Varanasi Gopalkrishna

Polymorphism of the p53 codon 72 arg/pro and the risk of HPV type 16/18-associated cervical and oral cancer in India

Infection of high risk human papillomaviruses (HPVs) specifically the types 16 and 18 has been strongly implicated in the development of cervical cancer. The E6 oncoproteins of these high risk HPVs are known to bind and induce degradation of p53 tumour suppressor protein through the ubiquitin pathways. This degradation is controlled by a common polymorphism of the p53 gene encoding either a proline or an arginine at its codon 72 in exon 4. Recently, it has been demonstrated that the presence of homozygous arginine at codon 72 renders p53 about seven times more susceptible to E6-mediated proteolytic degradation as well as to cervical cancer than those with proline homozygotes or proline/arginine heterozygotes. In India, prevalence of HPV as well as cancers of the uterine cervix and the oral cavity are highest in the world. We have examined this allele-specific predisposition in cervical and oral cancer which is associated with HPV as well as in a non-HPV-linked cancer of the breast. We have carried out investigation in women comprising whole spectrum of cervical lesions with 128 HPV 16/18 positive and 35 HPV negative invasive cervical carcinomas and 34 cases of HPV (16/18) positive and 16 HPV negative cervical dysplasias (mild, moderate and severe) and 104 age-group-matched healthy women as controls. Additionally, we have analysed p53Arg-Pro polymorphism in 13 high risk HPV positive and 31 HPV negative oral cancers along with 20 normal controls and 77 breast cancers with 41 age-matched healthy controls.We observed more than 2 fold higher risk for homozygous arginine (χ2 = 6.3, df = 2, p = 0.04; OR = 2.3; 95% CI: 1.08–5.16) for HPV 16/18-positive cervical carcinomas when comparison was made only between HPV positive cervical cancers and normal controls but most interestingly, no significant association either in the frequency of homozygous arginine or proline alleles or their heterozygotes could be observed when all the three groups i.e. HPV-positive, HPV-negative cervical cancers and controls were considered simultaneously. No difference was also observed for either arginine or proline polymorphism between women with precancerous lesions of the uterine cervix carrying HPV 16/18 infection and controls. Similarly, increased risk of oral or breast cancer could not be correlated with the polymorphism of arginine/proline allele.Thus the interaction between HPV oncoproteins and the p53 gene polymorphism specifically, homozygous arginine at codon 72 appears to play no role in the development of either cervical or oral cancer and also it can not serve as a biomarker for early identification of cervical, oral or breast cancer.

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.

Characterization of the non-polio enterovirus infections associated with acute flaccid paralysis in South-Western India.

Non-polio enteroviruses (NPEVs) have been reported frequently in association with acute flaccid paralysis (AFP) cases during Polio Surveillance Programs (PSPs) worldwide. However, there is limited understanding on the attributes of their infections. This study reports characteristics of NPEVs isolated from AFP cases, investigated during PSPs held in 2009–2010, in Karnataka and Kerala states of south-western India having varied climatic conditions. NPEV cell culture isolates derived from stool specimens that were collected from 422 of 2186 AFP cases (<1–14 years age) and 17 of 41 asymptomatic contacts; and details of all AFP cases/contacts were obtained from National Polio Laboratory, Bangalore. The distribution of NPEV infections among AFP cases and circulation pattern of NPEV strains were determined by statistical analysis of the data. Genotyping of all NPEV isolates was carried out by partial VP1 gene sequencing and phylogenetic analysis. NPEV positive AFP cases were significantly higher in children aged <2 years; with residual paralysis; in summer months; and in regions with relatively hot climate. Genotyping of NPEVs identified predominance of human enteroviruses (HEV)-B species [81.9%—Echoviruses (E): 57.3%; coxsackieviruses (CV) B: 15%; numbered EVs: 8.9%; CVA9: 0.7%] and low levels of HEV-A [14.5%—CVA: 6%; numbered EVs: 8.5%] and HEV-C [3.6%—CVA: 2.6%; numbered EVs: 1%] species, encompassing 63 genotypes. EV76 (6.3%) and each of E3, CVB3 and E9 (4.97%) were found frequently during 2009 while E11 (6.7%), CVB1 (6.1%), E7 (5.1%) and E20 (5.1%) were detected commonly in 2010. A marked proportion of AFP cases from children aged <2 years; presenting with fever; and from north and south interior parts of Karnataka state was detected with E/numbered EVs than that found with CVA/CVB. This study highlights the extensive genetic diversity and diverse circulation patterns of NPEV strains in AFP cases from different populations and climatic conditions.

Astrovirus associated acute gastroenteritis in western India: predominance of dual serotype strains.

A five-year (2004-2008) study was conducted on patients with acute gastroenteritis from different cities of Maharashtra, western India to detect and characterize astrovirus infections. A total of 1340 fecal specimens were collected from sporadic cases that included 1240 children (<or=8 years) and 100 adults (18-70 years) from Pune, Aurangabad and Nagpur cities. All specimens were subjected to astrovirus specific RT-PCR followed by sequencing and phylogenetic analysis. The overall positivity to astrovirus was found to be 3.1% with highest number of infections in winter months. A high prevalence of astrovirus was observed in children <or=1 year of age. Phylogenetic analysis of the partial ORF1a (serine protease) and ORF2 (capsid gene) regions showed the circulation of three probable recombinant types with different ORF1a/ORF2 specificities (HAstV-8/HAstV-1, HAstV-7/HAstV-2, HAstV-4/HAstV-5) along with HAstV-8 of a single specificity in the study population. HAstV-8/HAstV-1, specificity predominated (67.7%) in the region followed by HAstV-7/HAstV-2 (9.7%), HAstV-4/HAstV-5 (6.5%) and HAstV-8 (16%) types. This is the first report that highlights the genetic diversity of astrovirus strains circulating in Maharashtra state, western India.

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.

Circulation of Aichi virus genotype B strains in children with acute gastroenteritis in India.

Acute gastroenteritis (AG) is considered as one of the major health problems affecting humans of all ages. A number of viruses have been recognized as important causes of this disease. Recently, Aichi virus has been shown to play an aetiological role in sporadic infections and outbreaks of AG. A study on surveillance of enteric viruses was conducted during 2004-2008 in three cities in Maharashtra state, western India. A total of 1240 stool specimens from children aged ≤8 years hospitalized for AG were screened for the presence of Aichi virus by RT-PCR of the 3C-3D junction region followed by sequencing for the identification of genotype. Aichi virus was detected at a prevalence of 1·1% in the <5 years age group and characterized as genotype B. This is the first report on the circulation of Aichi virus genotype B in 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.

Molecular epidemiology and clinical severity of Human Bocavirus (HBoV) 1–4 in children with acute gastroenteritis from Pune, Western India

Although acute gastroenteritis is a major public health problem worldwide, ∼40% of the cases remain undiagnosed for any etiological agent. Human Bocavirus (HBoV) has been detected frequently in feces of diarrhoeic children suggesting its possible etiological involvement in the disease. HBoV has not been reported in association with acute gastroenteritis from India. Fecal samples (n = 418) collected from children (age ≤5 years) hospitalized with acute gastroenteritis, between January 2009 and December 2011, from three local hospitals were examined for presence of HBoV using PCR targeting the partial VP1/VP2 capsid region (∼575 bp) followed by phylogenetic analysis. HBoV was detected in 24/418 (5.7%) cases. Co‐infection was observed in 5/24 (21%) cases. HBoV infections occurred in children ≤12 months of age. Peak HBoV activity was observed in monsoon and post monsoon season. All four HBoV genotypes were detected in the study region. Major clinical symptoms of HBoV mono infections included diarrhoea (100%), fever (90%), dehydration (74%), and vomiting (58%). Dehydration was observed in all of the HBoV2–4 cases and in 50% of the HBoV1 cases. Clinical severity varied with genotype (HBoV2 > HBoV1 > HBoV3 > HBoV4). HBoV2 cases recorded severe and very severe infections. The study illustrates prevalence and vast genetic diversity of HBoVs in acute gastroenteritis. It highlights the clinical features of HBoV1–4 infections and sheds light on clinical impact of HBoV genotypes in gastroenteritis. J. Med. Virol. 89:17–23, 2017.

Full genome analysis of rotavirus G9P[8] strains identified in acute gastroenteritis cases reveals genetic diversity: Pune, western India

Group A rotaviruses (RVA) are the major enteric etiological agents of severe acute gastroenteritis among children globally. As G9 RVA now represents as one of the major human RVA genotypes, studies on full genome of this particular genotype are being carried out worldwide. So far, no such studies on G9P[8] RVAs have been reported from Pune, western part of India. Keeping in view of this, the study was undertaken to understand the degree of genetic diversity of the commonly circulating G9P[8] RVA strains. Rotavirus surveillance studies carried out earlier during the years 2009‐2011 showed increase in the prevalence of G9P[8] RVAs. Representative G9P[8] RVA strains from the years 2009, 2010, and 2011 were selected for the study. In general, all the G9 RVA strains showed clustering in the globally circulating sublineage of the VP7 gene and showed nucleotide/amino acid identities of 96.8‐99.7%/96.9‐99.8% with global G9 RV strains. Full genome analysis, of all three RVAs in this study indicated Wa‐like genotype constellation G9‐P[8]‐I1‐R1‐C1‐M1‐A1‐N1‐T1‐E1‐H1. Within the strains nucleotide/amino acid divergence of 0.1‐3.4%/0.0‐4.1% was noted in all the RVA structural and non‐structural genes. In conclusion, the present study highlights intra‐genotypic variations throughout the RVA genome. The study further emphasizes the need for surveillance and analysis of the whole genomic constellation of the commonly circulating RVA strains of other regions in the country for understanding to a greater degree of the impact of rotavirus vaccination recently introduced in India.