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PubMed2004

[Study on the distribution of human immunodeficiency virus-1 subtypes in different regions of China and mother-to-child transmission].

OBJECTIVE: To study the distribution of human immunodeficiency virus (HIV)-1 genotypes in major prevalent regions of China and to illustrate the relationship between HIV-1 subtypes and mother-to-child transmission in a retrospective cohort. METHODS: HIV-1 gag p17 and env C2-V4 region were amplified by nested-polymerase chain reaction (nPCR) and the sequences were obtained by sequencing gag nPCR products or clones of env gene. RESULTS: 60 HIV-1 positive individuals were subject to typing for gag p17 and 69 for env C2-V4 region. Single clade was only found in Henan (subtype B') and Xinjiang (subtype C), and subtypes C and E were demonstrated in Yunnan. These regions represented most of the HIV-1 infections in China. Multiple subtypes (A, B, C, E, etc.) were found in Beijing and Shanghai, where HIV infections were still in low level. The sequences of subtype C were less diversive in Xinjiang (p17: 0.0192 +/- 0.0078, C2-V4: 0.0455 +/- 0.0145) than in Yunnan (p17: 0.0279 +/- 0.0102, C2-V4: 0.0482 +/- 0.0171), but all of them clustered in "C" branch in phylogenetic trees. Trafficking of subtype C from Yunnan to Xinjiang was found but had already been reported by others. Compared to subtype C, subtype E was quite divergent (p17: 0.0473 +/- 0.0105, C2-V4: 0.1114 +/- 0.0112) in Yunnan, but no recombination was found in the C2-V4 region of env gene. Highe divergence of subtype B' was found in Henan and the peripheral provinces (p17: 0.0381 +/- 0.0101, C2-V4: 0.0691 +/- 0.0166), which might be attributed to the early epidemics of HIV-1 in these areas (early 1990's). In maternal-child cohort, subtypes B (7/21), C (11/21), E (1/21) and undefined types (2/21) were identified in non-transmitting HIV-1 positive mothers, while only subtype B (7/11) and C (4/11) appeared in transmitting HIV-1 positive mothers. The rate of transmission was 53.8% (7/13) in mothers infected with subtype B and 30.8% (4/13) in those infected with subtype C, but with no significant difference (P = 0.196). The imbalancing distribution of subtypes might be explained by the fact that transfusion or illegal blood would increased mother-to-child transmission on HIV-1 and most of mothers with clade B were infected by illegal blood transfusion in this cohort. In addition, most of the maternal-child pair's sequences clustered in gag or env phylogenetic trees but only a few did disperse among the unrelated patients because children were older (>/= 4 years). CONCLUSION: The characteristics of HIV-1 clade's distribution differed over most parts of China but no difference was demonstrated between subtype B and C in mother-to-child transmission on HIV-1.

Child, Preschool↗
PubMed2005

Determination of HIV-1 subtypes (A-D, F, G, CRF01_AE) by PCR in the transmembrane region (gp41) with novel primers.

HIV-1 has a huge genetic diversity. So far, nine subtypes have been isolated, namely, subtypes A, B, C, D, F, G, H, J, and K. Epidemiological study provides information which may help in the development of HIV-1 prevention programs or health policies. In the future, subtyping may also be critical for vaccine development, and an effective anti-viral drug will need to be effective for different subtypes of HIV virus. The analysis of the nucleotide sequence of the v3 region is considered the most reliable method for determining the HIV-1 subtype. However, the procedures for determining the v3 sequences are complicated and time consuming, requiring expensive reagents, equipment, and well-trained personnel. The polymerase chain reaction (PCR) method using subtype-specific primers for HIV-1 subtyping is easier and faster. The objective of this study was to develop subtype-specific primers for subtyping PCR. The specific primers were designed for subtypes A, B, C, D, F, G, and CRF01_AE, and these primers could be applied to assay for various HIV-1 subtypes in the clinical samples. The specific primers were designed for each subtypes in the gp41 region. The result of PCR was compared with the subtypes which was determined by the v3 sequence. The results of subtyping by PCR using the newly designed primers could detect 29 of 33 patients tested, and all matched those obtained by nucleotide sequencing of the env v3 region except for three subjects, which were differentiated as CRF02_AG. The newly designed primers functioned accurately and conclusively. In comparison with PCR as a method for the determination of subtypes, sequence analysis requires better-trained personnel, more expensive reagents, and more equipment and time.

Adolescent↗
PubMed2002

Muscarinic receptor subtypes are differentially distributed in the rat cochlea.

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Galpha(q/11)-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Galpha(i) and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters' cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype. From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters' cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.

Animals↗
PubMed1999

Comparison of three commercial assays for the quantification of HIV-1 RNA in plasma from individuals infected with different HIV-1 subtypes.

BACKGROUND: Commercial human immunodeficiency virus 1 (HIV-1) ribonucleic acid (RNA) quantification assays vary in their ability to quantify different subtypes of HIV-1, a problem in regions where multipte HIV-1 subtypes may be circulating. OBJECTIVES: To assess commercial HIV-1 RNA quantification assays on two plasma panels. Panel 1 consisted of HIV-1 seronegative plasma 'spiked' with a known amount of cultured virus of different subtypes, and Panel 2 comprised plasma collected from individuals infected with different HIV-1 subtypes. STUDY DESIGN: The comparison involved the Amplicor HIV-1 reverse transcriptase-polymerase chain reaction (RT-PCR), Quantiplex branched DNA, and NucliSens HIV-1 QT assays. Panel 1 consisted of 11 plasma 'spiked' with cultured viruses of HIV-1 subtypes A-F, and Panel 2 included 33 plasma samples from 16 patients infected with subtypes A, B, C, E and G. RESULTS: In Panel 1, the Quantiplex branched deoxyribonucleic acid (bDNA) assay quantified subtypes A-F efficiently, comparable to published results from two other laboratories. The Amplicor RT-PCR assay quantified subtypes B, C, and D but was relatively less efficient with subtypes E, F, and did not or poorly quantified subtype A. Testing of Panel 2 showed some inter-assay differences. In contrast to Panel 1, the Amplicor RT-PCR assay performed variably with subtype A when compared with the Quantiplex bDNA and NucliSens QT assays, and higher viral load levels were generated with subtype E using the Amplicor RT-PCR assay. Subtypes B and C showed some inter-patient differences but the Quantiplex bDNA generally gave a lower quantification than the Amplicor RT-PCR and NucliSens QT assays. CONCLUSIONS: These studies confirm that commercial HIV-1 load assays vary in their ability to quantify different HIV-1 subtypes. This may be more apparent with individual patient samples than with 'spiked' panels. This variability emphasizes that it is preferable for patient samples to be tested with the same assay, and care should be taken where infection with unusual subtypes is suspected.

DNA, Viral↗
PubMed2005

HIV-1 subtype diversity in Minnesota.

BACKGROUND: Genetic variation in human immunodeficiency virus (HIV)-1 poses significant public-health and clinical challenges. In North America, subtype B is most prevalent. HIV-1 subtyping is not integrated into routine HIV/acquired immunodeficiency syndrome surveillance in the United States. In 2003, the Minnesota Department of Health piloted HIV-1 subtyping with routine surveillance to describe the existence and variety of non-subtype B strains. METHODS: Targeted HIV-1 subtype surveillance was conducted on 98 African-born HIV-infected patients. Sentinel subtype surveillance was conducted in a Minneapolis sexually transmitted disease clinic on 28 newly diagnosed non-African HIV-positive patients. Subtype determination was based on a partial sequence of the gp41 region of the HIV-1 env gene. RESULTS: Subtyping was successful for 87 of 98 samples from African-born HIV-infected patients; 95% were non-B subtypes. The 7 subtypes observed were consistent with strains endemic in patients' birth regions. Subtyping was also completed for samples from 25 of 28 non-African-born patients; all were subtype B. CONCLUSIONS: Multiple HIV-1 subtypes are present in Minnesota. Our data suggest that most of the HIV cases in Minnesota among African-born patients are non-B subtypes. Population-based surveillance inclusive of groups at high risk for variant strains is needed to monitor the prevalence and variety of HIV subtypes in the United States.

Adult↗
PubMed2005

HIV-1 subtype C dissemination in southern Brazil.

OBJECTIVES: To describe the molecular and epidemiological profile of HIV-1 in patients followed at the University Hospital of Rio Grande, Brazil. DESIGN AND METHODS: A cross-sectional study was conducted from September to December 2002. Plasma viral RNA of 85 patients was extracted and protease and reverse transcriptase genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined to antiretroviral resistance mutations. Laboratory data and past history of antiretroviral treatment were also collected. RESULTS: Most viruses were either subtype B (42%) or subtype C (45%). No risk behaviour, sexual orientation or laboratory parameter was associated with any specific subtype, but subtype C tended to be more frequently found in women (P = 0.06). The prevalence of subtype C has increased over the HIV/AIDS epidemic, accounting for almost 60% of cases diagnosed in 2002. Intra-subtype genetic distances were smaller in subtype C than in subtype B, suggesting a more recent introduction of the former in the epidemic. Of patients under treatment, 60% had at least one antiretroviral drug resistance mutation, but no mutation was specifically associated with any HIV-1 subtype. Only one resistance mutation each was found in drug-naive patients with subtypes B and C. CONCLUSION: Despite the fact that subtype C appeared in southern Brazil more recently than subtype B, it is now the predominant strain in Rio Grande. The epidemic spread of subtype C could be taking place in Brazil, and possibly in south America, a phenomenon similar to that seen in other countries where this subtype is now totally dominant.

Adult↗
PubMed1995

Selectivity of the imidazoline alpha-adrenoceptor agonists (oxymetazoline and cirazoline) for human cloned alpha 1-adrenoceptor subtypes.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

Adrenergic alpha-Agonists↗
PubMed1996

Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization.

Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.

Gene Frequency↗
PubMed2004

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4(+) lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1alpha, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C' and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.

Amino Acid Sequence↗
PubMed2001

Subtyping HIV-1 infections in Taiwan using peptide-enzyme immunoassay, reverse transcription-polymerase chain reaction, and sequencing.

BACKGROUND AND PURPOSE: There are important questions about epidemiologic transmission patterns as well as the possibility that genetic and phenotypic differences in human immunodeficiency virus type 1 (HIV-1) affect transmissibility, infectivity, pathogenicity, and response to therapy and vaccines. To delinate the genetic heterogeneity of HIV-1 and the association of subtypes with risk factors and location of residence in Taiwan, subtypes of HIV-1 in Taiwanese patients were identified and a phylogenetic study was performed. In addition, the accuracy of peptide-enzyme immunoassay (EIA) using serum samples from Taiwanese patients infected with HIV-1 was investigated. METHODS: Peptide-EIA was used to give a preliminary subtype of HIV-1-positive serum samples collected from different areas of Taiwan. Reverse transcription (RT)-polymerase chain reaction (PCR) and genetic sequencing were used to confirm the peptide-EIA results and to construct a phylogenetic tree. RESULTS: Among the 149 serum samples, 98 were subtype B (66%), 38 subtype E (25%), two subtype Thai-B (1.3%), one subtype G (0.7%), and one subtype C (0.7%). Comparison of risk factors for HIV-1 infection and subtype revealed that most B subtype infections (59/98) occurred in homosexual or heterosexual patients, whereas 28 of 38 E subtype infections occurred in heterosexual patients. The B/E ratio was significantly different (p < 0.05) in Taipei than in other areas of Taiwan. CONCLUSIONS: These results suggest that the predominant subtype of HIV-1 infection in Taiwan is B, followed by E, and that the distribution of HIV-1 subtypes in Taiwan is similar to that of Thailand, although the genetic sequences are distinct. Homosexuality, heterosexuality, bisexuality, and intravenous drug use behaviors affect the distribution of different subtypes of HIV-1 infection. Peptide-EIA in conjunction with RT-PCR and sequencing can provide accurate subtyping of HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Characterization of surfactant subtypes of beractant and a synthetic peptide containing surfactant KL4 following surface area cycling and addition of fibrinogen.

Surfactant is not a homogeneous material and can be separated into subtypes. Subtype conversion is clinically important because it is thought to occur naturally and because surface activity varies depending on the subtype. Fibrinogen, a naturally occurring serum protein, is known to affect this conversion. In this study we studied two surfactants, beractant and KL4, to examine their subtype characteristics. Surface area cycling, an in vitro method, was used in conjunction with sucrose gradient ultracentrifugation to separate subtypes in both surfactants. Activity, expressed as minimum surface tension of these subtypes, was measured using a pulsating bubble surfactometer. The effect of fibrinogen on subtype conversion and subsequent change in activity was elucidated. Our results indicate that following surface area cycling, beractant and KL4 have different subtypes and different responses to fibrinogen. Cycling of beractant resulted in two bands, representing a heavy and a light subtype. In the presence of fibrinogen, cycling resulted in two separate heavy subtypes. Cycling of KL4 surfactant also yielded light and heavy subtypes. However, in the presence of fibrinogen, cycling of KL4 resulted in ultraheavy subtypes. These ultraheavy subtypes retained minimum surface tension comparable to that of native KL4 surfactant. We conclude that these two surfactant preparations have different subtype conversions when subjected to surface area cycling and in the presence of fibrinogen. These conversions result in different activities toward lowering surface tension. We speculate that endogenous fibrinogen will also affect these two surfactants differently in vivo and thus affect their clinical effectiveness.

Biological Products↗
PubMed1999

Changes in the expression of alpha(2)-adrenergic receptor subtypes during maturation of neuronal cells from fetal pig superior cervical ganglia.

The expression of presynaptic alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes was investigated in cultured neurons from fetal pig superior cervical ganglion (SCG). Cells were incubated with chicken antibodies against alpha(2)A-, alpha(2)B- or alpha(2)C-AR subtypes either alone or together with antibodies against dopamine-beta-hydroxylase (DbetaH, a marker for adrenergic neurons) or against choline acetyl transferase (ChAT, a marker for cholinergic neurons). We found immunoreactivity for all three alpha(2)-AR subtypes in SCG-cells when cultured for 8-11 days. The relative expression of the alpha(2)A-subtype was approximately 1/3 of that of alpha(2)B- and alpha(2)C-AR. Co-localisation of all three alpha(2)-AR subtypes was observed in cells expressing DbetaH or ChAT. Increasing the potassium concentration in the culture medium increased the expression of DbetaH and decreased the expression of the alpha(2)A- and alpha(2)C-subtype without altering the expression of the alpha(2)B-subtype. Co-culture of neurons with pig splenocytes enhanced the expression of ChAT and decreased the expression of the alpha(2)B-subtype without altering the expression of alpha(2)A- and alpha(2)C-subtypes. Our results indicate that the three alpha(2)-receptor subtypes are expressed on both noradrenergic and cholinergic nerves. Induction of the noradrenergic phenotype favours the expression of the alpha(2)B-subtype over that of the alpha(2)A- and alpha(2)C-subtype. Conversely, enhancement of the cholinergic phenotype favours the expression of the alpha(2)A- and alpha(2)C-subtypes over that of the alpha(2)B-subtype. Our results suggest that the alpha(2)B-receptor is preferentially associated with noradrenergic nerve endings.

Animals↗
PubMed2002

Prevalence and origin of HIV-1 group M subtypes among patients attending a Belgian hospital in 1999.

HIV-1 group M strains are usually subtyped based on gag and/or env gene sequences. In our lab, part of the pol gene sequence was available in order to determine the genotypic anti-HIV drug resistance profile. To estimate the prevalence of the different HIV-1 subtypes in patients visiting the University Hospitals in Leuven in 1999 and for whom a genotypic drug resistance test was needed, we tried to use the pol sequence for subtyping. Recombination was investigated by similarity plots and bootscanning and subtyping was performed by phylogenetic analysis. The overall region spanning the entire protease and 747 nucleotides of the reverse transcriptase proved very suitable for subtyping, although there was a low phylogenetic signal at the beginning of the reverse transcriptase (nucleotides 0-250), as we demonstrated by likelihood mapping. Of the 41 samples analyzed, 21 belonged to subtype B. Of the other 20 non-B strains, 9 belonged to subtype C, 2 to subtype D and 1 to subtype A, G, H and J, respectively, 3 were CRF_02 (Circulating Recombinant Form), 1 was recombinant with a novel breakpoint and 1 sample was untypable. Although subtype B is still the most prevalent subtype in Belgium, it seems to be responsible for only half of the infections in this study. We could also document that the prevalence of subtype C is high in the Belgian native patients, especially among the heterosexually infected population. This could possibly be an indication for an epidemic spread of HIV-1 subtype C in Belgium, as for one third of these patients, no link to an endemic region could be found. The other non-B subtypes and the recombinants are mainly introduced by immigrants or by Belgian citizens traveling abroad.

Belgium↗
PubMed1995

Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

Alternative Splicing↗
PubMed1998

HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996.

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.

Amino Acid Sequence↗
PubMed2005

In vitro generation of HIV type 1 subtype C isolates resistant to enfuvirtide.

Enfuvirtide (ENF) is the first in a new class of antiretroviral agents targeting the fusion process of the viral life cycle. ENF is a synthetic 36-amino acid peptide that binds to the HR-1 region of gp41 preventing fusion of viral and cellular membranes. With the introduction of ENF there are now four classes of antiretrovirals each with distinct and different resistance pathways. Resistance to ENF among subtype B HIV-1 isolates is associated with amino acid changes mainly in the HR-1 region, although other regions of envelope have also been implicated. To determine whether subtype C viruses developed resistance mutations similar to subtype B viruses, 11 subtype C and 4 subtype B viruses were passaged in the presence of increasing concentrations of ENF. The subtype C isolates showed varying levels of replication at 1 microg/ml ENF by day 18, but by day 29 all replicated efficiently at 10 microg/ml ENF. All subtype C isolates showed evidence of genotypic changes in gp41 HR-1 following exposure to ENF that included G36S/E/D, I37T, V38M/A/L/E, N/S42D, N43K, L45R/M, and A50T/V. Three subtype C viruses had compensatory changes in the HR-2 region, which corresponds to the ENF sequence, and two isolates had changes in the V3 region. Mutational patterns among the four subtype B viruses were similar to those for subtype C and those previously published in the literature. These data indicate that in vitro resistance to ENF develops rapidly among HIV-1 subtype C isolates. In general, mutational patterns for subtype C were similar to those described for subtype B, suggesting that the mechanism of action for ENF is similar for HIV-1 subtype B and C isolates.

Amino Acid Sequence↗
PubMed2003

Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis.

Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (A(s)), A-paired (A(pr)), A-aligned (A(al)), and A1-A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (approximately 1 microm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRalpha-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRalpha-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially A(s), A(pr), and possibly A(al) spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.

Animals↗
PubMed1999

Clinical disease associated with HIV-1 subtype B' and E infection among 2104 patients in Thailand.

BACKGROUND: Two HIV-1 envelope subtypes have accounted for virtually all infections in Thailand: subtype B' (Thai B), found mainly in injection drug users (IDU), and subtype E, found in over 90% of sexually infected persons and an increasing proportion of IDU in recent years. It remains unclear whether there are differences in pathogenesis associated with these HIV-1 subtypes. METHODS: From November 1993 to June 1996, demographic, risk, clinical, and laboratory data were collected by enhanced surveillance from HIV-infected inpatients (> or =14 years) at an infectious disease hospital near Bangkok. HIV-1 subtype was determined by V3-loop peptide enzyme immunoassay (EIA). Because of confounding, multivariate analyses were stratified by risk category and controlled for sex and age. RESULTS: The infecting HIV-1 subtype was determined for 2104 (94.9%) of 2217 HIV-infected patients with complete data: 284 (13.5%) were subtype B', 1820 (86.5%) were E. Specimens from 113 (5.1%) patients were non-reactive or dually reactive on peptide EIA and were excluded. Among IDU, 199 (67.2%) had subtype B', and 97 (32.7%) had E. IDU accounted for 70.1% (199/284) of patients with subtype B' and 5.3% (97/1820) of those with E. Patients infected with HIV-1 subtypes B' or E had similar spectrums of opportunistic infections (OI), levels of immunosuppression, and in-hospital mortality rates. Of patients who did not inject drugs, more patients infected with subtype E had extrapulmonary cryptococcosis than those with subtype B' (adjusted odds ratio, 2.6; 95% confidence interval, 1.28-5.37). CONCLUSION: HIV-1 subtypes B' and E seem to be associated with similar degrees of immunosuppression and, with one exception, with similar OI patterns. These data do not suggest an association between HIV-1 subtype and differences in pathogenicity.

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