Genetic characterization and phylogenetic analysis of HIV-1 subtype C from Uganda

To better understand the emergence of subtype C and its potential impact on vaccine efforts in Uganda, we have characterized subtype C sequences from Uganda (n = 13), Zimbabwe (n = 11), Mozambique (n = 5), South Africa (n = 4), and India (n = 3). Phylogenetic analysis of subtype C sequences in the env gp41 gene region revealed multiple subclusters within subtype C. Further, while most Ugandan specimen subclustered together, other subclusters did not reflect a clear geographic location. The nucleotide divergence within the Ugandan subset was 8.2% (6.1-9.8%) compared with 9.5% (2.5-15%) for the other subtype C gp41 sequences. The protein sequence alignment revealed marked sequence conservation of major immunodominant epitopes within the gp41 region.     

1例输入性HIV-1 G亚型毒株的检出及基因组序列分析

目的 对1例输入性HIV-l G亚型感染者病毒基因组进行序列测定,并进行系统进化分析.方法 采集在本地区艾滋病监测中发现的1例HIV-1 G亚型感染者的血清,提取病毒RNA,利用反转录巢式-PCR技术扩增并测定病毒近似全长基因序列.结果 获得此毒株近似全长基因组序列,长度为8695 bp.重组分析结果显示,此毒株序列中...     

Evidence for separation of HCV subtype 1a into two distinct clades

Summary. The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error‐prone RNA‐dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole‐genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star‐like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a‐specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain‐specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.     

HIV type 1 A/J recombinant with a pronounced pol gene mosaicism

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.     

Sequence features downstream of the primer-binding site of HIV type 1 subtype E shared by subtype G and a subset of subtype A

Two distinct sequence features downstream of the primer-binding site (PBS) were identified in a full-length HIV-1 subtype E clone amplified in this study. Both features are frequently found in HIV-1 subtypes A and G and in more than half of the full-length intersubtype recombinant clones. One of these is the absence of a trinucleotide sequence, which is located 14 nucleotides downstream of the PBS and found only in subtypes B, C, D, F, and H. The other is an insertion of 24 nucleotides immediately downstream of the PBS, which was previously reported as a sequence feature shared by subtypes A, E, and G. The analysis conducted here revealed that this 24-nucleotide insertion contained two sequence motifs duplicated in adjacent regions and was not found in all HIV-1 subtype A clones. Furthermore, our finding suggests that the PBS region of all known full-length subtype E clones, which are A/E intersubtype recombinants, is derived from the group of HIV-1 subtype A, which contains a similar insertion.     

Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand

In January 2005, a survey of intestinal parasitic infections was performed in a primary school, central Thailand. Of 675 stool samples, Blastocystis was identified with a prevalence of 18.9%. Genetic characterization of Blastocystis showed subtype 1 (77.9%) and subtype 2 (22.1%). Study of the water supply in this school was performed to find the possible sources of Blastocystis. Blastocystis from one water sample was identified as subtype 1, which had a nucleotide sequence of small subunit (SSU) ribosomal RNA (rRNA) gene that was 100% identical to that of Blastocystis infected in schoolchildren. Our information supports the evidence of water-borne transmission in this population.     

An HIV type 1 subtype A outbreak among injecting drug users in Kazakhstan

Kazakhstan experienced the start of the HIV-1 outbreak among intravenous drug users (IDUs) in 1997. To characterize genetically HIV-1 strains circulating in this country, peripheral blood mononuclear cells (PBMCs) DNA samples (1999-2002) derived from HIV-infected IDUs and their sexual partners in Pavlodar (n = 19), Shymkent (n = 6), and Qaraghandy (n = 18) regions were analyzed by the gag/env heteroduplex mobility assay (HMA). The 366-bp proviral env gene fragments encoding the gp120 C2-V3 region obtained from 16 individuals were sequenced. The results of HMA revealed that all 43 HIV-1 strains studied belonged to gag/env subtype A. The nucleotide sequence analysis showed a marked genetic homogeneity with the mean genetic distance being 3.63 +/- 2.39 (range 0.00-12.13). The mean genetic distance between each sequence within the Kazakhstan set and the East-European IDU subtype A consensus was 2.94 +/- 1.92 (range 0.79-8.48). The data presented thus confirm the spreading of the same IDU subtype A virus in the former Soviet Union.     

Characterization of a complete immunoglobulin heavy-chain variable region germ-line gene of rainbow trout.

A germ-line heavy-chain variable region (VH) gene (RTVH431) has been isolated from a rainbow trout (Salmo gairdneri) and characterized by complete nucleotide sequencing. It is characteristic of VH, as shown by the conserved octamer and TATA motif in the 5' region, the heptamernonamer recombination signal sequence in the 3' region, and the 18-amino-acid-long hydrophobic leader interrupted by an intron. The 98-amino-acid-long VH coding region has 50-70% nucleotide sequence homology and 40-60% amino acid sequence homology with VHS of various vertebrate species. We have also found unique or species-specific amino acid residues in the VHS of rainbow trout, amphibia (Xenopus), reptile (Caiman), and shark (Heterodontus) in our sequence analyses. The RTVH431 has an unusual amino acid in the conserved 34th position in complementarity-determining region 1 of VH. Southern hybridization results suggest the presence of a large gene family related to RTVH431 in the trout genome. The complex evolution of antibody V genes is discussed.     

2010--2012年福建省H3N2亚型流感病毒血凝素基因特征分析

目的通过对2010—2012年福建省分离的H3N2亚型流感毒株血凝素HA1基因进行分析,了解2010-2012年福建省H3N2亚型流感病毒的基因特征和变异规律。方法随机选择28株毒株,提取病毒RNA,采用RT-PCR扩增目的片段,纯化PCR产物后进行测序;利用DNAstar和Mega4．0等生物信患学软件进行核苷酸整理、拼接、校对、差异性比较以及基因进化树构建等分析研究。结果28株H3N2亚型流感病毒HAl区核苷酸同源性在96．4％～100％之间。基因系统进化分析显示,其进化规律都是沿着树枝主干随着时闻推移向,上延伸,序列的差异和年代成正比。2010—2012年间HAl区共出现43个氨基酸位点的变化,其中有10个氨基酸位点变异累计涉及4个抗原决定簇,2个变异位点发生在受体结合位点（RBS）及其附近,应予高度重视。结论相对于A／Perth／16／2009疫苗代表株,2012年的部分H3N2亚型流感出现了抗原漂移;部分毒株已出现氨基酸位点的改变,并涉及抗原决定簇及受体结合位点,提示2010—2012年福建省人群中流行的H3N2亚型流感正逐渐发生变异,应加强流感病原学监测,以及时发现新的流感变异株,为福建省流感防控提供科学依据。     

Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents

Among the major circulating HIV-1 subtypes, subtype C is the most prevalent. To generate full-length subtype C clones and sequences, we selected 13 primary (PBMC-derived) isolates from Zambia, India, Tanzania, South Africa, Brazil, and China, which were identified as subtype C by partial sequence analysis. Near full-length viral genomes were amplified by using a long PCR technique, sequenced in their entirety, and phylogenetically analyzed. Amino acid sequence analysis revealed 10.2, 6.3, and 17.3% diversity in predicted Gag, Pol, and Env protein sequences. Ten of 13 viruses were nonmosaic subtype C genomes, while all three isolates from China represented B/C recombinants. One of them was composed primarily of subtype C sequences with three small subtype B portions in gag, pol, and nef genes. Two others exhibited these same mosaic regions, but contained two additional subtype B portions at the gag/pol overlap and in the accessory gene region, suggesting ongoing B/C recombination in China. All subtype C genomes contained a prematurely truncated second exon of rev, but other previously proposed subtype C signatures, including three potential NF-kappa B-binding sites in the viral promoter-enhancer regions, were found in only a subset of these genomes.     

Isolation, characterization, and sequence analysis of a novel HIV type 1 subtype A1 strain from Russia

A new syncytia-inducing human immunodeficiency virus type 1 (HIV-1) strain designated PokA79 was first isolated from a peripheral blood mononuclear cells (PBMC) pool from 24 HIV-1-infected patients from Russia and adapted to growth in MT2, MT4, and U937 cell cultures. The full-length genome of this isolate was amplified by RT-two-round PCR and sequenced. The obtained sequence belongs to the A1 subtype and clustered with other A1 former Soviet Union (FSU) strains. Full-length genome analysis showed that some of the PokA79 genes contain insertions and frame shifts, and the gp120 V3 loop has the three amino acid insertion inside its apex, which is rare for subtype A1 sequences. Nonidentical insertions of the same size are also present in the V3 apex region of several HIV-1 sequences from Central Africa.     

A new subtype of 3′ region of cagA gene in Helicobacter pyloristrains isolated from Zhejiang Province in China

AIM: To isolate the subtypes of 3′ region of cagA gene in Helicobacter pylori (H pylon) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3′ region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3′ region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pyloriisolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes Ⅰ, Ⅱ, and Ⅲ according to their sizes. The sizes of subtypes Ⅰ, Ⅱ, and Ⅲ were 648-650bp, 705-707bp, and 815bp, respectively. Among the 132 cagA-positive H pylori strains, 123(93.2%) belonged to the group of subtype Ⅰ, 6 (4.5%) presented subtype Ⅱ, 1(0.8%) was subtype Ⅲ, and 2(1.5%) presented subtypes Ⅰ and Ⅲ both. The primary structure of subtype Ⅰ was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype Ⅱ possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3′ region of cagA gene which had not been reported before. The primary structure of subtype Ⅲ consisted of 4 repeats of R1, Ⅰ repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype Ⅰ strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype Ⅲ strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype Ⅱ strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%),respectively. There were no statistical differences in the distribution of subtypes of 3′ region of cagA gene among different H pylori-associated gastroduodenal diseases (x^2=11.544, P>0.05). CONCLUSION: There are three subtypes (Ⅰ,Ⅱ, and Ⅲ) of 3′ region of cagA gene in Hpylori strains isolated from Zhejiang Province, and subtype Ⅰ is predominant. Subtype Ⅱ is a newly found subtype of 3′ region of cagA gene. The result of this study does not support the view that the subtypes of 3′ region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.     

An infectious DNA clone of HIV type 1 subtype C.

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.     

Full-length gag sequences of HIV type 1 subtype C recent seroconverters from Pune, India

Although HIV-1 subtype C is the most prevalent subtype worldwide, data on subtype C viruses are rather limited. Very little information is available on the complete HIV-1 subtype C gag sequences from India. We report full-length gag (p55) sequences from six Indian early seroconverters. The samples were collected within few weeks of seroconversion and may represent immunologically naive viruses. The comparison of p55 sequences with other Indian and non-Indian subtype C sequences as well as with nonsubtype C sequences obtained from the Los Alamos database revealed gag as a well-conserved region of the HIV genome (range: 84-95%). The phylogenetic tree indicated that the sequences compared here cluster together within clade C. Two epitopes in the p24 region of the gag gene were subtype C specific while many epitopes in the same region were also present in other clades. The data on HIV-1 subtype C full-length gag sequences would be useful in the design and evaluation of effective subtype C-based HIV vaccines.     

Genetic analysis of human immunodeficiency virus in Abidjan, Ivory Coast reveals predominance of HIV type 1 subtype A and introduction of subtype G

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.     

Acquisition and analysis of lipophosphonoglycan 1 gene and the noncoding region of Leishmania donovani isolates from hilly foci of China

A fragment about 2.2 kb located at upstream of lipophosphonoglycan 1 (LPG1) gene of Leishmania donovani isolate from hilly foci of China was obtained by PCR. The nucleotide sequence of the fragment was determined by sequencing. The sequence of the LPG1 gene and its downstream fragment (about 2 kb) was determined by using genome walking method. The above two fragment splicing result showed that the nucleotide sequence of the LPG1 gene with the noncoding region was 4 121 bp (GenBank accession number: HMO27899). The BLAST results showed that the LPG1 gene of L. donovani isolate from hilly foci of China had 72%-78% nucleotide identity compared to that of other Leishmania spp. in GenBank.     

上海地区H9亚型禽流感病毒HA基因序列分析

目的对分离自上海活禽批发市场的7株H9AIV(2002年1株、2006-2008年每年各2株)进行了HA序列的测定,以阐明这7株毒株HA基因的遗传变异及分子特征。方法采集鸡气管和泄殖腔样品,将H9亚型荧光RT-PCR试剂盒检测的阳性样品处理后,经鸡胚尿囊腔接种分离病毒,HI进一步确定HA亚型,通过RT-PCR方法分别扩增了这7个毒株的HA基因,并将其克隆到pMD18-T载体后进行序列测定与同源性分析。结果HI试验表明这7株毒株均属于H9亚型禽流感病毒。核苷酸和其推导的氨基酸序列同源性比较结果表明,这7株毒株HA基因的核苷酸序列同源性为88.6%～99.6%,氨基酸序列同源性为89.1%～99.1%;7株分离株HA基因的裂解位点氨基酸顺序则均为RSSR↓GLF,为低致病性毒株;所有毒株的潜在糖基化位点均有7个;2002年的分离株与2006年早期的分离株在aa226和aa228位分别为Q和G,其余5株分离株在这两个位点分别为L和G。遗传发生关系分析,这7株分离株均属于Ck/Bei/1/94系。结论上海地区7株H9亚型分离株具有相同的起源,且分子特性相似,受疫苗免疫选择压力的影响不大。     

Intersubtype recombinant HIV type 1 involving HIV-MAL-like and subtype H-like sequence in four Norwegian cases

Suspected epidemiological links between three cases of human immunodeficiency virus type 1 (HIV-1) infection were verified by the finding of a shared unique virus genotype. A probable male index case was not available for testing. Case 1 was a female sexual partner of the index case. Case 2 was an adult son of case 1. Case 3 was a female sexual partner of case 2. The link to the index case was substantiated by the subsequent finding of another female sexual contact of the index case, harboring the same HIV-1 genotype as the three other cases. To characterize the genotype further, the complete provirus nucleotide sequence was obtained directly from blood cell DNA of case 3. HIV cultivated from case 3 demonstrated CCR5 dependence, an extreme slow-low phenotype, and some genotypic features not present in its directly sequenced counterpart. Most of the gag, pol, and vif genes of these viruses clustered with one of the earliest African HIV-1 strains, MAL, previously classified as a recombinant between the subtypes A, D, and I. Most of the rest of the genome was related to subtype H, albeit with less than 90% identity in most regions. These viruses are the only ones shown to display extensive similarity with MAL in the gag-pol region and among the first HIV-1 recombinants described involving subtype H. We postulate that the gag-pol genes of MAL and these viruses are derived from a common ancestor that is not necessarily intersubtype recombinant in the pol region.     

Sequence determination of the capsid protein gene and flanking regions of tobacco etch virus: Evidence for synthesis and processing of a polyprotein in potyvirus genome expression

The nucleotide sequence of the 3′-terminal portion of the tobacco etch virus (TEV) genome was determined. The 2324-nucleotide sequence represented approximately one-fourth of the TEV genome and included the capsid protein gene and flanking regions. An open reading frame of 2135 nucleotides and an untranslated region of 189 nucleotides adjacent to a polyadenylate tract were identified. The sequence began within an open reading frame, indicating that the initiation codon was upstream of the available sequence data. The sequence of the 20 NH₂-terminal amino acids of the TEV capsid protein was established chemically. An identical amino acid sequence, predicted from the nucleotide sequence, was located, commencing at amino acid - 263. These data indicated that maturation of the capsid protein required a post-translational cleavage of a larger protein precursor, with a probable cleavage site between the amino acids glutamine and glycine.