Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.     

Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates

Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.     

Molecular evolution and geographic origins of type 1 human lymphotrophic virus in Colombia detected by RFLP polymorphism

The human T-lymphotropic virus type I (HTLV-I) infection is a public health problem in many endemic areas of Colombia. The subtyping of HTLV-I was based on the analysis of restriction fragment length polymorphisms (RFLP) in 3'LTR proviral DNA. From 31 HTLV-I isolates collected throughout Colombia, a RFLP analysis in a 737 bp 3'LTR fragment was performed. Fifty-eight percent (18/31) were identified as the Cosmopolitan subtype a, 19.4% (6/31) in the West African subtype b, 12.9% (4/31) in the Cosmopolitan subtype b and 9.6% (3/31) in the West African subtype c. The phylogenetic analysis of 3'LTR nucleotide sequences indicated that all the isolates in the current study were in the subgroup B or Japanese, in contrast with the highly divergent isolates from native Amerindians grouped in subgroup a or Transcontinental. The supported hypothesis was that of a post-Columbus introduction of virus represented in the African-American communities of the Colombian South Pacific. Some viral isolates from Colombian native Amerindians exhibited a nucleotide variation compatible with a Paleolithic introduction of the virus. The genetic diversity of HTLV-I in Colombia is complex and probably represents several independent introductions of lymphotropic virus.     

A new subtype of the metalloprotease toxin gene and the incidence of the three bft subtypes among Bacteroides fragilis isolates in Japan

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.     

Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.     

rDNA-based genotyping of clinical isolates of Candida albicans

The study presents an analysis of the restriction pattern ofrDNA fragments of 95 C. albicans isolates previously classified on the basis of the presence of the intron in rDNA into genotypes A (62 isolates), B (28), and C (5). Most isolates (61) with genotype A were classified as "subtype a" and one as "subtype d" (Karahan and Akar; 2005). No differences were observed in the restriction patterns of the tested genotype B isolates. Similarly, most genotype C strains (4/5) showed the same restriction pattern. The results indicate low subtyping variations of the analyzed isolates, which is in contrast to published data obtained from a Turkish collection of yeasts.     

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

Diversity of the outer membrane protein P2 gene from major clones of Haemophilus influenzae type b

In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model. Outer membrane protein subtyping has been employed to subclassify type b Haemophilus isolates. Strain MinnA has the outer membrane protein subtype 1H and is representative of the dominant clonal group of disease-producing isolates in the United States. In the present study, the P2 gene from strain MinnA was employed to probe EcoRI- and Pvull-digested chromosomal DNA from 24 Haemophilus influenzae type b isolates representative of the common outer membrane protein subtype groups observed throughout the world. Restriction fragment length polymorphisms were identified for the members of the outer membrane protein subtype 3L group, but not for the other subtypes examined. The P2 gene from each of four prototype isolates was then cloned, sequenced and compared to the previously reported sequence of the strain MinnA gene. The P2 gene from each of two isolates with the outer membrane protein subtype 3L was identical to the MinnA P2 sequence. The P2 gene from a subtype 2L isolate differed by a single nucleotide and the gene from a subtype 6U isolate differed by 13 nucleotides. Thus, the P2 protein is highly conserved among type b isolates.     

Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.     

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.     

Virological surveillance,molecular phylogeny,and evolutionary dynamics of hepatitis C virus subtypes 1a and 4a isolates in patients from Saudi Arabia

Hepatitis C virus (HCV) subtypes are pre-requisite to predict endemicity, epidemiology, clinical pathogenesis, diagnosis, and treatment of chronic hepatitis C infection. HCV genotypes 4 and 1 are the most prevalent in Saudi Arabia, however; less consensus data exist on circulating HCV subtypes in infected individuals. This study was aimed to demonstrate the virological surveillance, phylogenetic analysis, and evolutionary relationship of HCV genotypes 4 and 1 subtypes in the Saudi population with the rest of the world. Fifty-five clinical specimens from different parts of the country were analyzed based on 5′ untranslated region (5′ UTR) amplification, direct sequencing, and for molecular evolutionary genetic analysis. Pair-wise comparison and multiple sequence alignment were performed to determine the nucleotide conservation, nucleotide variation, and positional mutations within the sequenced isolates. The evolutionary relationship of sequenced HCV isolates with referenced HCV strains from the rest of the world was established by computing pairwise genetic distances and generating phylogenetic trees. Twelve new sequences were submitted to GenBank, NCBI database. The results revealed that HCV subtype 4a is more prevalent preceded by 1a in the Saudi population. Molecular phylogeny predicts the descendants’ relationship of subtype 4a isolates very close to Egyptian prototype HCV strains, while 1a isolates were homogeneous and clustering to the European and North American genetic lineages. The implications of this study highlight the importance of HCV subtyping as an indispensable tool to monitor the distribution of viral strains, to determine the risk factors of infection prevalence, and to investigate clinical differences of treatment outcomes among intergenotypic and intragenotypic isolates in the treated population.     

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

H9N2亚型禽流感病毒NS1基因的进化分析

利用RT-PCR方法,扩增了1998～2005年间分离的9株H9N2亚型禽流感病毒的NS1基因,对其进行了序列测定和进化分析.序列分析表明,9株AIV NS1基因完整的阅读框均为654bp,编码217个氨基酸,其核苷酸和推导的氨基酸同源性分别为95.4%～99.8%和93.6%～100%;9株病毒的NS1蛋白的C端均有13个氨基酸的缺失;进化分析表明,9株AIV属于A群,且形成一个独立分支,在该分支中,只有Ck/HN/A3/98株属于Ck/HK/Y280/97-like亚类,且与Ck/BJ/8/98的进化关系最近,其余8株属于Ck/SH/F/98-like亚类,说明Ck/SH/F/98-like亚类的H9N2亚型AIV在中国大陆的鸡群中广泛存在.NS1基因的进化及其编码产物的特性分析,为AIV的毒力变异、致病机制、药物靶位点的设计及鉴别诊断的研究奠定了基础.     

Genetic variability of hepatitis B virus isolates among population of Shuryskarsky area of Yamal-Nenets autonomous region

The purpose of this work was to determine occurrence of serological markers of hepatites B and to describe subtypes of a superficial antigen and genotypes of hepatitis B virus (HBV) isolates among indigenous population of Yamal-Nenets Autonomous Region (YNAR), Russia. METHODS: We investigated 657 serum samples from inhabitants of Shuryskarsky area of YNAR. ELISA method was used to define the hepatitis B markers: HBsAg, anti-HBs (total) and anti-HBc (IgG and IgM). The HBsAg-positive samples were PCR-tested for the presence of HBV DNA. Genotyping of isolates was by sequencing of the Pre-Sl/Pre-82/S region of HBV genome and phylogenetic analysis. Definition of HBsAg subtypes was executed by two methods: ELISA with subtype-specific monoclonal antibodies and S-gene nucleotide sequence analysis. RESULTS: The following occurrence of hepatitis B markers was observed: HBsAg - 3.2%, anti-HBs (total) - 36.2%, anti-HBc IgG - 30.3%, anti-HBc IgM - 1.6%. Frequency of carrying even one of the markers in the observed population was 47.5%. HBV DNA was found in 17 HBsAg-positive samples. Pre-SI, Pre-S2 and S regions sequences were determined for all HBV DNA-positive samples. The phylogenetic analysis showed an accessory of all investigated HBV isolates to genotype D. HBsAg subtypes distribution appeared the following: ayw2 - 23.5%, ayw3 - 70.6%, adw2 - 5.9%. Results of definition of the subtype ELISA method and by the analysis of S gene nucleotide sequences have coincided in 10/11 (90.1%) cases. CONCLUSIONS: The indigenous population of Shuryskarsky area of YNAR belongs to groups with average HBV carrying. Absolute domination of genotype D (subtypes ayw2, ayw3 and adw2) was revealed. High percentage of concurrence of HBsAg subtypes detected by the ELISA method and method of the analysis of S gene primary structure (90%) was observed. Sequencing of HBV S-gene is preferable to define HBsAg subtypes.     

Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene

The entire nucleotide sequences of 70 hepatitis B virus (HBV) isolates of genotype B (HBV/B), including 38 newly determined and 32 retrieved from the international DNA database (DDBJ/EMBL/GenBank), were compared phylogenetically. Two subgroups of HBV/B were identified based on sequence divergence in the precore region plus the core gene, one with the recombination with genotype C and the other without it. The analysis over the entire genome of HBV/B by the SimPlot program located the recombination with genotype C in the precore region plus the core gene spanning nucleotide positions from 1740 to 1838 to 2443 to 2485. Within this genomic area, HBV/B strains with the recombination had higher nucleotide and amino acid homology to genotype C than those without the recombination (96.9 versus 91.1% in nucleotides and 97.0 versus 92.9% in amino acids). There were 29 HBV/B strains without the recombination, and they were all recovered from carriers in Japan. The remaining 41 HBV/B isolates having the recombination with genotype C were from carriers in China (12 strains), Hong Kong (3 strains), Indonesia (4 strains), Japan (3 strains), Taiwan (4 strains), Thailand (3 strains), and Vietnam (12 strains). Due to the frequency of the distribution of HBV/B without the recombination (29 of 32 isolates, or 91%) and the fact that it was exclusive to Japan, it was provisionally classified into the Bj (j standing for Japan) subgroup, and HBV/B with the recombination was classified into the Ba (a for Asia) subgroup. Virological differences between HBV/Bj and HBV/Ba may be reflected in the severity of clinical disease in the patients infected with HBV of genotype B, which seems to be under strong geographic influences in Asia.     

HIV-1 subtypes in Russia in 1987-1988

The subtyping of 350 isolates of HIV-1, isolated on the territories of 38 subjects of the Russian Federation, was carried out. The analysis was made by the method of the comparative heteroduplex mobility assay, as well as by the determination of the sequence of genes env [correction of ens] (gp 120) and gag (p17-p24). The study revealed that more than 50% of all cases of HIV-1 infection were caused by closely related variants of subtype A virus. The number of cases of HIV-1 infection caused by recombinant virus A/B was not less than 25%. The total number of cases caused by viruses of subtypes C, D, E, F and H was not more than 5%.