Hepatitis B virus genotypes and serotypes in western India: lack of clinical significance

To determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection.     

Molecular Studies on Dengue Viruses Detected in Patients from Central India

Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna. Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes. Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016. Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring.     

Genotype determination of hepatitis C virus from Northern India: Identification of a new subtype

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68–79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the nonstructural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5′-noncoding (5′ NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India. © 1996 Wiley-Liss, Inc.     

Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus

Using phylogenetic analysis and pairwise comparison of 670 complete hepatitis B virus (HBV) genomes, we demonstrated that nucleotide divergence greater than 7.5% can be used to separate strains into genotypes A-H. Strains can be separated into subgenotypes when two criteria are met: nucleotide divergence of about 4% but less than 7.5% and good bootstrap support. There is a highly statistically significant association between serological subtypes and genotypes (chi2-test for association, P < 0.0001): adw is associated with genotypes A, B, F, G, and H, adr with C and ayw with D and E. The logistic regression method showed that 1802-1803CG are characteristic of genotypes A, D, and E whereas 1802-1803TT are characteristic of genotypes B, C, and F. 1858C is positively associated with genotypes A, F, and H and 1858T with genotypes B, D, and E. Subgenotypes C2, F1/F4 can be differentiated from subgenotypes C1, F2/F3, respectively, because the latter have 1858C as opposed to 1858T in the former. 1888A was positively associated with subgenotype A1 and TAA at 1817 with genotype G. The Haploplot method revealed high linkage between loci 1858 and 1896 but strong evidence of recombination between loci 1862 and 1896. Loci 1809-1812, 1862, and 1888 may have co-evolved. Using a computer program, we showed that serological subtype deduced from the S region (position 155-835) and mutations/variations within the basic core promoter/precore region (1653-1900), allowed genotyping of HBV with 97% sensitivity and 99% specificity. Certain subgenotypes or subgenotype groups could also be differentiated.     

Molecular characterization of Chikungunya virus during an outbreak in South India

Introduction: Re-emergence of Chikungunya is a major public health problem in the southern states of India. Objectives: This study was undertaken to investigate an outbreak of Chikungunya, in June–August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. Materials and Methods: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. Results: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368–GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. Conclusion: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.     

The detection and characterization of hepatitis E virus in pig livers from retail markets of India

The zoonotic transmission of Hepatitis E virus (HEV) is a well-established fact and pigs are known reservoirs of the virus. The human and swine HEV from countries such as the USA, Japan and Taiwan, show a remarkable sequence identity. Swine liver samples from markets in Japan and US were shown to be HEV RNA positive. In contrast, in India, viruses belonging to different genotypes, that is, genotypes 1 and 4 circulate in humans and pigs respectively, at least for the last 20 years. To assess possible exposure of the Indian population to swine HEV, 240 pig liver samples were collected from retail markets of Pune, western India. Two (0.83%) samples were found positive for HEV RNA by nested RT-PCR. Nucleotide sequence-based phylogenetic analysis showed the presence of genotype 4 HEV with 90-91% similarity and clustering with Indian swine HEV sequences generated earlier. The data suggest the possibility of HEV infection in persons consuming infected pig liver. So far, it has not been possible to detect any type 4 HEV infection in humans. The absence of type 4 infections in humans may be attributed to cooking leading to the inactivation of the virus.     

Gaucher disease and parkinsonism: a phenotypic and genotypic characterization

Among the many phenotypes associated with Gaucher disease, the inherited deficiency of glucocerebrosidase, are reports of patients with parkinsonian symptoms. The basis for this association is unknown, but could be due to alterations in the gene or gene region. The human glucocerebrosidase gene, located on chromosome 1q21, has a nearby pseudogene that shares 96% identity. Immediately adjacent to the glucocerebrosidase pseudogene is a convergently transcribed gene, metaxin, which has a pseudogene that is located just downstream to the glucocerebrosidase gene. We describe a patient with mild Gaucher disease but impaired horizontal saccadic eye movements who developed a tremor at age 42, followed by rapid deterioration of her gait. A pallidotomy at age 47 was unsuccessful. Her motor and cognitive deterioration progressed despite enzyme replacement therapy. Sequencing of the glucocerebrosidase gene identified mutations L444P and D409H. Southern blot analysis using the enzyme SspI showed that the maternal allele had an additional 17-kb band. PCR amplifications and sequencing of this fragment demonstrated a duplication which included the glucocerebrosidase pseudogene, metaxin gene, and a pseudometaxin/metaxin fusion. Gene alterations associated with this novel rearrangement, resulting from a crossover between the gene for metaxin and its pseudogene, could contribute to the atypical phenotype encountered in this patient.     

Hepatitis B virus genotype D strains from Estonia share sequence similarity with strains from Siberia and may specify ayw4

The genotypes and subtypes of 205 HBV isolates collected during 1989-2002 in Estonia and 14 other regions of the former USSR were determined by sequencing and phylogenetic analysis of the S gene. The in Europe prevailing genotypes, A and D, were also circulating in the whole territory of the former USSR including Estonia and accounted for 18.5 and 81% of the strains, respectively. All genotype A strains specified adw2, and a single genotype C strain specified adrq+. Most genotype D strains specified ayw3 and ayw2, although, three strains from Estonia and Siberia specified ayw4. Due to unique substitutions, Ser122 and Ala127, four strains could not be classified according to the subtype. One strain specifying ayw3 encoded Leu143 and Ala145 and was possibly an immune "escape" mutant. At phylogenetic analysis 93% of the Estonian genotype D strains belonged to a cluster specifying mainly ayw3 and were more similar to isolates from Siberia and the Far-East of Russia than to isolates originating from Central Russia which belonged to another cluster of strains specifying mainly ayw2. This pattern might be explained by part of the Estonian population, has roots east of European Russia, based on linguistic evidence. Eight dominant HBV strains represented by identical S gene sequences were identified, one within genotype A and seven within genotype D, three of which included isolates from Estonia and Siberia. Some of these strains were collected over a period of at least 13 years indicating there are genetically stable variants of HBV that remain conserved over decades.     

Analysis of the full-length genome of a subgenotype IIIB hepatitis A virus isolate: primers for broadly reactive PCR and genotypic analysis

Among six known subgenotypes (IA, IB, IIA, IIB, IIIA, and IIIB) of human hepatitis A virus (HAV), the complete genomic sequence has not been determined for IIIB. In this study, the full-length genomic sequence of a IIIB HAV isolate (HA-JNG06-90F) recovered from a Japanese patient who contracted sporadic hepatitis A in 1990, was determined. The HA-JNG06-90F genome, which comprised 7462 nt excluding the poly(A) tail, was related most closely to NOR-21 of subgenotype IIIA with an identity of 89.1%, and was only 82.6-83.4% similar to human HAV isolates of genotypes I and II over the entire genome. Comparison of full-length genomic sequences of 20 reported isolates and HA-JNG06-90F generated optimal results for separation of different levels: the nucleotide identities were 80.7-86.6% at the genotype level, 89.1-91.9% at the subgenotype level, and 94.6-99.7% at the isolate level. Similar ranges of nucleotide identity were observed when comparing partial nucleotide sequences of the VP1-2B (481 nt; primer sequences at both ends excluded) and 3C/3D (590 nt) regions, which were amplifiable by PCR with primers designed from well-conserved areas of the HAV genome. All 66 samples with IgM-class HAV antibodies tested positive for HAV RNA by both VP1-2B (481 nt)-PCR and 3C/3D (590 nt)-PCR: subgenotype assignment was concordant in all samples tested (IA [n = 61], IB [n = 1], IIIA [n = 2] and IIIB [n = 2]). These results suggest that two broadly reactive PCRs using primers derived from the VP1-2B and 3C/3D regions, respectively, may be applicable to universal detection and phylogenetic analysis of various HAV strains.     

Molecular Characterisation and Phylogenetic Analysis of Dengue Outbreak in Pasighat,Arunachal Pradesh,Northeast India

Background and Objectives: Dengue is one of the most prevalent arboviral diseases in the world with 390 million dengue infections per year. In this study, we report the molecular characterisation of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India during 2015. Subjects and Methods: A total of 613 dengue-suspected cases were screened for dengue virus by dengue NS1 Ag and anti-dengue IgM antibody depending on the duration of sample collection and onset of symptom. Further, molecular characterisation was done by amplifying the C-PrM region by real-time polymerase chain reaction followed by phylogenetic analysis. Results: Molecular characterisation revealed that the dengue outbreak was predominantly due to dengue virus serotype-1 (DENV-1) (90.9%) while DENV-2 was detected in 7.5% of samples. Co-infection of DENV-1 and DENV-2 was detected in one case. Phylogenetic analysis of the DENV-1 strains with the prototype revealed that the DENV-1 strains were grouped within genotype III. Similarly, DENV-2 strains were clustered within genotype IV. The study revealed a change in the predominant serotype in recent years with DENV-3 in 2012 to DENV-1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in the study region. A unique L24M mutation was observed in the DENV-1 strains of Arunachal Pradesh which was absent in all the circulating strains in India except one strain from the state of Kerala in South India. Marked variation within the DENV-2 strains was observed at A102V and I163V in one strain similar to earlier circulating isolates in India. Conclusions: The present study reveals a shift in the serotype dominance in the study region. As serotype shifts and secondary infection with a heterologous DENV serotype are frequently associated with disease severity, there is an urgent need for sustained monitoring of the circulating serotypes and enhanced surveillance operations, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this region.     

Molecular characterization of clinical isolates of Cyclospora cayetanensis from patients with diarrhea in India

Purpose: Cyclospora cayetanensis is an intestinal coccidian protozoan that has emerged as an important cause of both epidemic and endemic protracted diarrhea worldwide. Though humans appear to be the only natural hosts; the role of animals as natural reservoir is uncertain but of increasing concern. The present study aimed to study the prevalence of coccidian in different groups such as immunocompromised, clinically apparent immunocompetent and healthy individuals. Also, the study isolates were assessed for heterogeneity among the sequences. Materials and Methods: Stool samples from different groups of patients were collected. The parasite was detected in stool by different diagnostic tools such as light microscopy and nested PCR-restriction fragment length polymorphism using 18S ribosomal RNA as the target gene. Results: The prevalence of C. cayetanensis was 2.4% (19/800) in the present study. The PCR assay amplified Cyclospora cayetanensis DNA in only 89% (17/19) isolates. Further, sequencing revealed no significant difference among the study isolates and the non-primates. Phylogenetic analysis of the study isolates however, formed two clusters. While one cluster showed close evolutionary association with the C. cayetanensis strains, the other cluster showed evolutionary association with the two non-primate species. Conclusion: The methods described here for detection of C. cayetanensis oocysts are simple, efficient, specific, and sensitive and therefore can be effectively applied for laboratory diagnosis and environmental assessment of fresh produce and water sources. Clinicians should include Cyclospora infection in the differential diagnosis of prolonged or relapsing diarrheal illness even in clinically apparent immunocompetent individuals.     

Molecular and epidemiological profiles of hepatitis C virus genotype 4 in Denmark

The prevalence of hepatitis C virus (HCV) genotype 4 has increased throughout Europe. This is an epidemiological study of patients infected chronically with HCV genotype 4 in Denmark. The HCV strains analyzed originated from patient samples collected between 1999 and 2007 as part of the national Danish hepatitis B and C network, DANHEP. Sequence analyses were based on the envelope 1 region of HCV. Results from a total of 72 patients indicated a high degree of genetic heterogeneity. Fifty‐six patients (78%) were infected with one of the three dominating subtypes: 4d, 4a, or 4r. The remaining 16 patients (22%) were infected with subtypes 4h, 4k, 4l, 4n, 4o, or 4Unclassified. Three epidemiological profiles were identified: (1) patients infected with HCV by intravenous drug use were infected solely with subtype 4d. They were all of European origin, and 15 of the 16 patients were ethnic Danes. No single transmission event could be confirmed, but the pairwise nucleotide identity within the patients of Danish origin was relatively high (～95%), suggesting a recent introduction into Denmark. (2) The 21 patients infected with subtype 4a all came from Northern Africa, Egypt, Pakistan, or the Middle East. (3) Patients from Southern Africa dominated among patients infected with subtype 4r (10 of 12 patients). This study demonstrates that HCV genotype 4d has been introduced in and spread among Danish intravenous drug users. The remaining subtypes show restricted distribution, infecting almost exclusively patients from geographical areas with a relatively high prevalence of HCV genotype 4 infections. J. Med. Virol. 82:1869–1877, 2010. © 2010 Wiley‐Liss, Inc.     

Prevalence and genotypic distribution of TT virus in Athens, Greece

The prevalence of TT virus (TTV) infection in various population groups from Athens, Greece, was assessed by the polymerase chain reaction (PCR) using two primer sets from distinct regions of the genome: the conventional set derived from the open reading frame-1 (ORF-1) and the new, highly sensitive set targeting the region that includes the TATA signal localized upstream of ORF-2. Based on both primer sets, TTV DNA was detected in 42/50 (84.0%) healthy individuals, 42/50 (84.0%) chronic hepatitis C patients, 31/39 (79.5%) acute non-A-E hepatitis patients (group I), 14/16 (87.5%) renal failure patients with acute non-A-E hepatitis (group II), 47/50 (94.0%) intravenous drug users (IVDU), 36/50 (72.0%) hemophiliacs, and 21/31 (67.7%) hemodialysis patients. The presence of TTV was not associated with any particular risk group, and no differences were observed in relation to demographic, biochemical and virological characteristics between TTV DNA-positive and -negative patients. TTV did not seem to have a profound effect on the course of chronic C or acute non-A-E hepatitis either. Phylogenetic analysis revealed that TTV strains circulating in the greater metropolitan area of Athens belong not only to the G1 and G2 genotypes that are encountered worldwide, but also to G3 and to G5 that are found mainly in Europe and Asia, respectively. Further studies will shed light on the role of this highly prevalent virus.     

Serological and molecular studies on subclinical hepatitis E virus infection using periodic serum samples obtained from healthy individuals

Subclinical hepatitis E virus (HEV) infection among healthy individuals was studied serologically and molecularly. Serum samples collected at screening between March and April 2004 (or just before retirement) from 266 medical staff members (35 males, 231 females) who had been working for 8.8 +/- 8.5 (mean +/- standard deviation, range, 0.3-35.1) years in a city hospital in Japan and serum samples that had been collected from these staff members at the start of employment were tested for IgA, IgM, and IgG antibodies to HEV (anti-HEV) by in-house enzyme-linked immunosorbent assays. Overall, six subjects (2.3%) tested positive for anti-HEV IgG at the screening; among them, four subjects (1.5%) had already been positive for anti-HEV IgG at the start of employment and two subjects (0.8%) seroconverted after initiation of employment. Periodic serum samples that had been collected from the two seroconverted subjects were tested for HEV antibodies and HEV RNA. The two subjects became positive for anti-HEV IgG in 1978 or 2003, respectively, with no discernible elevation in alanine aminotransferase (ALT) level, and continued to be seropositive up through the screening date. Although anti-HEV IgM was not detectable in the two subjects, one was infected transiently with Japan-indigenous HEV strain of genotype 3 and the other was positive transiently for anti-HEV IgA. The present study indicates that even an individual with subclinical HEV infection had evidence of transient viremia in the absence of ALT elevation and that anti-HEV IgA detection may be useful for serological diagnosis of recent subclinical HEV infection.     

Molecular characterization of hepatitis A virus isolates from Argentina

Hepatitis A, a vaccine preventable disease, is now of transitional or intermediate endemicity in Argentina, as the epidemiologic pattern of the disease has shifted with improvements in living conditions in some parts of the country. Increase in the susceptibility of older children and adults has led to increasing disease incidence. Molecular epidemiology has played an important role in the understanding of HAV infection by identifying modes of spreading and by permitting the monitoring of changes in circulating virus brought about by prevention programs. South American isolates characterized are limited. Eighty-two sporadic and outbreak isolates from Argentina were sequenced in the VP1/2A region of HAV genome over a 9-year period. All the isolates belonged to subgenotype IA. All our sequences grouped into two big clusters. Apparently, at least two lineages have been co-circulating in the same place at the same time. Despite great genetic variability, few point amino acid changes could be deduced. Four sequences showed an Arg --> Lys substitution at 1-297 which characterized the genotype IB at the amino acid level. Many isolates carried a conservative amino acid substitution Leu --> Ile at position 42 of the 2A domain, previously described as a possible fingerprint of HAV sequences in Brazil. The other rare changes have been found before, except for a 1-277 Asn --> Ser substitution displayed in two isolates that has not been previously reported. Argentina recently implemented universal vaccination in 1-year-old children. Molecular tools would be useful in an active surveillance program.     

Comparison of hepatitis B virus subgenotypes in patients with acute and chronic hepatitis B and absence of lamivudine-resistant strains in acute hepatitis B in Japan

Hepatitis B virus (HBV) has been classified into eight genotypes and can be further divided into several subgenotypes that have different geographic distributions. Because of increased human migration, the prevalence of rare subgenotypes is increasing in Japanese patients with acute hepatitis B. Lamivudine-resistant strains of HBV have begun to emerge in association with chronic hepatitis B. The aim of this study was to investigate the distribution of HBV subgenotypes and lamivudine-resistant strains in patients in Japan with acute hepatitis B. One hundred twenty-three patients with acute hepatitis B and 123 with chronic hepatitis B were studied. HBV subgenotypes and lamivudine-resistance mutations were determined by direct sequencing of the preS and polymerase region, respectively. HBV subgenotypes Aa (n=3), Ae (n=23), Ba (n=7), Bj (n=3), Cs (n=7), Ce (n=76), D (n=2), and H (n=2) were detected in patients with acute hepatitis. In patients with chronic hepatitis, HBV subgenotypes Ae (n=4), Ba (n=1), Bj (n=18), and Ce (n=100) were found. Non-common Japanese subgenotypes, that is, non-Bj and non-Ce, were detected more frequently in patients with acute hepatitis (35.8%) than in patients with chronic hepatitis (4.1%) (Odds ratio, 0.076; 95%CI, 0.029-0.200; P<0.0001). Lamivudine-resistance mutations were detected in chronic hepatitis patients with breakthrough hepatitis but not in other patients. In conclusion, the prevalence of uncommon Japanese HBV subgenotypes is expected to increase, although lamivudine-resistant strains have not yet been found in patients with acute hepatitis B.     

Characterization of seven genotypes (A to E, G and H) of hepatitis B virus recovered from Japanese patients infected with human immunodeficiency virus type 1

To investigate the prevalence of hepatitis B virus (HBV) genotypes and characteristics of HBV isolates among Japanese patients infected with human immunodeficiency virus type 1 (HIV), serum samples collected between September 1990 and March 2002 from 471 HIV-infected patients (age, 38.8 +/- 11.4 [mean +/- standard deviation] years; male, 90%) were tested for hepatitis B surface antigen (HBsAg) and HBV DNA. Positivity for HBsAg and HBV DNA was seen in 42 patients (8.9%), 41 of whom had contracted HIV infection through sexual activity and 1 had hemophilia. Genotypes of HBV were determined by comparative and phylogenetic analyses of the S gene sequence (396 nucleotides [nt]). The distribution of HBV genotypes among the 42 HBV-viremic patients was: A (50%), B (5%), C (24%), D (5%), E (2%), H (10%), A plus D (2%), A plus G (2%). The hemophilia patient had HBV genotype D. Genotypes E, G, and H which had not been reported in Japan, were found in one patient each who had traveled to Zambia, the US, and South America, respectively. Genotypes A and D, which are rare in Japan, were found in patients who had no history of traveling abroad. The entire genome of the HB-JI411 (genotype E [3,212 nt]), HB-JI444G (genotype G [3,248 nt]), and HB-JI260 (genotype H [3,218 nt]) isolates had the highest identity of 98.3%, 99.9%, and 98.5%, respectively, with reported HBV isolates of the same genotype. Most Japanese patients coinfected with HIV and HBV had HBV genotypes that are found rarely or had not been reported in Japan.