Genetic analysis of HAV strains isolated from patients with acute hepatitis in Korea, 2005-2006

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis, which represents a significant public health problem. HAV is usually transmitted by oral-fecal route and prevalent not only in developing countries but also in developed countries worldwide. To characterize the HAV wild type strains circulating in Korea, the VP3/VP1 and VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives during 2005 and 2006. Among 160 HAV IgM positive sera, 30% (n = 48) were positive for HAV RNA. Additionally, the VP3/VP1 junction regions were detected all six stools, which collected from outbreak in Gyeonggi province. Phylogenetic analysis of the sequences obtained from 54 distinct HAV isolates revealed that most of the strains (n = 45) belonged to genotype IA and the others including nine strains belonged to genotype IIIA. Interestingly, a Q --> S amino acid change was dominantly observed at position 810 of the VP1/P2A junction region in 14 isolates. The molecular epidemiology of HAV infection in Korea has changed with the co-circulation of at least two genotypes and 810Q --> S amino acid substitutions were found to be prevalent. These results strongly suggest that various HAV strains, including genotype IIIA, might be imported from high-endemic countries into Korea.     

Partial sequence analysis of VP1 of Indian isolates of foot-and-mouth disease virus type Asia-1

Nucleotide sequence of 3' end of VP1 (1D region) was determined using RT-PCR amplified DNA of 31 foot and mouth disease virus (FMDV) type Asia-1 field isolates originating from 11 different geographically distinct states of India during the period 1987-2000. These field strains exhibited an average of 7.5% divergence among them and were found to be divergent from the Indian vaccine strains Asia-1 WBN 117/85, IND 8/79, and IND 63/72, by an average 5.9, 14.8, and 7.4% divergence, respectively. Phylogenetic analysis of these 31 field isolates including 3 of the vaccine strains of India and sequences of 22 Indian field isolates obtained from the GenBank revealed that all the Indian FMDV type Asia-1 isolates belonged to a single genotype comprising of two distinct lineages (Lineages A and B). All the field isolates under study belonged to the Lineage-B comprising 8 different clusters, which also includes the vaccine strains WBN-117/85 and IND 8/79. Surprisingly, another vaccine strain IND 63/72 formed Lineage-A. Phylogenetic analysis of sequences of another 23 exotic type Asia-1 isolates from 15 different countries obtained from the GenBank along with the 56 Indian isolates revealed the existence of three distinct genotypes. The prototype strain Asia-1 PAK 1/54 belongs to a separate genotype. Two strains from India along with one strain each from China and Russia belongs to another genotype. The third genotype is formed by the remaining isolates including all the 31 isolates from the present study and exotic viruses from 14 other different countries. Comparison of deduced amino acid (aa) sequence indicated that majority of the mutations were found within two distinct regions corresponding to amino acid positions 130-160 and 193-211. The motif at aa positions 138-141 in vaccine strains WBN 117/85, IND 8/79 and in all the field isolates was ETTS/P; however, the same motif in IND 63/72 was TQPT. The motif 153-156 in majority of Indian isolates including vaccine strains WBN 117/85 and IND 8/79 was LSGQ/R whereas the same motif seen in IND 63/72 was VSNR. The study revealed that the FMDV type Asia-1 isolates circulating in the country are not highly heterogeneous, but showed considerable genetic variations. Certain mutations were also observed in the residues, which have been proved to be contributing to the formation of neutralizing epitopes. In neutralization studies employing polyclonal antisera, type Asia-1 WBN 117/85 revealed broader serological spectrum than other vaccine strains of India used in this study.     

Genetic and antigenic analysis of type A foot-and-mouth disease viruses isolated in India during 1987-1996

Twenty-three foot-and-mouth disease virus (FMDV) type A field isolates, recovered from different outbreaks during 1987-1996 in India, were subjected to antigenic and genetic analysis. The isolates showed a close antigenic relationship to the current vaccine strain (IND 17/77) in micro-neutralization test conducted using a vaccine strain (IND 17/77) antiserum and a peptide (aa 136-151 of VP1 protein of the A22/Azerbaijan/65 strain) antiserum. However, the isolates revealed minor antigenic differences in their reactivity with three neutralizing monoclonal antibodies (MAbs) recognizing trypsin-sensitive conformation-independent epitopes of the vaccine virus strains. Phylogenetic relationship between the isolates was carried out employing a part of the 1D gene (168 nucleotides at the 3'-end). Additional seven type A Indian field isolates reported earlier were included in the analysis. The percent similarity among the Indian isolates varied from 82.7% to 99.4% at nucleotide level, and from 83.9% to 100% at amino acid level. These observations clearly demonstrate genetic heterogeneity of the field isolates. The current vaccine strain IND 17/77 showed divergence of 9.7% at nucleotide level and 5.6% at amino acid level from the A22 Iraq 24/64 isolate. The field strains were divergent from the vaccine strain IND 17/77 by 5.6%-14.6% and 3.7% 13.7% at nucleotide and amino acid level, respectively. In the phylogenetic tree, the isolates were distributed into 21 genetic groups. The clustering pattern of the isolates in the phylogenetic tree revealed no specific distribution pattern of the foot-and-mouth disease (FMD) outbreaks in relation to their geographical locations, caused by unrestricted animal movement and endemic nature of the disease.     

Genotyping of acute hepatitis a virus isolates from China, 2003-2008

Hepatitis A virus (HAV) is usually transmitted by an oral–fecal route and is prevalent not only in developing countries but also in developed countries. In the present study, the phylogenetic characterization of the VP1/2A junction region (321 nucleotides) of China HAV isolates was examined. Anti‐HAV IgM‐positive serum samples were collected from 8 provinces, including 20 cities or counties in China from 2003 to 2008; 337 isolates from 406 HAV patients' serum samples were amplified by RT‐PCR, sequenced at the VP1/2A junction region and aligned with the published sequences from GenBank to establish phylogenetic analysis. All China HAV isolates in this study belonged to genotype I, with 98.8% (333/337) of samples clustering in sub‐genotype IA and 1.2% (4/337) in sub‐genotype IB. In addition, sub‐genotype IA isolates clustered into four groups (92.7–100% nucleotide identity), and the samples collected from all China HAV isolates in this investigation showed 87.5–100% nucleotide identity, but the amino acids in this region were more conserved (95.2–100% identity). Few unique amino acid changes could be deduced (VP1‐253: Glu → Gly; 2A‐34: Pro → Ala; 2A‐33: Leu → Phe). Genetically identical or similar HAV strains existed in some investigated areas in China during different years, suggesting that an indigenous strain has been circulating in those regions. This report provides new data on the genetic relatedness and molecular epidemiology of HAV isolates from China as well as the distribution of sub‐genotype IA and IB in this part of the world. J. Med. Virol. 83:1134–1141, 2011. © 2011 Wiley‐Liss, Inc.     

Nucleotide sequence and phylogenetic analysis of a segment of a highly virulent strain of infectious bursal disease virus

The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.     

Full-length sequences of subgenotype IIIA and IIIB hepatitis A virus isolates: characterization of genotype III HAV genomes

To elucidate the extent of genomic heterogeneity of human hepatitis A virus (HAV) strains and to characterize genotype III HAV strains over the entire genome, the full-length sequence of three subgenotype IIIA isolates (HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F) and one IIIB isolate (HAJ85-1F) was determined. The HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F genomes which comprised 7463 or 7464 nt excluding the poly(A) tail, were closest to a reported nearly entire sequence of a IIIA isolate (NOR-21) with identities of 94.4-97.8% over the entire ORF sequence, and the HAJ85-1 genome (7462 nt) to HA-JNG06-90F of IIIB with an identity of 98.6%. The phylogenetic trees constructed based on the complete ORF sequence or the 168-nt VP1/2A junction sequence and comparative analysis with reported HAV isolates suggested the presence of three distinct clusters within IIIA represented by HA-JNG04-90F, HA-JNG08-92F, and HAJ95-8F. The extreme 5' end sequences of IIIA and IIIB were well-conserved, beginning with the sequence UUCAAGAGGG. A single base deletion of G at nt 20, which is involved in the formation of a small loop in domain I, was characteristic of both IIIA and IIIB. Conserved and divergent amino acid sequences as well as amino acids unique to genotype III, IIIA or IIIB were recognized.     

Sequence analysis of both genome segments of two very virulent <Emphasis Type="Italic">Infectious bursal disease virus</Emphasis> field isolates with distinct pathogenicity

Summary. The deduced amino acid sequences of segment A and B of two very virulent Infectious bursal disease virus (vvIBDV) isolates, UPM94/273 and UPM97/61 were compared with 25 other IBDV strains. Twenty amino acid residues (8 in VP1, 5 in VP2, 2 in VP3, 4 in VP4, 1 in VP5) that were common to vvIBDV strains were detected. However, UPM94/273 is an exceptional vvIBDV with usual amino acid substitutions. The differences in the divergence of segment A and B indicated that the vvIBDV strains may have been derived from genetic reassortment of a single ancestral virus or both segments have different ability to undergo genetic variation due to their different functional constraints.     

South African G4P[6] asymptomatic and symptomatic neonatal rotavirus strains differ in their NSP4, VP8*, and VP7 genes

Over the past decade, a G4P[6] strain has been found to be circulating in different neonatal wards in the Pretoria area. This endemic strain was associated with both asymptomatic and symptomatic infection, providing the opportunity to undertake a molecular study of some of the putative "virulence" genes. The genes encoding NSP4, VP8*, and VP7 of two asymptomatic and two Symptomatic strains were sequenced and compared with ST3. Within each of these genes, amino acid substitutions unique to South African strains were recorded. Four conserved amino acid differences between asymptomatic and symptomatic strains at aa 82 (serine to leucine), aa 114 (aspartic acid to glutamic acid), aa 138 (proline to threonine), and aa 169 (leucine to serine) were identified within the NSP4 gene. The hypervariable region of VP8* exhibited 10 specific amino acid differences (at aa 73, 78, 98, 111, 116, 142, 145, 167, 169, and 188) between asymptomatic and symptomatic strains, while three amino acid substitutions within VP7 were noted. These changes to VP7 occurred within the glycosylation site at aa 70 (leucine to serine), at antigenic region A (aa 96, asparagine to threonine), and at aa 318 (aspartic acid to glycine). It may be speculated that these changes are specific to G4P[6] strains. Furthermore, the observed substitutions may also be particular to South African strains. NSP4, VP8*, and VP7 have been associated with virulence and the amino acid substitutions within these genes correlate with both asymptomatic and symptomatic infection observed in neonates.     

Detection of hepatitis A virus genotype IB variants in clams from Maputo Bay, Mozambique

Clams provide an important source of food and income for the population of Maputo, Mozambique, where conditions of poor water supply and inadequate sanitation favor endemic infection with hepatitis A virus (HAV). To determine the role of bivalves in an endemic area, clams gathered from Maputo Bay were bought from market and examined for HAV. Four batches, total 150 clams, were sampled over the year. RNA extracted from individual digestive glands was assayed by nested RT-PCR and sequencing of HAV 5' noncoding region (5' NCR). Specific HAV signals were detected in one batch, 23 of 34 clams (67%) testing positive. Phylogenetic analyses of VP3/VP1, VP1/P2A, and 5' NCR determined clustering of clam strains as genotype I, subtype B. In addition to identifying HAV IB strains with predicted conserved amino acid sequence, IB variants exhibiting novel amino acid substitutions at the VP1/P2A junction were detected. HAV strains from clams showed 93%-99% homology with wild-type IB strains from South African outbreaks and from a panel of HAV IgM positive Swedish patients. DNA from enteric human adenovirus 40/41 was found in a limited number of clams from two batches, 6/34 (17%) and 4/35 (11%). Detection of HAV subgenotype IB in bivalves provided indirect evidence of the strains circulating in a densely populated coastal region where HAV is presumed to be hyperendemic. The results suggest that clams may be an important source of HAV in Maputo region, and indicate the need for further molecular study of strains circulating in the indigenous population.     

Increased circulation of hepatitis A virus genotype IIIA over the last decade in St Petersburg, Russia

The current study, covering the period 2004–2009, is a part of long‐term monitoring for hepatitis A virus (HAV) strains circulating in St Petersburg, Russia. The HAV RNA was isolated directly from the sera of hepatitis A patients and RT‐PCR was carried out using primer pairs for VP1/2A and VP1 genomic regions. PCR products were sequenced and 324 nucleotides from VP1/2A and 332 from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common circulating subtype during the follow‐up period, as found in the previous study: almost 90% of the isolated HAV strains belonged to the IA subtype. The large hepatitis A food‐borne outbreak in St Petersburg in 2005 was caused by HAV IA. However, the proportion of HAV isolates belonging to subtype IIIA significantly increased in the period 2001–2009 (7.9%) compared to the period 1997–2000 (none found). The subtype IIIA was first found in St Petersburg in 2001 among a group of intravenous drug users. The increase in its circulation during the decade suggests that this previously unusual genotype has been permanently introduced into the general population of St Petersburg. These results indicate the usefulness of molecular epidemiological methods for studying changes in the circulation of HAV strains. J. Med. Virol. 84:1528–1534, 2012. © 2012 Wiley Periodicals, Inc.     

Genetic diversity of hepatitis A virus in Siberia

The nucleotide sequences of a region of VP1/2A genes of a large group of hepatitis A virus (HAV) isolates circulating in Siberia (the Altai Territory, the Irkutsk and Novosibirsk Regions) were determined. Comparison of these sequences with those of prototype HAV of genotypes IA, IB, and IIA revealed their high similarity to prototype genotype IA strains. The above domains were shown to contain the types of viruses, which were close to both the European subtypes of HAV genotypes IA (78.3%) and the Far Eastern subtypes of this genotype (21.7%). The similar comparison of the derived amino acid sequences suggests that VP1 and 2A contains the amino acid substitutions that are typical of this geographical region.     

Comparative sequence analysis of the VP7 genes of G6, G8 and G10 bovine group A rotaviruses and further characterization of G6 subtypes

Summary.  We previously reported the relatively high prevalence (15%) of bovine G6 subtypes (G6s) in the field using RT-PCR and restriction fragment length polymorphism (RFLP) analysis (Chang et al., Arch. Virol. 141: 1727–39). In the present study, we report the nucleotide and antigenic characterization of a G6s strain (C-8336). We also sequenced the VP7 genes of four additional bovine rotavirus (BRV) strains: another G6s (MC27), G6 (IND), G8 (C-8008) and G10 (2292B) and compared these with other bovine and human rotavirus strains. The C-8336 and MC27 strains were confirmed as P[11]G6s by RT-PCR and RFLP analysis. The VP7 genes of the C-8336 and MC27 strains showed high homology to each other (∼98%) and with other bovine G6s strains (greater than 95% homology in nucleotide and amino acid sequence with KN-4{P[11]G6s}) and also showed lower, but substantial sequence homology with human G6s strains and prototype G6 BRV (79–87% in nucleotide and 88–91% in amino acid). Serologic analysis of the cell culture adapted C-8336 strain showed that it was neutralized by a G6 monoclonal antibody (MAb IC3) to similar titers as the reference NCDV and IND G6 strains. In two-way cross-neutralization tests, strain C-8336 showed 4- to 16-fold differences in antibody titers with NCDV and IND G6 BRV. Moreover polyclonal antiserum against strain C-8336 neutralized the NCDV and IND strains weakly. Genetic variability was also observed among G8 and G10 bovine and human group A rotaviruses: the VP7 genes of the bovine C-8008 (P[5]G8) and 2292 B (P[11]G10) strains showed from 10 to 17% nucleotide divergence with those of Cody I801 (P[1]G8, bovine), A5 (P[1]G8, bovine), 69 M (P[10]G8, human) and Hal 1166 (P[14]G8, human), and I321(P[11]G10, human) and MC35 (P[14]G10, human) rotaviruses, respectively. The divergence of VP7 genes among bovine and human G6, G8 and G10 strains appears related to host species origin and their combination with VP4 (P type). The data presented in this report confirms the genetic variability among homotypic bovine and human strains and highlights the importance of continued monitoring of BRV G and P types circulating in the field for the future development and monitoring of effective vaccines. Received July 4, 1999/Accepted October 31, 1999     

Molecular epidemiology of hepatitis A virus in patients in the Ahwaz region of Iran

Hepatitis A virus (HAV) is one of the etiologic agents of acute viral hepatitis, an important public health problem worldwide. The aim of this study was to investigate the genetic diversity of HAV in Southwest Iran (Ahwaz). A total of 59 sera were collected from acutely ill patients with anti-HAV IgM antibodies during 2009 and 2010 were tested also by RT-PCR targeting the 5' NCR for molecular diagnosis and examined in the VP1-2A and VP3-VP1 regions for genotyping. Twelve (20%) patients were detected VP1-2A by RT-PCR and 10 patients had VP3-VP1. The resulting amplicons were sequenced for genotype identification. All HAV strains were identified as subgenotype IB. Phylogenetic analysis revealed an extensive genetic heterogeneity among the strains. Seven hundred sixty-five S→F and 788 K→R amino acid substitutions in IRI49 isolate were found. It is concluded that subgenotype 1b is the sole genotype HAV in this region.     

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains

A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4-6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.     

Isolates of infectious bursal disease virus from India are of very virulent phenotype

Four Indian field isolates, a classical virulent and an attenuated vaccine strains of Infectious bursal disease virus (IBDV) have been characterized by sequence analysis of part of the VP1 gene (from nucleotide 1538-1979) comprising one of viral RNA dependent RNA polymerase motifs. Sequence alignment of these viruses with reported viruses of other countries revealed Indian IBDV field isolates to be 100% similar to very virulent Japanese (OKYM), European (UK661) and Bangladesh (BD3/99) IBD viruses at amino acid level, whereas they had 0.2-0.9% divergence at nucleotide level. Out of the total 24 nucleotide changes found in the Indian field isolates, as well as reported very virulent viruses, only one resulted in amino acid change S-P at 562 position. The Indian field isolates displayed nucleotide divergence of 10.6-11.6% and amino acid divergence of 2.8-3.5% from the classical virulent and attenuated vaccine strains. The RNA dependent RNA polymerase motif from amino acid 528-541, present in the sequence analyzed, was conserved among all the viruses, irrespective of pathotype and serotype. In the phylogenetic tree, based on nucleotide sequence, Indian field viruses were grouped with reported very virulent viruses in one lineage whereas, classical virulent, attenuated vaccine and serotype 2 strains formed part of the second lineage. But in the phylogenetic tree based on amino acid sequence alignment, the serotype 2 strain OH grouped with Indian field isolates and reported very virulent viruses in one lineage and classical virulent and attenuated vaccine strains formed the second lineage.     

Molecular epidemiology of hepatitis A in St. Petersburg, Russia, 1997-2003

The molecular epidemiology of hepatitis A virus (HAV) strains circulating in the St. Petersburg and Karelia regions was studied during 1997-2003. Hepatitis A virus RNA was isolated from both clinical samples (stools or sera) and environmental samples (sewage water). RT-PCR was carried out using different primer pairs from the VP1/2A and VP1 genomic regions, the variable parts of the HAV genome. PCR products were sequenced and 306 nucleotides from the VP1/2A and 332 nucleotides from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common during the follow-up period: >90% of the isolated HAV strains belonged to that subtype. The HAV strains found in intravenous drug users belonged to subtypes IA and IIIA. Only one out of a total of 88 sequenced strains was of the IB subtype. The subtypes IB and IIIA were found only in 2001-2003, which suggests that new strains were introduced into the endemic situation. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes.     

Hepatitis A virus genotype and its correlation with the clinical outcome of acute hepatitis A in Korea: 2006-2008

Korea has recently experienced a nationwide outbreak of hepatitis A. This study aimed to investigate hepatitis A virus (HAV) genotypes and to compare clinical features between patients infected with HAV genotype IA and those with genotype IIIA. From September 2006 to August 2008, 595 patients with symptomatic hepatitis A were enrolled prospectively in four hospitals in Korea. Among them, 556 patients participated in this study by providing serum or stool samples for genotypic analysis. HAV RNA was detected in 499 patients (89.7%). Major genotypes included IA (n = 244, 48.9%) and IIIA (n = 244, 48.9%), and the remaining genotype was IB (n = 11, 2.2%). From September 2006 to August 2007, the distribution of genotypes IA and IIIA were 64.6% and 35.6%, respectively, which changed to 42.3% and 54.6%, respectively, from September 2007 to August 2008, indicating change of circulating HAV genotypes in the study period from IA to IIIA. Major patterns of amino acid substitution in the VP3/VP1 junction region were observed at position 512 (P → L) in genotype IA and at 520 (R → K) in genotype IIIA. Patients with genotype IIIA infection showed significantly higher aminotransferase levels, prothrombin time, and leukocyte count, with more severe symptoms than those with genotype IA at the time of admission. These results suggest the occurrence of a change of circulating HAV genotypes in recent community‐wide outbreaks of hepatitis A in Korea, and genotype IIIA infection, compared with genotype IA infection, might show more severe clinical manifestations. J. Med. Virol. 83:2073–2081, 2011. © 2011 Wiley Periodicals, Inc.     

Nucleotide sequence variation of the VP7 gene of two G3-type rotaviruses isolated from dogs

The sequence of the VP7 gene of two rotaviruses isolated from dogs in southern Italy was determined and the inferred amino acid sequence was compared with that of other rotavirus strains. There was very high nucleotide and amino acid identity between canine strain RV198/95 and other canine strains, and to the human strain HCR3A. Strain RV52/96, however, was found to have about 95% identity to the G3 serotype canine strains K9, A79-10 and CU-1 and 96% identity to strain RV198/95 and to the simian strain RRV. Therefore both of the canine strains belong to the G3 serotype. Nevertheless, detailed analysis of the VP7 variable regions revealed that RV52/96 possesses amino acid substitutions uncommon to the other canine isolates. In addition, strain RV52/96 exhibited a nucleotide divergence greater than 16% from all the other canine strains studied; however, it revealed the closest identity (90.4%) to the simian strain RRV. With only a few exceptions, phylogenetic analysis allowed clear differentiation of the G3 rotaviruses on the basis of the species of origin. The nucleotide and amino acid variations observed in strain RV52/96 could account for the existence of a canine rotavirus G3 sub-type.     

Altered hepatitis A VP1 protein resulting from cell culture propagation of virus

The published sequence of hepatitis A virus (HAV), strain HAS-15, after 20-30 cell culture passages contains an 18 nucleotide deletion (Ovchinnikov et al., 1985) within the VP1 genome region. This results in a significant amino acid difference of the VP1 protein when this strain of HAV is compared with other published HAV sequences. Comparison of the polyacrylamide gel electrophoretic migration of HAS-15 HAV and two other strains of HAV revealed that the HAS-15 VP1 molecule migrated faster than the VP1 molecule of the other two strains. Enzymatic amplification of viral RNA derived from the original stool suspension and cell culture adapted HAS-15 using the polymerase chain reaction followed by hybridization analyses with selected synthetic oligonucleotide probes revealed that the original wild type virus did not contain the deletion. These results confirm that cell culture adapted HAS-15 contains an eighteen nucleotide deletion which apparently was selected during cell culture adaptation.     

Characterization of a genetic variant of human hepatitis A virus.

Human isolates of hepatitis A (HAV) are a single serotype; however, recent genetic surveys using limited nucleotide sequencing have provided evidence that more than one genotype is responsible for HAV infection in different parts of the world (Jansen et al. [1990]: Proc Natl Acad Sci USA 87:2867-2871; Robertson et al. [1991] J Infect Dis 163:286-292). One of these genotypes was originally isolated from Panamanian owl monkeys (strain PA21), but has subsequently been found associated with human cases of HAV from Sweden in 1979 (H-122) and the United States of America in 1976 (GA76). The nucleic acid sequence of the exposed capsid polypeptide region of GA76 differs from other human HAV sequences by approximately 20%, yet differs by only 2.4% when compared with P1 sequence of the PA21 strain. The 20% nucleic acid variability between GA76 and other human HAV results in limited amino acid changes (3%), while a comparison with PA21 revealed only four homologous amino acid substitutions within VP2, VP3, and VP1 polypeptides. HAV infected stool specimens from Nepal and northern India during 1989 and 1990 were found to contain virus whose genetic makeup was related to the PA21 and GA76 isolates. This genotype of HAV appears to be circulating in some parts of the world where HAV is hyperendemic, and is a potential cause of hepatitis A infection within a susceptible population.