Global spatiotemporal transmission dynamics of measles virus clade D genotypes in the context of the measles elimination goal 2020 in India.

Measles viruses (MeV) circulating in India mainly belong to genoypes D4 and D8 of clade D. In the context of measles elimination goal 2020 in India, molecular clock and phylogeography studies would help to identify the timescales of evolution and track the transmission pathways of MeV. We used nucleoprotein gene sequences (n = 756) from GenBank, representing 86 countries (1973-2016), to study the spatiotemporal transmission dynamics of clade D. Genotype D4 was introduced into India around 1991 and genotype D8 around 1994. Recent transmissions of the D4 genotype of measles virus (MeV) were noted from India to the United States of America and East Asia region while D8 genotype importations from North America were noted in recent years.

Genetic characterization of the glycoprotein G of Chandipura viruses in India with emphasis on an outbreak of 2015.

Chandipura virus (CHPV) is found to be associated with sporadic encephalitis outbreaks in humans in India since 1965. We report here, the investigation of CHPV activity during the period of June-August 2015 in the state of Gujarat, which revealed 24.44% positivity among 45 referred encephalitis cases. Phylogenetic study of the G gene sequences of strains from Gujarat 2015 along with available sequences of additional strains from different geographical locations and isolation years (1965-2015), indicated the relatedness of the 2015 strain to a group of the CHPV prototype strain of 1965 and the earliest outbreak strains of 2003. Analyses of selection pressure in the G gene revealed positively selected sites within the signal peptide region and a putative CHPV epitope. These results indicate a probable role of G protein-based immune selection and underline the need for continued surveillance to monitor genetic and antigenic variations in the CHPV.

A meta-analysis of the diagnostic accuracy of dengue virus-specific IgA antibody-based tests for detection of dengue infection.

Immunoglobulin A (IgA)-based tests have been evaluated in different studies for their utility in diagnosing dengue infections. In most of the studies, the results were inconclusive because of a small sample size. Hence, a meta-analysis involving nine studies with 2096 samples was performed to assess the diagnostic accuracy of IgA-based tests in diagnosing dengue infections. The analysis was conducted using Meta-Disc software. The results revealed that IgA-based tests had an overall sensitivity, specificity, diagnostic odds ratio, and positive and negative likelihood ratios of 73·9%, 95·2%, 66·7, 22·0 and 0·25, respectively. Significant heterogeneity was observed between the studies. The type of test, infection status and day of sample collection influenced the diagnostic accuracy. The IgA-based diagnostic tests showed a greater accuracy when the samples were collected 4 days after onset of symptoms and for secondary infections. The results suggested that IgA-based tests had a moderate level of accuracy and are diagnostic of the disease. However, negative results cannot be used alone for dengue diagnosis. More prospective studies comparing the diagnostic accuracy of combinations of antigen-based tests with either IgA or IgM are needed and might be useful for suggesting the best strategy for dengue diagnosis.

Phylogeographic analysis of Japanese encephalitis virus in India (1956-2012).

Japanese encephalitis virus (JEV) isolates from India phylogenetically belong to two genotypes, III and I. We used envelope gene sequences from GenBank, representing different states of India and other countries, to study the spatiotemporal transmission histories of these two JEV genotypes separately. Genotype III was found to have been successively introduced in the 1930s, 1950s and 1960s, followed by genotype I twice around 2003-2006. Changes in JEV disease patterns in India over the last five decades could thus be attributed to multiple introductions of JEV strains from neighboring Asian countries along with increased transmission potential due to altered ecological settings.

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses.

During 1960-80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences. All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A-D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782-1934) and DENV-4 (1719-1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938-1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India. The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.

Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962-2005).

Dengue is a major health problem in India with all four serotypes represented. Recently there has been an increase in the occurrence of dengue-1 outbreaks. It is possible that there have been changes in the genetics of dengue virus-1 (DENV-1), either by fresh introductions or by evolution in situ. The studies on DENV-1 evolution so far have no Indian sequences included. To gain insight into the dynamics of DENV-1 in India, the envelope (E) gene of thirteen virus isolates representative of the period 1962-2005 were sequenced and analyzed together with the available sequences of 40 globally representative isolates. All the Indian DENV-1 isolates were found to belong to the American African (AMAF) genotype. With the addition of 13 Indian isolates, the AMAF genotype can now be called Cosmopolitan. The Indian isolates were distributed into four lineages, India I, II, III and the Africa lineage, now called Afro-India. Of these, India III was the oldest and extinct lineage; the Afro-India was a transient lineage while India I, imported from Singapore and India II, evolving in situ, were the circulating lineages. Despite the extinction and introduction of lineages, no specific codon site was observed to be under selection pressure. The rate of nucleotide substitution estimated for DENV-1 was 6.5 × 10(-4) substitutions/site/year, and the time to the most recent common ancestor (tMRCA) was estimated to be 78-180 years (1825-1925), similar to previous estimates. The tMRCA for the AMAF/Cosmopolitan genotype was 56-98 years (1907-1949), a period that covers World War I and II. The two imports from Africa (1953-1968) and Singapore (1964-1975) and an export to the Americas (1955-1965) prove that there have been changes in the lineage of the DENV-1 viruses circulating in India which has contributed to the global dynamics of DENV-1 evolution and perhaps to the changing epidemiology of dengue in India.

A novel HBV recombinant (genotype I) similar to Vietnam/Laos in a primitive tribe in eastern India.

Genotyping of 20 strains of Hepatitis B virus (HBV) from the Idu Mishmi primitive tribe of northeast India identified multiple genotypes and the presence of a unique cluster grouping with strains from Vietnam and Laos identified as novel recombinants/genotype I. Sequence analysis (similarity and bootscan plots) of three complete HBV genomes from the tribe provided evidence of recombination. Phylogenetic analyses supported recombination between genotypes A, G and C. The Pre-S gene between nt 2943 and 397 was clearly of genotype A origin, whereas nt 397-1397 represented genotype G and nt 1397-2943 represented genotype C. Percentage divergence from genotypes B, D, E, F, G and H varied from 9.2 +/- 0.45% to 13.8 +/- 0.53%, whereas genotype A and C differed by 7.9 +/- 0.42% and 7.4 +/- 0.39% respectively. The identification of similar recombinant viruses in three countries, especially in a primitive tribe with no contact with the outside world suggests that these viruses do not represent recent recombination events, but circulation of closely related viruses highly divergent from known HBV genotypes and should be classified as members of genotype 'I'.

Full length genomes of genotype IIIA Hepatitis A Virus strains (1995-2008) from India and estimates of the evolutionary rates and ages.

With the changing epidemiology, outbreaks of Hepatitis A Virus (HAV) have been reported from different parts of India. To characterize HAV strains circulating in India (1995-2008), 6 full genome sequences of the predominant genotype, IIIA, were determined. Further, applying the Bayesian Markov Chain Monte Carlo (MCMC) framework to the full genomes of Indian HAV strains as well as other global strains (human as well as simian), we derived the mean nucleotide substitution rate and evolutionary timescales with emphasis on the age of genotype III and IIIA strains. The genomic length of all the 6 HAV isolates was 7464 nt excluding the poly A tract. Phylogenetic analysis confirmed that all the Indian isolates were close to Nor-21 (AJ299464) and HMH (AY644337) of subgenotype IIIA. The ORF of the isolates when compared within genotype III at amino acid level showed a highly conserved pattern. Under the best fit expansion population relaxed molecular clock model, the estimated mean substitution rate of the HAV full genomes (human and simian strains) was 1.73 x 10(-4) substitutions/site/year based on which the earliest transmission of HAV from simian to humans is estimated to have occurred about 3564 years ago. The mean substitution rate within human HAV full genomes under the same model was estimated to be 1.99 x 10(-4) substitutions/site/year. With this the mean age of genotype III strains was estimated to be 592 years while that of genotype IIIA was estimated to be 202 years. The time to the most common recent ancestor (tMRCA) of the Indian genotype IIIA isolates was calculated to be 116 years.

Hepatitis B virus: prevalence of precore/core promoter mutants in different clinical categories of Indian patients.

To determine the association of precore (Pre-C)/basal core promoter (BCP) mutants with clinical outcome of hepatitis B in Western India, 192 hepatitis B virus (HBV) infected individuals were investigated. HBV-DNA PCR positivity among asymptomatic hepatitis B surface antigen (HBsAg) positive carriers (61/100) was lower (P < 0.0001) than chronic hepatitis B (CHB), acute (P = 0.0001), and fulminant hepatitis B patients (P = 0.047). Pre-C status was based on restriction fragment length polymorphism (RFLP, n = 153) and sequencing (n = 118). Prevalence of Pre-C mutants was higher among carriers (23/61) than CHB (10/62, P = 0.0071) or acute (3/22; P = 0.037) patients. Children from carrier and CHB categories showed significantly higher circulation of Pre-C-wild than mutant HBV. Clinical manifestations were independent of BCP mutations (1762/64-T/A). Hepatitis B e antigen (HBeAg) negative CHB patients [62.5% (15/24)] were circulating wild HBV. Higher HBV-DNA levels were associated with chronic hepatitis and HBeAg positivity, whilst Pre-C mutant positives had lower levels. BCP mutations did not affect HBV-DNA levels. Multivariate regression analysis identified HBeAg (OR = 4.3) and Pre-C mutants (OR = 3.1) to be associated with chronic hepatitis and carriers respectively. In a separate sub-set analysis (n = 59), HBV-DNA level was identified as the only variable. In conclusion, chronic or fulminant hepatitis B was not associated with Pre-C or BCP mutants and switching over to Pre-C mutant was beneficial for the infected individual in maintaining disease free status for extended periods.

Swine HEV infection in south India and phylogenetic analysis (1985-1999).

Hepatitis E is endemic in India. It was recently noted that although all the Indian human hepatitis E virus (HEV) isolates (1976-2001) were placed in genotype I, the swine HEV recovered from western India (2000) belonged to genotype IV. This was in contrast to reports from the United States and Taiwan wherein both human and swine HEV belonged to the same genotype, i.e., genotypes III and IV, respectively. In order to validate these findings further, we retrospectively examined serum samples collected from pigs from southern India. Sequential serum samples from 45 (1985-1987) and 12 (1999) pigs from Karnataka state, south India, were screened for the presence of HEV RNA (nested PCR) and IgG-anti-HEV (ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. In this study, 42/45 (1985-1987) and 12/12 (1999) pigs showed seroconversion to IgG anti-HEV antibodies, with a mean age at seroconversion of 4.8 +/- 1.6 months. Four samples collected in 1999 and two samples collected during 1985 were HEV RNA positive. All swine HEV sequences clustered with genotype IV, demonstrating that swine HEV was prevalent among south Indian pigs for at least for 16 years and, similar to western India, belonged to genotype IV. Thus, genotype I and IV HEV continue to circulate in humans and pigs, respectively, from India. Whether swine HEV infects humans remains to be determined.

Hepatitis E antibody profiles in serum and urine.

The aim of this study was to evaluate anti-HEV antibody profiles in urine specimens in comparison to corresponding serum samples to assess the utility of urine as a clinical specimen. Paired serum and urine specimens from 71 hepatitis E patients, 33 non-E hepatitis patients, 63 patients with nonhepatic diseases, and 26 healthy individuals were tested by recombinant HEV protein (55 kD)-based indirect enzyme-linked immunosorbent assay (ELISA). Uronegativity for anti-HEV IgM was noted in 71 (100%) serologically confirmed patients with hepatitis E. Hepatitis E patients (10/10) showed urinary absence or very low levels of total IgM by capture ELISA, suggesting absence or low levels of filtration, and/or local synthesis, and/or transudation of IgM in urine during infection. When these patients were tested for total IgG and IgA, microquantities of immunoglobulins were noted in all urine samples (10/10 for each). However, the proportions of uropositivity for anti-HEV IgG and IgA in hepatitis E patients were low and indicated only 21.42% and 49.33% concordance with seropositivity, respectively. Control groups also showed low and variable uropositivity for anti-HEV IgG and IgA. Overall, HEV-specific antibodies exhibited by serum in recent and past infections were not found in urine. The study demonstrated the inadequacy of urine specimens for detection of hepatitis E antibodies.

Changing epidemiology of hepatitis A and hepatitis E in urban and rural India (1982-98).

The epidemiology of hepatitis A virus (HAV) and hepatitis E virus (HEV) was assessed among age-stratified urban high socioeconomic, lower middle socioeconomic status and rural populations from western India in 1998. When compared with previous surveys, a clear shift from high to intermediate endemicity of HAV was evident only for higher socioeconomic population (1982-98), raising the possibility of outbreaks of hepatitis A in this category. A decrease in anti-HAV positivity was noted in rural children aged 6-10 years. Lower circulation of HEV was noted among < 25-year-old urban higher socioeconomic and rural individuals. For both viruses, the lower middle socioeconomic populations were comparable in 1982 and 1998. Socioeconomic status and family size (odds ratio = 23 and 1.6, respectively) were independently associated with anti-HAV positivity. Age, lower middle socioeconomic status and well water were significant independent variables for HEV infection (odds ratio = 5.7, 2.4 and 1.9, respectively). Hence, vaccination policy for hepatitis A needs to be reviewed.

Hepatitis G virus infection in India: prevalence and phylogenetic analysis based on 5' non-coding region.

OBJECTIVES: To determine the prevalence of hepatitis G virus (HGV) infection in western India and to carry out phylogenetic analysis of HGV isolates. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect HGV RNA in serum samples obtained from paid plasma donors, patients with hemophilia and voluntary blood donors. Nine Indian and one Kenyan HGV RNA-positive samples were sequenced in the 5' non-coding region (5'-NCR). Phylogenetic analysis based on the comparison of a 101 nucleotide fragment from a large number of HGV isolates from 22 countries (including Indian and Kenyan sequences obtained during the present study) was carried out. RESULTS: HGV RNA positivity rates among paid plasma donors from a commercial plasmapheresis unit (7/43, 16.3%) and patients with hemophilia (5/44, 11.4%) were significantly higher than that in voluntary blood donors (0/51; p=0.003 and 0.019, respectively). Among patients with acute non-A to E hepatitis and fulminant hepatic failure, 1 of 50 and 1 of 28 were HGV RNA-positive, whereas 6 of 49 (12%) patients with chronic liver disease had circulating HGV RNA. All Indian isolates belonged to genotype 2, whereas the Kenyan isolate formed a distinct branch within genotype 1 consisting of African isolates. CONCLUSION: Our results suggest existence of parenteral transmission of HGV in the Indian population. HGV was not an important cause of acute non-A to E hepatitis or fulminant hepatic failure among the patients investigated. Genotype 2 seems to be the most prevalent genotype in western India.

Prevalence and phylogenetic analysis of TT virus DNA in Western India.

In western India, TT virus (TTV) DNA positivity varied from 6.7% (5 of 75) in chronic hepatitis patients to 24.4% (10 of 41) in hemophiliacs; 7.4% (4 of 54) of voluntary blood donors had circulating TTV DNA. Phylogenetic analysis revealed a predominance of genotype 1a. In India, TTV is transmitted mainly by nonparenteral routes and is not an important cause of chronic liver diseases.

Retrospective serological analysis of hepatitis E patients: a long-term follow-up study.

The aim of this study was to evaluate the persistence and protective role of antibodies to hepatitis E virus (anti-HEV) after natural hepatitis E infection. A retrospective analysis of immunoglobulin G (IgG) anti-HEV was performed in 37 patients followed-up for 5 years after epidemics of HEV. Two patients with sporadic hepatitis E (HE) were followed-up for 12 and 8 years. All patients infected during epidemics of HE were positive for IgG anti-HEV at 5 years of follow-up (geometric mean titre: 174.75). The two patients with sporadic HE were positive for IgG anti-HEV at the end of 12 and 8 years of follow-up (the IgG anti-HEV titre was 1: 200 in each patient). This study showed protection against disease by antibodies to HEV. It was therefore concluded that hepatitis E may be preventable by an efficacious vaccine.

Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993).

Seventeen Indian hepatitis E virus (HEV) isolates, representing epidemic and sporadic hepatitis E cases during 1976-1991, were sequenced in the RNA polymerase (RNAP) region. Five isolates were also sequenced in the non-structural hypervariable region of open reading frame 1. Open reading frames 2 and 3 were sequenced only for the prototype isolate. On the basis of the comparison of all the available sequences of the conserved RNAP region, the HEV isolates were divided into three genotypes, differing from each other by >15%. Genotype I included African and Asian isolates, whereas II and III were represented by Mexican and US isolates, respectively. Genotype I was further divided into four sub-genotypes. The majority of the Indian isolates (15/20), along with the Burmese and Nepali isolates, belonged to genotype IA. Genotype IB included HEV isolates from China, Pakistan and the former USSR and 2/20 Indian isolates, which represented the oldest (1976) HEV sequenced so far. Genotype IC included both the African isolates, whereas 3/20 Indian isolates formed genotype ID. Nucleotide sequence analysis of other regions of the HEV genome also placed isolates in the same genotypes. Both the Indian cities experiencing second HEV epidemics, after intervals of 8 and 10 years, showed shifts in the sub-genotypes found; from IB (Ahm-76) to IA (Ahm-84) and from IA (Kol-81) to ID (Kol-91). However, no major shift in the genotypes was noted. Overall, HEV genotypes appear to be segregated geographically.

Potency testing of inactivated tissue culture vaccine against Japanese encephalitis using antigen capture ELISA.

The potency of vaccines against Japanese encephalitis (JE) is usually determined by a mouse challenge test. In the present study, an antigen capture enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (MoAbs) was used to screen inactivated tissue culture JE vaccine lots. This test is simple, quick and reliable besides being very sensitive and specific.