Shobha D. Chitambar

Multicenter, Hospital-Based Surveillance of Rotavirus Disease and Strains among Indian Children Aged <5 Years

BACKGROUND Current, nationally representative data on rotavirus disease burden and rotavirus strains in India are needed to understand the potential health benefits of rotavirus vaccination. METHODS The Indian Rotavirus Strain Surveillance Network was established with 4 laboratories and 10 hospitals in 7 different regions of India. At each hospital, children aged <5 years who presented with acute gastroenteritis and required hospitalization with rehydration for at least 6 h were enrolled. A fecal specimen was obtained and was tested for rotavirus with use of a commercial enzyme immunoassay, and strains were characterized using reverse-transcription polymerase chain reaction. RESULTS From December 2005 through November 2007, rotavirus was found in approximately 39% of 4243 enrolled patients. Rotavirus was markedly seasonal in northern temperate locations but was less seasonal in southern locations with a tropical climate. Rotavirus detection rates were greatest among children aged 6-23 months, and 13.3% of rotavirus infections involved children aged <6 months. The most common types of strains were G2P[4] (25.7% of strains), G1P[8] (22.1%), and G9P[8] (8.5%); G12 strains were seen in combination with types P[4], P[6], and P[8] and together comprised 6.5% of strains. CONCLUSIONS These data highlight the need for development and implementation of effective prophylactic measures, such as vaccines, to prevent the large burden of rotavirus disease among Indian children.

Disease and economic burden of rotavirus diarrhoea in India

We used published and unpublished studies and national statistics to estimate the number of deaths, hospitalizations, and outpatient visits due to rotavirus diarrhoea and the associated national economic burden of disease in India. Annually in India, rotavirus diarrhoea causes an estimated 122,000-153,000 deaths, 457,000-884,000 hospitalizations, and 2 million outpatient visits in children <5 years of age. India spends Rs 2.0-3.4 billion (US

Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area

41-72 million) annually in medical costs to treat rotavirus diarrhoea. The use of specific interventions against rotavirus, such as newly available vaccines, would help prevent much of this large disease and economic burden.

Epidemiological, clinical, and molecular features of norovirus infections in western India.

BACKGROUND/AIMS Hepatitis A and E viruses (HAV, HEV) are transmitted enterically and are highly endemic in India. This study aims to evaluate prevalence of these and TT virus (TTV) in the sewage. METHODS Influent and effluent samples from a sewage treatment plant from Pune, India were collected twice a week for 1 year and subjected to nested polymerase chain reaction (PCR) for the detection of HAV RNA, HEV RNA and TTV DNA. HAV and HEV PCR products were sequenced. Effluent samples were not collected for 5 months as the plant was non-functional. RESULTS The overall prevalence was 24.42% (21/86, HAV), 10.98% (9/82, HEV) and 12.7% (8/63, TTV). Prevalence of HAV was significantly higher than HEV (P=0.023). During summer months, significantly higher HAV RNA positivity was noted (P<0.01). A substantial reduction in HAV RNA positivity (15/48 vs. 2/48, P=0.0008) was recorded for treated sewage samples. However, HEV RNA or TTV DNA positivity did not reduce significantly. Of the 17 HAV and HEV RNA negative sewage samples concentrated using ultracentrifugation, 13 and none were positive for HAV and HEV RNA, respectively. Phylogenetic analyses grouped these viruses in IB and Ia, respectively, the genotypes most prevalent in India. CONCLUSIONS Sewage may play an important role in maintaining hyper-endemicity of these infections. Sustained efforts are obligatory to render sewage less/non-infectious.

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

The study was conducted to investigate the molecular epidemiology of noroviruses (NoVs) from western India. A total of 830 fecal specimens were collected during July 2005–June 2007 from children, ≤7 years of age suffering from acute gastroenteritis in Pune, Nagpur, and Aurangabad cities. All the specimens were subjected to RT‐PCR, sequencing and phylogenetic analysis for detection and characterization of Genogroup I (GI) and GII NoVs. NoV positivity varied between 6.3% and 12.6% in different cities with the predominance of GII (96.6%). NoV infections were very common in the patients ≤2 years of age. A majority (55%) of the patients suffered from severe disease, however, vomiting was not experienced in 35%. Coinfections with rotaviruses were found in 10% cases. Summer month seasonality supported NoV infections in western India. The phylogenetic analysis of partial RNA polymerase and VP1 (capsid) genes identified 2 GI (GI. 2 and GI.6) and 5 GII (GII.4, GII.6, GII.7, GII.8, and GII.14) genetic clusters with possible occurrence of “2007 new‐variant” of GII.4. Six different combinations of RdRp and capsid genes (GII.b/GII.3, GII.b/GII.4, GII.d/GII.3, GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13) were also identified. GII.4 (52%) prevailed in 2005–2006 while the predominance of probable recombinant NoV strains (58%) was noted in 2006–2007 with the contribution of GII.b/GII.3 at 79% level. GII.b/GII.18 type identified in 37% infections in 2005–2006 was completely replaced by GII.b/GII.3 type in 2006–2007. This is the first report that highlights the norovirus epidemiology and strain diversity demonstrating possible circulation of new variants in patients with acute gastroenteritis from western India. J. Med. Virol. 81:922–932, 2009.

Characterization of the non-polio enterovirus infections associated with acute flaccid paralysis in South-Western 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 group A rotavirus infections in adolescents and adults from Pune, India: 1993-1996 and 2004-2007.

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.

Molecular characterization of three novel intergenotype norovirus GII recombinant strains from western India.

A total of 1,591 fecal specimens were collected in 1993–1996 and 2004–2007 from adolescents and adults with acute gastroenteritis in Pune, India for detection and characterization of rotavirus. At the two time points, group A rotavirus was detected in 8.6% and 16.2% of the adolescents and 5.2% and 17.2% of the adults, respectively. Reverse transcription‐PCR with consensus primers followed by multiplex genotyping PCR detected common strains G1P[8], G2P[4], G3P[8], and G4P[8] in a total of 53.1% of the samples from 1993 to 1996, while the only prevalent strain identified in 2004–2007 was G2P[4] (23.5% of total). Uncommon rotavirus strains (G1P[4], G2P[8] G9P[6]/P[4]) increased from 7.8% (1993–1996) to 41.2% (2004–2007), while the prevalence of mixed rotavirus infections was high (39%/35%) at both time points. Mixed infections detected by multiplex PCR were confirmed by sequencing two or more individual genotype‐specific PCR products of the VP7 and VP4 genes from the same sample. Phylogenetic analysis of the sequences showed circulation of a heterogeneous rotavirus strain population comprising genotypes G1 (lineages I and IIb), G2 (lineages I and IIb), G4 (lineage Ia), P[4] (lineages P[4]‐5 and P[4]‐1), P[8] (lineages P[8]‐II and P[8]‐III), and P[6] (M37‐like lineage). The VP6 gene sequences of the nontypeable strains were most homologous to animal strains. This study documents the molecular epidemiology of rotavirus strains in adolescents and adults in India, and suggests that it may be important to monitor these strains over time for the potential impact on rotavirus vaccines under development for use in the Indian population. J. Med. Virol. 82:519–527, 2010.

Full genomic analysis of Indian G1P[8] rotavirus strains

The phenomenon of recombination has been widely described among noroviruses (NoVs) in the past few years. In a NoV surveillance study conducted in western India, 3 novel and 3 known combinations of RNA-dependent RNA polymerase (RdRp) and capsid genes were identified in genogroup (G) II NoV strains. The present study pertains to the characterization of three novel intergenotype NoV GII recombinant strains. RT-PCRs were carried out for the amplification of nearly complete RdRp and complete capsid genes spanning ORF1/2 overlap of three strains followed by sequencing of the amplicons. The recombination event was confirmed by phylogenetic analysis using Bayesian MCMC approach, SimPlot analysis and Maximum chi(2) method. Three novel intergenotype (GII) recombinations of GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13 specificities were identified respectively in the strains PC03, PC24 and PC25 for the first time. The breakpoint in the novel recombinants was placed in the vicinity of the 20 bp ORF1/2 overlap, a common hotspot known to exist in NoV recombinants. The capsid genes of all of the 3 recombinants were closely related to their counter parts in reference strains however, a high degree of variation emerged in the polymerase genes especially of PC24 and PC25 in comparison to the reference strains.

Identification and characterization of enteric adenoviruses in infants and children hospitalized for acute gastroenteritis.

Rotavirus G1P[8] strains are the most predominant cause of rotavirus diarrhea, worldwide and are an important component of currently licensed RotaTeq and Rotarix vaccines. Despite a significant contribution of these strains in causing diarrhea in Indian children, none of them has been characterized completely, to date. This issue was addressed in the present study by sequencing and phylogenetic analysis of complete genomes of 3 Indian rotavirus strains (06361, 0613158 and 061060) of G1P[8] specificity. Genotype of G1P[8] I1R1C1M1A1N1T1E1H1 respectively, for the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 genes was assigned to all of the three strains. The sequence analysis of structural and nonstructural genes indicated genetic relatedness (94-99.5%) with recently circulating strains and divergence (2.4-15.6%) with old prototype strains. Phylogenetic analysis revealed that new strains (Western Indian rotavirus strains and recently isolated strains--Dhaka16-03 (G1P[8]), Dhaka25-02 (G12P[8]), Matlab13-03 (G12P[6]), B3458 (G9P[8]), Matlab36-03 (G11P[8]), and B4633-03 (G12P[8]) and old prototype strains (KU and Wa) clustered in the same lineages of VP1, VP2, VP3, NSP2 and NSP4 genes however, grouped separately in VP6, NSP1 and NSP5 genes with 10-11%, 15.6-16.7% and 6.3-7.5% nucleotide sequence divergence, respectively. These results suggest that the rotavirus VP6, NSP1 and NSP5 genes of Wa-like rotaviruses are more prone to temporal mutations. Both structural and nonstructural genes of the Western Indian rotavirus strains shared nucleotide and amino acid substitutions with the Bangladeshi strain, Dhaka16-03 (G1P[8]) in the year 2003. This study documents for the first time the phylogenetic and evolutionary relationships of Indian G1P[8] rotavirus strains with the rotavirus strains from other parts of world and provides data useful for the evaluation of rotavirus vaccine programs.