice. Organs became negative for virus isolation after 5 days p.i., except for brain and heart from CV-B4 E2-infected mice, which remained positive for up to 10 and 15 days p.i., respectively. Negative viral RNA strand was detected mainly in brain and spinal cord of infected mice until 30 and 60 days p.i. CONCLUSION: This is the first report on the persistence of CV-B4 and E-9 in the CNS of intraperitoneally inoculated mice.

Enteroviral central nervous system infections in children of the region of monastir, Tunisia: diagnosis, laboratory findings of cerebrospinal fluid and clinical manifestations.

Human enteroviruses (HEV) are one of the major causes of central nervous system (CNS) infections in pediatrics. A prospective study was conducted to assess the epidemiological, clinical, and laboratory characteristics of enterovirus (EV) infections of the CNS in children under 15-years-old, suspected of having viral CNS infections and admitted to the Pediatric Department of Monastir University Hospital, Tunisia. Enteroviral RNA was detected by 5' NCR nested RT-PCR assay in 33 % (20 out of 60) of cerebrospinal fluid specimens, whereas only six samples (10 %) were EV positive in cell culture. EV-positive patients were clustered according to their clinical manifestations, predominantly diagnosed as aseptic meningitis (65 %) and meningoencephalitis (20 %). Fever, headache, vomiting, and neck stiffness were the most pronounced symptoms. Pleocytosis with the predominance of lymphocytes was observed in 60 % of EV positive specimens. Although patients suffering from EV infections were encountered throughout the year, most occurred during spring and summer months. Using VP1-2A nested RT-PCR and sequence analysis, three of the 20 positive HEV were identified as Echovirus (E)-9. This is the first report of a cluster of aseptic meningitis cases caused by E-9 in Monastir.

Role of GNRA motif mutations within stem-loop V of internal ribosome entry segment in coxsackievirus B3 molecular attenuation.

The lengthy 5' nontranslated region of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure containing an internal ribosome entry segment (IRES), which plays an important role in controlling viral translation and pathogenesis. The stem-loop V (SL-V) of this IRES contains a large lateral bulge loop which encompasses two conserved GNRA motifs. In this study, we analyzed the effects of point mutations within the GNRA motifs of the CVB3 IRES. We characterized in vitro virus production and translation efficiency and we tested in vivo virulence of two CVB3 mutants produced by site-directed mutagenesis. The GNAA1 and GNAA2 RNAs displayed decreased translation initiation efficiency when translated in rabbit reticulocyte lysates. This translation defect was correlated with reduced yields of infectious virus particles in HeLa cells in comparison with the wild type. When inoculated orally into Swiss mice, both mutant viruses were avirulent and caused neither inflammation nor necrosis in hearts. These results highlight the important role of the GNRA motifs within the SL-V of the IRES of CVB3, in directing translation initiation.

Nucleotide sequences of IRES domains IV and V of natural ECHO virus type 11 isolates with different replicative capacity phenotypes.

ECHO viruses (ECV) belong to the enterovirus genus of the Picornaviridae family and are the most frequently isolated from clinical and environmental samples. They are responsible for a wide variety of clinical syndromes involving most organs of the human body. We previously postulated that some of the variations in the recognition of ECHO virus type 11 (ECV 11) strains by a group specific monoclonal antibody (Mab) which we have studied could be explained by variations in their replicative capacity in cell culture and variations within the 5' nontranslated region (5' NTR) of their genomes. To support this hypothesis, the replicative capacity in cell culture and the nucleotide sequences of domains IV and V of the IRES of the genome of five ECV11 strains (the Gregory reference strain and four wild isolates) were determined, and analysed. Our results indicate that the replicative capacity of wild ECV11 isolates studied by one-step growth cycle in both HEp-2 and Vero cell cultures showed variations among strains in comparison with the Gregory reference strain. The clinical ECV11 strains replicated as well as the reference strain, however environmental strains displayed a phenotype with a significant reduction of replication. The sequences of ECV 11 strains showed significant conservation with that of the poliovirus (PV1) Mahoney strain The comparative examination of the predicted secondary structures revealed, that the nucleotide variations did not affect the secondary structure of stem-loop structure IV and V in the IRES element, however differences were especially observed in the apical stem region (nucleotides 483 to 509) of the domain V of the ECV11 strains and resulted in modification of the central stem structure.

Epidemiological study of non-polio enterovirus neurological infections in children in the region of Monastir, Tunisia.

The epidemiological, virological, and clinical syndromes of 86 children younger than 13 years suspected of having neurological diseases and admitted to Fattouma Bourguiba Hospital of Monastir from January 2002 to November 2003 were analyzed. The presence of enterovirus was studied in cerebrospinal fluid (CSF) samples by reverse transcription-polymerase chain reaction (RT-PCR) and by isolation on cell culture. Sixty-one (71%) specimens were positive by RT-PCR, whereas 45 (52.3%) were detected by cell culture. Eighty percent (n = 36) of cultured enteroviruses were identified, whereas 20% were untyped. Echoviruses were isolated most frequently, with 32 cases (71.1%) in children. Echovirus 6 was the most commonly identified serotype (22.2%), followed by echovirus 13 (20%). The highest incidence on neurological infection (19.6%) occurred in children less than 6 months of age. The infected children were predominantly male (62.3%). Enteroviruses were detected in all the period of the year with the highest rate in the spring and summer months. Aseptic meningitis was the most commonly diagnosed disease (49%).