井戸 栄治

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

BACKGROUND/AIM: Sindbis virus (SINV) is a naturally occurring oncolytic virus that kills cancer cells and is less harmful to normal cells. In this study, a recombinant SINV, which expressed green and blue fluorescent proteins, was used to precisely analyze SINV infection and replication. MATERIALS AND METHODS: Antiviral responses, including IFN-β mRNA, protein kinase R (PKR), NF-B, and caspase 3/7, were analyzed in SINV-infected cancerous HeLa cells and normal human fibroblast TIG-1-20 cells. RESULTS: SINV could infect, replicate, and proliferate both in HeLa and TIG-1-20 cells, causing lytic infection only in HeLa cells. SINV grew preferentially in HeLa cells causing remarkable apoptosis. IFN-β mRNA expression was suppressed in SINV-infected HeLa cells compared to that in TIG-1-20 cells. Further analyses of PKR and NF-B upstream of IFN-β induction revealed that the compromised response in the PKR-NF-B pathway during early infection coincided with IFN induction suppression in HeLa cells. CONCLUSION: Dysregulation of PKR in HeLa cells is the determinant of SINV oncolysis.

The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for human immunodeficiency virus (HIV) RNA quantification in patients on antiretroviral treatment (ART) in Ghana, where recombinant strains including CRF02_AG are prevalent. The primers and TaqMan probe concentrations as well as reaction temperatures were optimized to establish an efficient in-house quantitative assay system for HIV RNA, a tool for HIV viral load measurement in patients. Then an already established HIV-specific PCR amplicon (HIV-1 NL4-3) was used as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the in-house real time quantitative assay. Finally, the assay was applied to quantify the viral load in clinical samples of HIV patients on ART. The real time quantitative assay was shown to have good linearity (R2=1.0), high amplification efficiency (E=1.91), high sensitivity (180 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Analytical specificity and sensitivity of the assay in clinical samples was 96.7% and 95.0%, respectively. The established tool is reliable and covers all relevant genotypes including rare and recombinant forms that circulate in the sub-region. It could therefore allow general monitoring of antiretroviral therapy in patients living in resource-limited settings due to its simplicity, rapidity and less-labour intensiveness. © 2014 Barnor JS, et al.

Compelling evidence appeared in 2002 of human exposure to a plethora of primate lentiviruses through hunting, handling of bushmeat, and/or animals kept as pets in Cameroon. To determine SIV prevalence in pet animals, an analysis of 28 sera of nonhuman primates found no SIV infection in greater spot-nosed monkeys (0/5) or chimpanzees (0/10), and a prevalence rate of 23.1% (3/13) in mandrills; kept as household pets in southern Cameroon. Phylogenetical analysis based on pol-integrase region and mitochondrial cytochrome b gene showed that the newly found SIV from Mandrillus sphinx (SIVmndCM-202, SIVmndCM-211, and SIVmndCM-218) clustered significantly with SIVmnd-2. Questionnaire data were also collected to assess whether owners had experienced bites, scratches, or exposure to blood and/or body fluid. Risk to human health from cross-species transmission of the newly identified SIVmnd-2 to infect humans remains unknown. (AIDS Rev. 2009;11:135-9)

Expanding the HIV-1-derived regions in the SHIV genome may help to clarify the viral restriction factors determining the host range. In this study, we constructed a new SHIV having the reverse transcriptase and integrase-encoding regions of HIV-1 in addition to the 3' half genomic region of HIV-1. This SHIV, termed SHIVrti/3rn, could replicate in a monkey CD4(+)T cell line, HSC-F, although its replication in monkey PBMCs was very weak. After SHIVrti/3rn was passaged in HSC-F cells for 26 weeks, it gradually began to replicate in monkey PBMCs. This monkey-cell-adapted virus, termed SHIVrti/3rnP, could replicate in rhesus macaques. The whole genome of SHIVrti/3rnP was sequenced and was found to differ from SHIVrti/3rn at eleven positions. We constructed a series of mutants having some or all of these mutations and investigated their replication kinetics. The mutational analysis revealed that all of the mutations, but mainly the mutations in env, were responsible for the adaptation in HSC-F cells and were enough to replicate in rhesus PBMCs. Of all the SHIVs reported so far that can infect rhesus monkeys in vivo, SHIVrti/3rnP is the one that is genetically the closest to HIV-1. (C) 2008 Elsevier Masson SAS. All rights reserved.

We generated a novel SHIV (termed SHIV-pr) that possesses the HIV-1-derived protease (PR) gene in the corresponding position in the SIVmac genome. SHIV-pr is replication-competent in human and monkey CD4(+) T lymphoid cell lines as well as rhesus macaque PBMCs. The viral growth of SHIV-pr was completely blocked in the presence of a peptide-analog PR inhibitor at the tissue culture level. When SHIV-pr was intravenously inoculated into two rhesus macaques, it resulted in a weak but long-lasting persistent infection in one monkey, whereas the infection of another was only temporary. To enhance the viral growth competence by adaptation, we then passaged the virus in vivo from a monkey up to the fourth generation. The initial peak values of plasma viral loads as well as the setpoint values increased generation by generation and reached those of a parental virus SIVmac. When a medication using the content of Kaletra capsule (a mixture of two PR inhibitors, lopinavir and ritonavir) was orally given to three SHIV-pr-infected monkeys for 4 weeks, plasma viral loads dropped to near or below the detection limit and quickly rebounded after the cessation of medication. The results suggest that SHIV-pr can be used to evaluate PR inhibitors using monkeys. (c) 2007 Elsevier Masson SAS. All rights reserved.

In 2002, compelling evidence highlighted human exposure to a plethora of primate lenti-viruses through hunting, handling of bushmeat and/or animals kept as pets in Cameroon. To determine SIV prevalence in pet animals, sera from 28 nonhuman primates (3 species), kept as household pets in southern Cameroon, was analyzed. Results showed there was no SIV infection among greater spot nosed monkeys (0/5) or chimpanzees (0/10). However, there was a prevalence rate of 23.1% (3/13) in mandrills. Phylogenetical analysis based on pol-integrase (IN) region and mitochondrial (mt) cytochrome b gene showed that the newly found SIVs from Mandrillus sphinx (SIVmndCM-202, SIVmndCM-211 and SIVmndCM-218) clustered significantly with SIVmnd-2. Questionnaire data were also collected to assess whether owners had experienced bites, scratches or exposure to blood and/or body fluid. Risk to human health from cross-species transmission of the newly identified SIVmnd-2 to infect humans remains unknown.

We previously reported that a mutant full-sized plasmid DNA vaccine regime in macaques was effective against a homologous challenge [Akahata W, Ido E, Shimada T, Katsuyama K, Yamamoto H, Uesaka H, et al. DNA vaccination of macaques by a full genome HIV-1 plasmid which produces non-infectious virus particles. Virology 2000;275:116-24; Akahata W, Ido E, Akiyama H, Uesaka H, Enose Y, Horiuchi R, et al. DNA vaccination of macaques by a full genome SHIV-1 plasmid that produces non-infectious virus particles. J Gen Virol 2003;84:2237-44]. In this study, to evaluate the DNA vaccination regime against a heterologous challenge, a novel plasmid named pSHIV-ZF1*IL-2 was constructed. Four monkeys were intramuscularly and intradermally injected four times with the pSHIV-ZF1*IL-2. Vaccinated monkeys were intravenously challenged with a highly pathogenic, heterologous SHIV at I I weeks post vaccination. All the vaccinated monkeys suppressed the challenge virus rapidly under the detectable level by 16 weeks post challenge. One vaccinated monkey was protected from a loss of CD4+ T cells. These results suggest pSHIV-ZF1*IL-2 alone seems partially effective even against a challenge with a heterologous, pathogenic virus. (c) 2005 Elsevier Ltd. All rights reserved.

The hereditary conservation in the genetically encoded CD1D sequences of various primates was analyzed. Genomic CD1D sequences of 17 rhesus macaques with distinct origins, eight Indian and nine Chinese, were examined and differences of only one or two nucleotides were detected and the consensus sequence of rhesus CD1D was determined. CD1D consensus sequences of three African green monkeys (AGMs) and the rhesus monkeys were then compared to study the evolutionary differences among interspecies. The CD1D consensus sequence determined from AGMs apparently differed by seven nucleotides from the rhesus consensus sequence, and nucleotide difference induced only three amino acid changes within Exon3, corresponding to the alpha(2) domain of CD1d having a hydrophobic ligand-binding pocket. Such changes in the alpha(2) domain may alter the characteristics of the SIV-derived glycolipid/lipid antigens presented by each CD1d molecule to innate natural killer T cells. In addition, the CD1D genomic sequences of three chimpanzees (chimps) were determined. To our surprise, although Exon2 and Exon3 reflecting antigen-binding alpha(1) and alpha(2) domains in chimps' CD1D were identical to that in humans except one amino acid, three amino acids within Exon4, reflecting alpha(3) domain, were distinct from humans, and one of them was identical to those in rhesus and AGM CD1D. On the basis of the findings, the evolutionary relationship of the CD1d molecules among the various primates and their HIV-1/SIV susceptibility will be discussed.

CD4-bearing T cells are the primary targets for human immunodeficiency virus type 1(HIV-1)/simian immunodeficiency virus (SIV) infection. However, it is unclear whether the susceptibility of CD4-bearing T cells including CD4 single positive and CD4/8 double positive T cells to HIV/SIV infection is the same or not. In this study, we compared the susceptibility to SIV infection between CD4(+) and CD4(+)8(+) T cells, using Herpesvirus saimiri (HVS)-transformed CD4(+) and CD4(+)8(+) T cells established from peripheral blood mononuclear cells (PBMC) of rhesus macaques. Although there was little difference between the two CD4-bearing T cell population in the expression level of CD4 molecules and chemokine receptors such as CXCR4 and CCR5, SIV replicated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells. Moreover, we found that reverse transcription initiated more efficiently in CD4(+)8 T cells than in CD4(+) T cells and that the cell lysates from CD4(+) T cells impaired the RT activity more strongly than that from CD4(+)8(+) T cells. These findings suggest that intracellular environment in CD4(+)8(+) T cells is better for reverse transcription and that the infection of those CD4(+)8(+) T cells might play critical and different roles in HIV-1/SIV infection and dissemination.

Blood samples (n=544) from two different populations (Pygmies and Bantus) in Cameroon, West Africa, were analysed. Serological tests indicated that the anti-hepatitis C virus (HCV) prevalence in Bantus (20-3%) was higher than that in Pygmies (2(.)3%, P < 0.0001), whereas the distribution of hepatitis B virus (HBV) serological markers was equally high in both populations: in total, 9(.)4, 17(.)3 and 86(.)8 0/b for HBsAg, anti-HBs and anti-HBc, respectively. HBV genotype A (HBV/A) and HBV/E were predominant (43(.)5% each) in both populations, and HBV/D was found in a minority (13%). The preS/S region was sequenced in nine cases (five HBV/A and four HBV/E) and the complete genome in six cases (four HBV/A and two HBV/E). Subsequent phylogenetic analysis revealed that the HBV/A strains were distinct from the subtypes (subgenotypes) described previously, Ae (A2) and Aa (A1), and in the preS/S region they clustered with previously reported sequences from Cameroon. Based on the nucleoticle difference from Aa (A1) and Ae (A2), more than 4% in the complete genome, the Cameroonian strains were suggested to represent a new subtype (subgenotype), designated HBV/Ac (A3). A high (3(.)9 %) nucleoticle divergence in HBV/Ac (A3) strains suggested that the subtype (subgenotype) has a long natural history in the population of Cameroon. One of the HBV/Ac (A3) strains was found to be a recombinant with an HBV/E-specific sequence in the polymerase reverse transcriptase domain. Further cohort studies will be required to assess detailed epidemiological, virological and clinical characteristics of HBV/Ac (A3), as well as its recombinant form.

To monitor the presence of genotypic HIV-1 variants circulating in eastern Cameroon, blood samples from 57 HIV-1-infected individuals attending 3 local health centers in the bordering rural villages with Central African Republic (CAR) were collected and analyzed phylogenetically. Out of the 40 HIV-1 strains with positive polymerase chain reaction (PCR) profile for both gag and env-C2V3,12 (30.0%) had discordant subtype or CRF designation: 2 subtype B/A (gag/env), 1 B/CRF01, 2 B/CRF02, 1 CRF01/CRF01.A, 2 CRF11/CRF01, 1 CRF13/A, 1 CRF13/CRF01, 1 CRF13/CRF11, and 1 G/U (unclassified). Twenty-eight strains (70.0%) had concordant subtypes or CRF designation between gag and env: 27 subtype A and 1 F2. Out of the remaining 17 HIV-1 strains negative for PCR with the env-C2V3 primers used, 10 (58.8%) had discordant subtype or CRF, and 7 (41.2%) had concordant one based on gag/pol/env-gp41 analysis. Altogether, a high proportion (22/57, 38.6%) of the isolates were found to be recombinant strains. In addition, an emergence of new forms of HIV-1 strains, such as subtype B/A (gag/env), B/CRF01 and B/CRF02, was identified. The epidemiologic pattern of HIV-1 in eastern Cameroon, relatively low and high prevalence of CRF02 and CRF 11, respectively, was more closely related to those of CAR and Chad than that of other regions of Cameroon, where CRF02 is the most predominant HIV-1 strain. These findings strongly suggest that this part of Cameroon is a potential hotspot of HIV-1 recombination, with a likelihood of an active generation of new forms of HIV-1 variants, though epidemiologic significance of new HIV-1 forms is unknown.

To investigate the prevalence of subtypes A and C, and the existence of recombinants of both subtypes in the southeast of the Democratic Republic of Congo (DRC), blood samples were collected from 27 HIV-infected individuals in Likasi, located in an area bordering close to Zambia, and analyzed phylogenetically. Out of the 24 strains with a positive PCR profile for pol-IN and env-C2V3, 15 (62.5%) had a discordant subtype or CRF designation: one subtype A/G (pol/env), four A/U (unclassified), three G/A, one G/CRF01, three H/A, one J/C, one CRF02 (G)/A, and one U/A. Nine (37.5%) strains had a concordant subtype or CRF designation: five subtype A, two C, one D, and one CRF02/G. The remaining three samples negative for PCR with env-C2V3 primers used in this study were further analyzed with env-gp41 primers and revealed the presence of two profiles: two J/J (pol-IN/env-gp41) and one C/G. These data highlight the presence of a high proportion (16/27, 59.3%) of recombinant strains and a low prevalence (4.1 and 7.4%) of subtype C based on env-C2V3 and pol-IN analyses, respectively, in Likasi. In addition, this is the first report that CRF02_ AG exists in DRC, though the epidemiological significance of the existence of CRF02_ AG in DRC remains unknown.

The objective of this study was to assess the impact of highly active antiretroviral therapy (HAART) by an oral route on the peripheral blood CD8 subset in the monkeys infected persistently with a pathogenic strain, SHIV89.6P. Two rhesus macaques were inoculated intravenously with SHIV89.6P, then treated with the combination of AZT, 3TC and Lopinavir/Ritonavir (LPV/RTV) as recommended in humans by the oral route with confectionery continued for 28 days. In one of two chronically infected macaques, MM260, the viral load was maintained in the range of 10(4) -10(5) copies/ml before HAART. The plasma viral load and proviral DNA decreased dramatically during the treatment, and cessation of this therapy the viral load rebounded to the pre-treatment level but the proviral DNA rebound was delayed. The other monkey, MM242, had low viral loads (1.2 x 10(3) < 5 x 10(2) copies/ml) both before and after HAART. CD4(+) and CD8(+) T cell counts and proviral DNA level were not significantly changed after the treatment. The percentages of CD8(+) CD45RA(-) Ki67(+) cells increased during (MM260) or after (MM242) HAART and the subset was maintained at a high percentage until 18 weeks post HAART in MM242. These findings suggest that this primate model might serve an important role in testing the virological and immunological efficacy of novel therapeutic strategies combined with HAART. (C) 2003 Elsevier B.V. All rights reserved.

A DNA vaccination regime was investigated previously in rhesus macaques using a full-genome human immunodeficiency virus type 1 (HIV-1) plasmid, which, due to mutations in the nucleocapsid (NC) proteins, produced only non-infectious HIV-1 particles (Akahata et al., Virology 275, 116-124, 2000). In that study, four monkeys were injected intramuscularly 14 times with the plasmid. All of them showed immunological responses against HIV-1 and partial protection from challenge with a simian immunodeficiency virus/HIV (SHIV) chimeric virus. To improve this DNA vaccination regime, the plasmid used for vaccination was changed. In the present study, four macaques were injected intramuscularly eight times with a full-genome SHIV plasmid that produces non-infectious SHIV particles. CTL activities were higher than those observed in monkeys vaccinated previously with the HIV-1 plasmid. In all macaques vaccinated, peak plasma virus loads after homologous challenge with SHIV were two to three orders of magnitude lower than those of the naive controls, and virus loads fell below the level of detection at 6 weeks post-challenge. This suggested that the vaccination regime in this study was partially effective and better than the previous regime.

A new simian/human immunodeficiency virus (SHIV) chimera with the reverse transcriptase (RT)-encoding region of pol, in addition to the 3' region encoding vpr, vpu, tat, rev, env and nef of HIV-1, on an SIVmac (SIV from a macaque monkey) background was constructed. This new SHIV chimera, named SHIVrt/3rn, could replicate in monkey peripheral blood mononuclear cells (PBMCs) as well as in the human and monkey CD4(+) T-cell lines M8166 and HSC-F. Since SHIVrt/3rn contains the RT gene of HIV-1, replication of the virus in M8166 cells was inhibited by an HIV-1 -specific non-nucleoside RT inhibitor, MKC-442, with a sensitivity similar to that of HIV-1. To investigate the replication competence of SHIVrt/3rn in vivo, two rhesus monkeys were inoculated intravenously with the virus. At 2 to 4 weeks post-inoculation (p.i.), plasma viral RNA loads of both monkeys showed a peak value of more than 10(4) Copies ml(-1). Infectious virus was isolated from the PBMCs of one monkey at 2 and 3 weeks p.i. and from the other at 4 weeks p.i. Moreover, proviral DNA was detected constantly throughout the observation period, starting from 3 weeks p.i. An antibody response, detected first at 3 weeks p.i., was maintained at high titres. These results indicate that SHIVrt/3m can infect and replicate in vivo. SHIVrt/3rn, having part of HIV-1 pol in addition to the 3' part of the HIV-1 genome is genetically more close to HIV-1 than any of the other monkey-infecting SHIVs reported previously.

To clarify the physiological function of two zinc-finger (ZF) motifs in the nucleocapsid (NC) protein of simian immunodeficiency virus (SIV), we constructed three mutant viruses with alterations in either or both motifs using a molecular clone of SIVmac (SIVmac239). An immunoblot analysis of the cell lysates transfected with DNA mutated in either the first (ZF1) or second (ZF2) motif showed that the amount of partially processed Gag products (Pr46) was greater than that produced by the wild-type (WT). The genomic RNA contents in the viral particles released from the transfected cells were measured by quantitative RT-PCR. Values for the ZF1 and ZF2 mutants and the double mutant were 26, 20 and 7% that of the WT, respectively, indicating that the two ZF motifs of SIVmac239 NC protein function almost equivalently with respect to RNA encapsidation and processing of Gag precursors. Despite the presence of some genomic RNA in the mutant viruses, they lost all viral infectivity. To determine the reason for this, we examined (using PCR) to which step viral DNA synthesis proceeded in the mutant viruses. We did not see any block up to the step of minus-strand DNA synthesis. However, plus-strand DNA synthesis after plus-strand transfer did not occur in any of the mutant viruses. These findings indicated that the mutations in the ZF motifs of SIVmac led to a loss of infectivity due partly to impairment of DNA synthesis, in addition to inefficient encapsidation of genomic RNA.

To investigate the route of zoonotic transmission of HIV-1, we isolated three and seven HIV-1 strains from 449 Pygmy hunter gatherers and 169 neighboring Bantu, respectively, in southern Cameroon. Phylogenetic analysis based on pol-integrase and env-C2V3 sequences revealed that strains from Pygmies were 1 CRF02_ AG/CRF02_AG, 1 subtype G/CRF02_AG (pol/env), and 1 CRF11_cpx/CRF11_cpx, and that those from Bantu were 2 CRF02_ AG/CRF02_AG, 1 CRF02_ AG/CRF01_AE/A, 1 CRF02_ AG/subtype A, 1 G/A, 1 G/CRF02_AG, and 1 unclassified/H. CRF02_ AG and CRF11_cpx have been identified in Cameroon. The results suggest that HIV-1 has been introduced into Pygmies through their neighboring Bantu rather than directly from nonhuman primates.

Human immunodeficiency virus (HIV) infects lymphocytes and macrophages via CD4 and chemokine receptors. In this study, the infectivity of a chimeric simian and human immunodeficiency virus (SHIV) having a CCR5-specific HIV-1 envelope gene was examined. A SHIV strain termed SHIV-JRFL could enter cells via CD4 with a chemokine receptor CCR5, not CXCR4, and the viral replication was suppressed by recombinant human RANTES, one of beta-chemokines. The intravenous inoculation of SHIV-JRFL into two rhesus macaques resulted in a systemic infection, though it was rather weak. During the early infection, the production of RANTES from Con A-stimulated PBMCs of the infected monkeys increased. These results suggested that beta-chemokine has the potential to limit the infectivity of an R5-type SHIV.

To investigate the HIV-1 subtypes prevalent in the Republic of Congo, we isolated 28 HIV-1 strains from Congolese AIDS patients in 1996 and 1997, and analyzed them phylogenetically. Phylogenetic analysis based on part of the 5' tat-env (vpu) and env sequences revealed that only 13 (46.4%) of the 28 isolates belonged to the same subtype in the vpu tree as in the env tree; the remaining 15 (53.6%) strains showed discordant subtypes between vpu and env with 6 different profiles; that is, 1 A/A (vpalenv), 1 D/D, 5 G/G, 4 H/H, 2 unclassified (U)/U, 9 G/A, 2 G/H, 1 G/J, 1 H/G, 1 U/A, and 1 U/J. Thus, 9 of the 15 discordant HIV-1s were of the G/A (vpu/env) type, and did not form any subcluster within the subtype G lineage in the vpu-based phylogenetic tree. In addition, CRF02_AG (IbNG), which is a G/A (vpu/env) type, was not found in the Republic of Congo. These data suggest that the majority of HIV-1 subtypes circulating in the Republic of Congo have mosaic structures and may have been derived from independent recombinational events.

In this study, we tried a DNA vaccination regime in rhesus macaques using a full genome HIV-1 plasmid. The HIV-I genome is under the control of its original LTR promoter, but has a mutated zinc finger motif gene in the nucleocapsid region. Due to the lack of genomic RNA packaging, the plasmid produces only noninfectious viral particles. We repeatedly injected four macaque monkeys intramuscularly with the naked DNA over a period of 40 weeks. To evaluate the humoral and cell-mediated immunity provided by this DNA vaccination, no other booster or other recombinant viral vectors were used. Immunological responses against HIV-I were elicited in all of the vaccinated monkeys: stable anti-HIV-l Env antibodies were raised in two monkeys and CTL activities were induced in the other monkeys. The macaques were intravenously challenged at 54 weeks with 100 TCID5C of SHIV-NM-3rN, which possesses an envelope gene homologous to the one in the vaccinated plasmid. In all of the vaccinated macaques, the peak plasma viral loads induced by the challenge virus were two to three orders of magnitude lower than those of the naive controls. These results suggest that a DNA vaccination regime with a full genome plasmid alone is potentially efficacious and provides a new possibility for the development of an AIDS vaccine, (C) 2000 Academic Press.

The development of highly efficient and safe gene transfer methods suitable for clinical use is required for human gene therapies. We have developed a novel lentiviral vector system, based on the nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm), that carries a unique dual gene expression system, This system utilizes the lentivirus Rev responsive element (RRE), Self-inactivating vectors were also developed by deleting a U3 region in the 3' long terminal repeat (3' LTR) of the virus. When pseudotyped with a vesicular stomatitis virus envelope glycoprotein G (VSV-G), the SIVagm-based vectors could transduce both growth-arrested human cells and terminally differentiated neuronal cell lines, Using these vectors, two reporter genes could be expressed simultaneously at equal level, and expression levels of both genes could be altered by modifying the length of the RRE sequence. These SIVagm-based vectors might offer safety advantages over other lentivirus-based vectors, Furthermore, the novel dual gene expression system described here could increase the usefulness and value of both viral and nonviral vectors in gene therapy.

To assess the molecular epidemiology of HIV-1 in Republic of Congo (Congo), we investigated 29 HIV-1s obtained from 82 Congolese AIDS and ARC patients in 1996 and 1997, Part of the env region including the V3 loop was phylogenetically analyzed. The genotypes observed were varied: of 29 specimens, 12 (41%) were subtype A, 1 (3%) was subtype D, 6 (21%) were subtype G, 6 (21%) were subtype H, 2 (7%) were subtype J, and 2 (7%) could not be classified as any known subtypes (U, unclassified), The heterogeneous profile of HIV-1 infection was different from the profiles of neighboring Central African countries. These data show that subtypes G and H as well as subtype A mere circulating with high prevalence. The fact that new genetic subtypes (J and U) are circulating indicates a need for a greater surveillance for these subtypes both in Congo as well as in other parts of the world.

Protective immunity induced by a recombinant vaccinia virus expressing the envelope (Env) protein of a simian immunodeficiency virus strain, SIVagm, (SEN-RVV), was evaluated in cynomolgus monkeys (Macaca fascicularis). Three monkeys were immunized twice with SEN-RVV and boostered with the purified SIVagm Env protein. These monkeys developed high titers of anti-SIVagm Env antibody, especially after boostering. After challenge with polyclonal SIVagm, no virus was recovered from two of the monkeys and no provirus DNA was detected in one of these two. After autopsy, however, proviral DNA was detected in the spleen of this monkey. These results suggest that this immunization regimen could not completely protect the monkeys from SIV infection but that it did reduce the replicability of the challenged virus. Copyright (C) 1999 Elsevier Science Ltd.

To determine newly identified lentiviruses, termed simian immunodeficiency virus (SIV)cpz97CG4 and SIVcpz97CG6. from two wild-captured juvenile brother chimpanzees in the Republic of Congo, subgenomic pol (integrase, 288 bp), 5'tat/rev-env C1 (including vpu, 354 bp) and env (C2-C4, 544 bp) gene fragments were amplified and sequenced. The analysis revealed significantly discordant phylogenetic positions of SIVcpz97CG in each genomic region. In the trees derived from partial env sequences (V3), both SIVcpz strains clustered in human immunodeficiency virus type 1 (HIV-1) subtype A. However. in the trees derived from partial pol (integrase) and 5'tat/rev-env C1 (including vpu) sequences, they clustered independently from any of the known HIV-1 subtypes. Especially, in the 5'tat/rev-vpu tree, they branched before the root of HIV-1 group M. These findings suggest that these Congolese SIVcpz genomes are mosaic, probably due to a recombinational event in the recent past, and it provides evidence for a rather recently occurring cross-species transmission between humans and chimpanzees.

Here we describe, for the first time, recombinants between two highly divergent major groups of human immunodeficiency virus type 1 (HIV-1), M and O, within a Cameroonian woman infected with three different HIV-1 strains, a group O virus, a subtype D virus, and a recently reported IBNG (A/G)-like recombinant virus. Using nested extra-long PCR amplification, we sequenced from the pol region to the env region including accessory genes of the viral genome obtained from the patient's uncultured peripheral blood mononuclear cells and examined the phylogenetic position of each gene. Compared with sequential blood samples obtained in 1995 and 1996, there were multiple segmental exchanges between three HIV-1 strains (O, D, and IBNG) and all the recombinants appeared to be derived from a common M/O ancestor. Importantly, recombination between groups M and O occurred, even though the homology between these two groups is 69, 76, 68, and 55% in the gag, pal, vif-vpr, and env regions, respectively. Recombination between strains with such distant lineages may contribute substantially to generating new HIV-1 variants.

In 1995, 53 blood samples from Muslim patients with AIDS, or who were thought to have AIDS, were collected in the main hospitals of Adamaoua Province, in the northern part of Cameroon, The variable env C2V3 region of HIV-1 was amplified by nested PCR and phylogenetically analyzed. The results indicated that of 15 amplified samples, 1 belonged to HIV-1 group O, 1 to HIV-1 subtype D, 1 to subtype G, 2 to subtype H, and 10 to subtype A. Furthermore, the northern Cameroonian subtype A could be divided into at least two subclusters as shown by the env tree as well as by two remarkably conserved hexameric amino acid sequences in the apex of V3 (GPGQAF in one subcluster and GPGQTF in the other). This distinction suggests that the HIV-1 subtype A circulating in northern Cameroon evolved from two main sources. More recently, three HIV-1 strains from Nigeria (IBNG) and Djibouti (DJ263 and DJ264), previously reported on the basis of their env C2V3 sequences as subtype A, were found to have a similar A/G mosaic structure alongside their full-length sequence and were tentatively designated as members of a new subtype called "IBNG," Interestingly, within the northern Cameroonian subtype A described, the isolates of the second subcluster clustered distinctly with these A/G mosaic strains, strongly suggesting that they may be members of the IBNG subtype.

To investigate the role of apoptosis in the early phase of HIV infection, we used macaques infected with sinian immunodeficiency virus strain mac (SIVmac) as a primate model and examined sequentially the characteristics of apoptosis of lymphocytes in peripheral blood mononuclear cells (PBMCs) and lymph nodes in the early phase of SIVmac infection. Five macaques infected with a pathogenic strain of SIV, SIVmac239, were analyzed during the first 4 weeks after infection, Peripheral CD4(+) and CD8(+) cells transiently decreased at 1 week postinfection, The percentage of apoptotic cells in cultured PBMCs increased from about 2 weeks postinfection, The number of apoptotic cells in lymph node sections was higher on days 13 and 28 postinfection than before infection and on day 5 postinfection, Fas antigen expression on peripheral lymphocytes was upregulated from day 8 postinfection, These results indicate that apoptosis is induced about 2 weeks after SIVmac239 infection, following the upregulation of Fas antigen expression on lymphocytes, Since apoptosis was induced about 1 week after the decrease in peripheral CD4(+) and CD8(+) cell counts, it appears that the apoptosis induction does not play an important role in the transient lymphopenia in the early phase of SIVmac infection, In macaques infected with a nonpathogenic derivative of SIVmac239, SIVmac Delta nef, apoptosis of lymphocytes was induced as it was in SIVmac239-infected macaques, but to a lesser degree, suggesting a correlation between the extent of apoptosis induction in lymphocytes in the early phase of SIVmac infection and the pathogenicity of SIVmac.

We analyzed 57 HIV-1 isolates from Cameroon and the Republic of Congo, with respect to the env C2V3 and/or the pol integrase regions. The results indicated that the topology of the pol tree correlated well with that of the env tree for four clusters of subtype D, F G and H, suggesting that these trees reflect the true evolution of the overall genome structures of these subtypes. However, of 22 Cameroonian isolates that were classified as subtype A based on env, 20 of them diverged in their pol sequence into two lineages that were completely different from the prototypical subtype A, tentatively designated as subtypes A1 and A2. The subtype A1 isolates (6 out of 22) were related in their env C2V3 regions with prototypical subtype A strain, but in their pol regions, they formed an independent cluster that diverged from known HIV-1 subtypes so far reported (except for subtypes I and J). The subtype A2 isolates (14 out of 22), which represent the major epidemic type of HIV-1 in Cameroon, clustered distinctly in both the env and pol trees with the recently described A/G mosaic strains from Nigeria and Djibouti. These two lineages were not spreading in the neighboring Republic of Congo.

In order to assess the incidence of HIV mixed infection as well as to clarify the molecular epidemiology of HIV in central Africa, we investigated 43 HIVs obtained from 211 Cameroonian AC, ARC, and AIDS patients in 1994 and 1995. Part of the pol region and part of the env region were phylogenetically analyzed. The genotypes observed were varied: of 43 specimens, 28 (65%) were subtype A, 1 (2%) was subtype B, 2 (5%) were subtype D, 3 (7%) were subtype F,and 2 (5%) were group O. Of the remaining 7 specimens, 3 were mixed infections with HIV-1 subtypes A and C, HIV-1 subtypes C and F, and HIV-2 subtype A and HIV-1 subtype A; 1 was a mixed infection with HIV-1 subtypes A and D and the highly divergent group O (triple infection); another 3 appeared to consist of mosaic genomes (A/G, A/E, and B/A recombinant). These data show that various types of mixed infection, such as between different subtypes of HIV-1 group M, between HIV-1 and HIV-2, and even between HIV-1 groups O and M, were confirmed at a rather high frequency (approximately 10%). The mixed infection is particularly significant where there is a greater variety of HIV-1 subtypes circulating, since it results in new genetic diversity generated by intersubtype recombination. (C) 1998 Academic Press.

We previously constructed a simian immunodeficiency virus/human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4(+) lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.

We constructed two new T-vectors called pUCTA119 and pUCTA18, derived from pUC18. The vectors were designed to produce single thymidine (T)-overhangs when digested with a restriction enzyme Eam11051. The use of the vectors provides a very rapid system for direct TA cloning and subsequent sequencing of unmodified PCR products since Taq DNA polymerase preferentially adds an adenosine (A) residue to the 3' end of the products under standard PCB conditions.

Our previous analysis of an HTLV-I isolate (CMR229) from a Cameroonian Pygmy demonstrated that the isolate is distinct from typical HTLV-Is of the ''Central African group,'' which has a close similarity to HTLV-I-related simian viruses (STLV-I) in Africa. In this study, we analyzed six new HTLV-Is from Cameroon consisting of three isolates from the Pygmy and three from the Bantu to examine further the genetic features of HTLV-I in Cameroon, especially in the Pygmy. A phylogenetic tree based on the long terminal repeats (LTR) region showed that all the new HTLV-Is belong to the Central African group. On the other hand, an env-based analysis of CMR229 confirmed the previous finding derived from LTR-based analysis that CMR229 has a similarity to African STLV-Is, but is distinct from the typical Central African group of HTLV-I. This suggests that multiple interspecies transmissions from non-human primates to humans have occurred in Central Africa, resulting in the presence of two distinct HTLV-I strains in this area. In addition, it seems likely that the Pygmy harbors the heterogeneous HTLV-I strains from which the main HTLV-I population spread into the Bantu.

Eleven human immunodeficiency virus 1 (HIV-1) isolates from Ghanaian acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients obtained by our serosurvey in 1986-1994 were genomically analyzed and phylogenetically compared with other known strains. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-I strains were circulating in Ghana and the majority of them (9 of 11 isolates) belonged to clade (subtype) A which is now furiously epidemic in Africa. Another isolate (1 of 11) belonged to clade D, and the remaining one (1 of 11) belonged to ''clade G''. This ''clade G'' virus grouped by the env analysis belonged to clade A by its pol sequence, suggesting an A/G intersubtype recombinant. The characteristic sequences in the V3 tip which have not yet been reported were observed in these Ghanaian isolates, which should be taken into account for future vaccine programs.

Six human T-cell leukemia virus type I (HTLV-I) and eight human T-cell leukemia virus type II (HTLV-LI) cases newly isolated from the South American countries of Colombia and Chile were analyzed together with the two Amerindian HTLV-I isolates previously reported. All of the HTLV-I isolates belonged to the transcontinental subgroup of the ''cosmopolitan'' group, and Colombian isolates, including those from native Amerindians and Negroes, formed a single tight cluster within this subgroup. The transcontinental subgroup consisted of isolates from various regions such as the Caribbean basin, India, Iran, South Africa, Sakhalin, and Japan, and included isolates from the ''Ainu'' and ''Okinawa'' people, regarded as relatively pure Japanese descended from the prehistoric ''Jomon'' period which began more than 10,000 years ago. This implied a dis semination of the subgroup associated with the movement of human beings in ancient times. On the other hand, all of the HTLV-II isolates from native Amerindians in Colombia and Chile belonged to the HTLV-IIb subtype which has previously been reported to be mainly endemic in certain populations of native Amerindians. The southernmost isolate from Chile, showing wide distribution of the IIb subtype in native South Amerindians and largest heterogeneity of the subtype in Colombian isolates, supported the idea that the HTLV-IIb subtype has been endemic for a long time in native Indians of South America.

In order to examine whether the viral population is affected by intramucosal transmission, we analyzed the viral genotypes first detected in peripheral blood mononuclear cells (PBMCs) after intravaginal inoculation, before virus antibodies were detectable, and compared them with those in the inoculum. Three female cynomolgus macaques were inoculated intravaginally and a fourth was inoculated intravenously with polyclonal simian immunodeficiency viruses (SIVmac32H). The provirus genomes which first appeared in PBMC were sequenced in the V1 to V2 region of the SIV envelope gene. A comparison of the sequences obtained from each monkey revealed a homogeneous or heterogeneous viral population depending on the infection route. In the intravenously inoculated monkey, the viral population was heterogenous and was similar to that in the virus inoculum. On the other hand, in the intravaginally inoculated monkeys, single genotypes (in two monkeys) and one genotype with a slight variation (in one monkey) were found, but they were different from each other, having no characteristic sequences in the V1 to V2 region in common. None of the genotypes found in the PBMC were major genotypes in the virus inoculum. These results suggest that some selective mechanism, which differs among individuals, restricts the viral population during mucosal transmission.

To study the evolutionary origin of human T-lymphotropic virus type I/simian T-lymphotropic virus type I (HTLV-I/STLV-I), we isolated and characterized STLV-I from orangutans (Pongo pygmaeus). Plasma samples from 3 out of 41 animals examined were reactive by particle agglutination and immunofluorescence, and one of these three was confirmed to be anti-HTLV-I antibody-positive by western blotting (WE). Cultured peripheral blood mononuclear cells from the WE-positive orangutan were reactive to anti-STLV-I-positive rhesus monkey plasma. The proviral long terminal repeat region was amplified by polymerase chain reaction and sequenced. A phylogenetic analysis indicated that orangutan STLV-I is related to the Melanesian group of HTLV-Is and other Asian STLV-Is, but the degree of divergence is considerable.

We previously reported that an HIV-1/SIVmac chimeric virus (designated as NM-3rN) having HIV-1 env efficiently infected macaque monkeys by intravenous inoculation. In this study, this chimeric virus was atraumatically inoculated into the vaginal cavity of two rhesus and one cynomolgus monkeys. Although antibody response and detection of proviral genome by PCR were observed in both rhesus monkeys, virus recovery was only once from PBMC in one of them. In the cynomolgus monkey, no virus was recovered and proviral DNA detection was rare. Thus, vaginal inoculation with NM-3rN resulted in poor systemic infection, implying the presence of selective pressure while passing through mucosal membranes.

To develop effective vaccines against infection with human T cell leukaemia virus type I (HTLV-I), we constructed a recombinant vaccinia virus (WR-SFB5env) synthesizing the HTLV-I envelope (Env) gp46 protein under the control of a strong promoter, termed the ATI hybrid promoter. WR-SFB5env expressed a large quantity of gp46. In cynomolgus monkeys (Macaca fascicularis) immunized with WR-SFB5env, anti-HTLV-I Env antibody, including neutralizing antibody, was induced and remained at a high level until 136 weeks (2 . 6 years) post-infection (p.i.). These immunized monkeys had HTLV-I Env-specific cytotoxic T lymphocyte activity. At 136 weeks p.i., the immunized monkeys were challenged with an HTLV-I-producing cynomolgus T lymphocyte cell line. Neither HTLV-I antigen nor HTLV-I proviruses were detected in peripheral blood mononuclear cells, lymph nodes or spleens of the WR-SFB5env-immunized monkeys, in contrast to non-immunized control monkeys. These results indicate that a single immunization with WR-SFB5env induced prolonged humoral and cellular immune responses to HTLV-I and protected the monkeys against virus challenge.