Epidemiology and genetic diversity of human parechoviruses circulating among children hospitalised with acute gastroenteritis in Pune, Western India: a 5-years study.

Human parechoviruses (HPeVs) are known to cause various clinical manifestations including acute gastroenteritis. Although HPeV infections and their genotypes have been detected in human patients worldwide, no such reports are available from India to ascertain the association of HPeVs in acute gastroenteritis. The present study was conducted to determine the clinical features and genetic diversity of HPeVs detected in children hospitalised for acute gastroenteritis. Stool specimens (n = 979) collected from children aged ⩽5 years hospitalised for acute gastroenteritis in Pune, western India during January 2006-December 2010 were included. HPeV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) (5'UTR) followed by genotyping using VP1 gene-based PCR and phylogenetic analysis. HPeV was detected in 13·9% (136/979) of the cases, co-infections with other enteric viruses were found in 43·4%. HPeV was more frequent in children ⩽1 year age with infections reported throughout the year. A total of 102/136 (75%) HPeV strains were genotyped, which comprised 13 different HPeV genotypes. Of these, HPeV1 was the most predominant genotype detected and phylogenetically clustered with the Harris strain which is rarely reported. The study documents circulation of heterogeneous HPeV genotypes. Two variant strains of HPeV4 and 'RGD absent' HPeV5 and 6 strains were also detected. This is the first report of HPeV with diversified genotypes identified in acute gastroenteritis patients from India.

Antiviral activity of the Indian medicinal plant extract Swertia chirata against herpes simplex viruses: a study by in-vitro and molecular approach.

PURPOSE: The antiviral activity of Indian Medicinal plant extract Swertia chirata was tested against Herpes simplex virus (HSV) type-1, using multiple approaches both at cellular and molecular level. METHODS: Cytotoxicity, plaque reduction, virus infectivity, antigen expression and polymerase chain reaction (PCR) assays were conducted to test the antiviral activity of the plant extract. RESULTS: Swertia plant crude extract (1 gm/mL) at 1:64 dilution inhibited HSV-1, plaque formation at more than 70% level. HSV antigen expression and time kinetics experiments conducted by indirect immunofluorescence (IFA) test, revealed a characteristic pattern of small foci of single fluorescent cells in Swertia extract treated HSV-1 infected cells at 4 hours post infection dose, suggested drug inhibited viral dissemination. Infected cell cultures treated with Swertia extract at various time intervals, tested by PCR, failed to show amplification at 12, 24-72 hours. HSV-1 infected cells treated with Acyclovir (antiviral drug) did not show any amplification by PCR. CONCLUSIONS: In this preliminary study, the Indian medicinal plant extract, Swertia chirata showed antiviral properties against Herpes simplex virus type-1.

Detection of Mycoplasma species in cell culture by PCR and RFLP based method: effect of BM-cyclin to cure infections.

PURPOSE: A two-stage nested polymerase chain reaction (PCR) assay system was described that amplifies the 16S-23S rRNA spacer region sequences of Mycoplasma and Acholeplasma infections in cell cultures and virus stocks. METHODS: Established cell lines and virus stocks were screened for the presence of Mycoplasma by using nested PCR using two sets of outer and inner primers, amplifies 16S-23S rRNA. PCR and restriction fragment length polymorphism (RFLP) assay was used to detect and identify most of the species-specific Mycoplasmas involved in cell cultures and virus stock contaminants. Infected cultures detected by PCR-RFLP were further treated with BM-cyclin (5 microg/mL) and passaged for three times and tested for Mycoplasma infections by PCR-RFLP. RESULTS: Mycoplasma pirum and Mycoplasma orale infections were detected by nested PCR. Species specificity was identified by using RFLP of Vsp I, Cla I and Hin dIII restriction enzymes. Mycoplasma infections were cured by treatment with BM-cyclin. This was further confirmed by non-amplification of PCR amplimers in BM-cyclin treated vs. non-treated cultures. CONCLUSIONS: Regular monitoring of cell cultures for Mycoplasma infections and identification of species-specific Mollicutes will identify the source of contaminations. This approach can be used for quality control of the biological reagents used in cell culture and virology laboratories.

Outbreak of acute hemorrhagic conjunctivitis in Maharashtra and Gujarat states of India, caused by Coxsackie virus A-24 variant.

Acute hemorrhagic conjunctivitis is associated with enteroviruses. Among these, Coxsackie A-24 variant (CA-24) and Enterovirus-70 (EV-70) are known to cause epidemics and pandemics. An outbreak of acute hemorrhagic conjunctivitis occurred in August-September 2003 in Maharashtra and Gujarat states of India. The present investigation was carried out to determine the viral etiological agent associated with the epidemic. Virus isolates were obtained from 11 eye swabs of conjunctivitis patients using HeLa/ Hep-2 cell lines. The isolates were characterized by serological and mouse pathogenecity tests, RT-PCR using enterovirus common primers (VP4-VP2), CA-24 specific primers (3C-proteinase region), EV-70 primers (VP-3) followed by sequencing, and phylogenetic analysis. The virus was characterized as a Coxsackie A-24 variant (CA-24v) and none of the isolates were found to be positive for EV-70. Sequencing of the PCR products derived from all the 11 isolates revealed 98.4% (SE 0.20) nucleotide identity within the Indian strains and 98.6% (0.50) and 94.4% (0.30) nucleotide identity respectively with the West Indies and Asian strains reported worldwide. The findings suggest that the outbreak of acute hemorrhagic conjunctivitis that occurred in Maharashtra and Gujarat states of India during August-September 2003 was caused by the Coxsackie A-24 variant (CA-24v).