Pooja R. Patil

Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India

Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, can be caused by enteroviruses. Coxsackievirus A16 (CV-A16) and enterovirus 71(EV-71) are the major aetiological agents of HFMD. Other EV serotypes, CV-A4-7, CV-A9-10, CV-B1-3, CV-B5, E-4 and E-19, have also been found associated with both sporadic infections and outbreaks of HFMD. In India, outbreaks of HFMD have been documented; however, molecular characterization of the aetiological agents has rarely been reported. Cases of HFMD were identified during 2009-2010 on the basis of clinical features in southern and eastern parts of India. The aim of the present study was to detect and characterize the aetiological agents associated with the disease. A total of 89 specimens consisting of 41 sera, 24 vesicular fluids, 18 stools and 6 throat swabs were collected from 61 clinically diagnosed HFMD cases from southern and eastern parts of India. RT-PCR followed by sequencing of PCR amplicons and phylogenetic analysis were performed on all specimens for detection of EV RNA and identification of EV types. EV RNA was detected in 47.1 % (42/89) of the specimens collected from 57.4 % (35/61) of the HFMD cases. Thirty-six of 42 EV strains showed amplification of the VP1/2A junction or VP1 regions. Sequence analysis of the amplicons identified the presence of CV-A16 (54.8 %), CV-A6 (38.1 %), EV-71 (2.4 %), CV-A10 (2.4 %) and E-9 (2.4 %) serotypes in the HFMD cases. The study documents CV-A16 and CV-A6 as major and CV-A10, EV-71 and E-9 as rare viral pathogens of HFMD in 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.

Diversity in the Enteric Viruses Detected in Outbreaks of Gastroenteritis from Mumbai, Western India

Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Molecular surveillance of non‐polio enterovirus infections in patients with acute gastroenteritis in Western India: 2004–2009

Acute gastroenteritis is a major cause of childhood morbidity and mortality worldwide. Rotavirus (RV) and Norovirus (NoV) are the leading cause of the disease. Despite the use of improved diagnostic methods a significant proportion of gastroenteritis cases remained undiagnosed. Though nonpolio enteroviruses (NPEVs) have been reported frequently in children with acute gastroenteritis, their etiologic role has not been established. To investigate the epidemiology of NPEVs in gastroenteritis cases which remained negative for leading causative agents, 955 RV and NoV negative stool specimens from children hospitalized for acute gastroenteritis were included in the study. A case control study was conducted which includes stool specimens from 450 children with gastroenteritis and 162 asymptomatic control subjects to determine the association of NPEVs with the disease. NPEV detection and typing was carried out by RT‐PCR and sequencing. Presence of RV, NoV, Adenovirus, and Astrovirus was confirmed by ELISA or PCR/RT‐PCR. Overall 14% NPEV prevalence was noted. The percentage of children with NPEV infection differed significantly between gastroenteritis and non‐gastroenteritis patients (13.7% vs. 4.9%). NPEV was more prevalent among patients with gastroenteritis of undetectable etiology as compared to those detected positive for other viruses (17.9% vs. 7%) (P < 0.01). Genotyping of NPEV identified predominance of EV‐B species (56.5%) followed by EV‐C (16.7%), EV‐A (13.8%) species and mixed NPEV infections (13%). These data support the association of NPEVs with acute gastroenteritis and highlights the clinical and epidemiological features of NPEV infections in patients with acute gastroenteritis from western India. J. Med. Virol. 87: 154–161, 2015.

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.

Sequence analysis of VP4 genes of wild type and culture adapted human rotavirus G1P[8] strains.

OBJECTIVE To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361, 0613158, 061060 and 0715880) and cell culture adapted (06361-CA, 0613158-CA, 061060-CA and 0715880-CA) G1P[8] rotavirus strains. METHODS Full-length VP4 genes of each of the four wild type G1P[8] rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing. RESULTS All four cell culture adapted G1P[8] rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains. The number of substitutions however, varied from 1-64 and 1-13 respectively. The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilization and hemagglutinating activity. The presence of unique amino acid substitutions was identified in two of the four wild type (V377G, S387N in 061060 and I644L in 0715880) and all four cell culture adapted (A46V in 0613158-CA, T60R in 06361-CA, L237V, G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8] specificity. Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains. CONCLUSIONS Amino acid substitutions detected in the VP4 genes of G1P[8] rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation, immunogenicity and conformation is needed for the development of newer rotavirus vaccines.

Identification and molecular characterization of adenovirus types (HAdV-8, HAdV-37, HAdV-4, HAdV-3) in an epidemic of keratoconjunctivitis occurred in Pune, Maharashtra, Western India.

Epidemic keratoconjunctivitis (EKC) is a highly contagious infectious disease of the ocular surface and is caused mainly due to adenoviruses species D, B, and E. The present study was carried out to identify and characterize the viral etiological agents associated with the keratoconjunctivitis cases reported from Pune (Maharashtra), Western India between November–December 2013 and January, October–November 2014. Conjunctival swab specimens (n = 23) obtained from keratoconjunctivitis patients were subjected to detection of Adenovirus (AdV) and Enterovirus (EV) by PCR/RT‐PCR using hexon and 5′ NCR gene specific primers, respectively. Molecular typing of AdV and EV positive specimens was carried out by amplifying penton, fiber, and VP1 genes, respectively followed by sequencing and phylogenetic analysis. In this study, human adenovirus (HAdV) was identified as an etiological agent. None of the clinical specimens were found positive for enterovirus. AdV positivity in keratoconjunctivitis cases was found to be 60.9% (14/23). Fourteen of the HAdV positive strains, all of them were amplified by hexon gene, nine strains by fiber gene, and all 14 strains by penton gene specific primers. Sequencing of all HAdV positive samples revealed the presence of HAdV‐8, HAdV‐37, HAdV‐3, and HAdV‐4. All Indian strains showed highest nucleotide identity with the reference strains reported worldwide. The study revealed the circulation of HAdV‐8 (78.6%) as predominant AdV strain followed by HAdV‐37, HAdV‐3, and HAdV‐4 (7.2%) identified in the epidemic keratoconjunctivitis. Multiple types of AdVs in EKC reported for the first time in Western India. J. Med. Virol. 88:2100–2105, 2016.