Strains of measles vaccine differ in their ability to replicate in an damage human thymus.

A number of strains of live attenuated measles virus are in use worldwide as measles vaccines. All effectively induce protective immunity, but many differences in immunogenicity and, potentially, in safety are seen between strains in young infants. However, no system for assessing biologic differences between vaccine strains has been available. The SCID-hu thymus model system was used to compare AIK-c, Edmonston-Zagreb (EZ), and Moraten-3 common vaccine strains derived from the Edmonston prototype virus-for replication and pathologic changes in human thymus. EZ replicated best and induced substantial thymocyte death; AIK-c replicated poorly but induced moderate thymocyte death; Moraten replicated moderately well but did not affect thymic architecture. Thus, live attenuated measles vaccines differ in replicative capacity and pathogenicity in vivo. These differences may account for strain-dependent variations in immunization.

[Overview of sleep medicine and sleep respiratory disorders centers in Japan].

Sleep medicine in Japan evolved gradually, beginning with sleep research being conducted in the late 1960's exclusively on the university basis. Japan Sleep Society was founded in 1977 mainly by psychiatrists. Recently public awareness regarding sleep related breathing disorders has been raised as a result of a media campaign in Japan. However, the medical service system is unable to appropriately meet the needs of caring for sleep-disordered patients due to insufficient reimbursement and the lack of well-trained technical personnel. So far only several sleep centers existed in Japan. To make sleep be included in the mainstream of the health care system, the educational system and throughout society, many tasks remains to be done by healthcare professionals and government.

Differing functional recovery of donor-derived immune cells after purified haploidentical and fully mismatched hematopoietic stem cell transplantation in mice.

Functional recovery of the immune system is critical for long-term survival in hematopoietic stem cell transplant recipients. In this study, two donor-recipient allogeneic transplant settings (haploidentical and fully mismatched) are used to investigate the functional activity of donor-derived B and T cells in animals grafted with purified c-kit(+), Thy 1.1(lo), Lin(-/lo), and Sca-1(+) hematopoietic stem cells (KTLS HSC).Ovalbumin-specific immunoglobulin G, polyclonal immunoglobulin isotypes, and B- and T-cell proliferation were examined on the recipients who received haploidentical or fully mismatched HSC.A severe deficiency of antigen-specific immunoglobulin response occurs in fully engrafted mice that received KTLS HSC from fully mismatched, but not haploidentical, donors. This lack of B-cell-specific immunity is not due to a deficiency of polyclonal immunoglobulins in serum. B cells from both fully mismatched and haploidentical recipients proliferate normally after stimulation with anti-mu and the percentage of mature B cells is normal. The T-cell response to anti-CD3 in fully mismatched recipients was much weaker than that of their untransplanted controls. However, T cells from haploidentical recipients respond normally to anti-CD3. This study demonstrates that numerical recovery of donor-derived cells in the periphery of recipients does not represent a functional reconstitution, particularly in animals that receive fully mismatched transplants. Defects of specific B-cell immunity and T-cell proliferation are observed in fully mismatched, purified HSC transplant recipients with a quantitative recovery within the normal range of donor-derived lymphocytes.

Altered virulence of vaccine strains of measles virus after prolonged replication in human tissue.

To understand the molecular determinants of measles virus (MV) virulence, we have used the SCID-hu thymus/liver xenograft model (SCID-hu thy/liv) in which in vivo MV virulence phenotypes are faithfully duplicated. Stromal epithelial and monocytic cells are infected by MV in thymus implants, and virulent strains induce massive thymocyte apoptosis, although thymocytes are not infected. To determine whether passage of an avirulent vaccine strain in human tissue increases virulence, we studied a virus isolated from thymic tissue 90 days after infection with the vaccine strain Moraten (pMor-1) and a virus isolated from an immunodeficient child with progressive vaccine-induced disease (Hu2). These viruses were compared to a minimally passaged wild-type Edmonston strain (Ed-wt) and the vaccine strain Moraten. pMor-1, Hu2, and Ed-wt displayed virulent phenotypes in thymic implants, with high levels of virus being detected by 3 days after infection (10(5.2), 10(2.8), and 10(3. 4), respectively) and maximal levels being detected between 7 and 14 days after infection. In contrast, Moraten required over 14 days to grow to detectable levels. pMor-1 produced the highest levels of virus throughout infection, suggesting thymic adaptation of this strain. Similar to other virulent strains, Ed-wt, Hu2, and pMor-1 caused a decrease in the number of viable thymocytes as assessed by trypan blue exclusion and fluorescence-activated cell sorter analysis. Thymic architecture was also disrupted by these strains. Sequence analysis of the hemagglutinin (H) and matrix (M) genes showed no common changes in Hu2 and pMor-1. M sequences were identical in pMor-1 and Mor and varied in H at amino acid 469 (threonine to alanine), a position near the base of propeller 4 in the propeller blade/stem model of H structure. Further study will provide insights into the determinants of virulence.

Induction of MHC class I expression on immature thymocytes in HIV-1-infected SCID-hu Thy/Liv mice: evidence of indirect mechanisms.

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.

Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells.

Post HIV-1 entry, productive HIV-1 infection of primary T cells requires overcoming several cellular blocks to provirus establishment and replication. Activation of unknown host intracellular events overcomes such inhibitory steps and is concomitant with HIV-1 replication. We show that the transcription factor NFATc was sufficient as a cellular factor to induce a highly permissive state for HIV-1 replication in primary CD4+ T cells. NFATc overcame a blockade at reverse transcription and permitted active HIV-1 replication. Pharmacologic blockade of endogenous NFAT activity by FK506 or CsA inhibited synthesis of reverse transcription and also potently blocked HIV-1 replication. T cells therefore can become competent for HIV-1 replication by control of regulated host factors such as the NFATc transcription factor. The host mechanisms regulated by such permissivity factors are potential targets for anti-HIV-1 therapy.

The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse.

The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine/threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus/liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity in vivo, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable in vitro, has a critical role within differentiated cells that are essential targets for VZV pathogenesis in vivo.

Recombinant measles viruses with mutations in the C, V, or F gene have altered growth phenotypes in vivo.

An understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734-3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5' untranslated region of the F gene (F 5'UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but had different replication kinetics. Abrogation of V expression led to delayed and markedly prolonged replication. Additionally, thymocyte survival was prolonged and implant architecture was preserved throughout the course of infection. In contrast, massive bystander thymocyte death occurred after infection with EdTag and all other mutants. A mutant which overexpressed V in Vero cells (V+) had the opposite phenotype of the A mutant not expressing V (V-). V+ grew more rapidly than EdTag with 100-fold-greater levels of virus production 3 days after infection. These results suggest that C, V, and the F 5'UTR are accessory factors required for efficient virus replication in vivo. In addition, thymocyte survival after V- infection suggests this protein may play multiple roles in pathogenesis of MV infection of thymus. Since these recombinant mutant viruses grew identically to the parent virus in Vero cells, the data show that thy/liv implants are an excellent model for investigating the determinants of MV virulence.

[The correlation between the outbreaks of asthma attack and meteorologic parameters in Okinawa].

The correlation between the outbreaks of asthma attack and meterologic parameters was analyzed in Okinawa island which belongs to the subtropics. The epidemiologic outbreaks of asthma was investigated for 2 years from the asthma diaries described by 27 patients. The severity of asthma attack was expressed as the asthma score on the basis of asthma diaries. The number of patients carried to hospitals by ambulance on asthma attacks was investigated for 3 years. Two-by-two contingency tables were computed for the meteorologic parameters and analyzed with the method of chi-square test. From the view point of asthma scores, the total scores of 27 patients were increased when a mean and a minimum temperature were respectively higher than each mean value in the period of investigation (p < 0.05, respectively). From the view point of the number of patients carried to hospitals by ambulance on asthma attacks, it was suggested that asthma attacks tended to occur when a mean, a maximum and a minimum temperature, and a vapor pressure were lower than each mean value (p = 0.0001, p = 0.0001, p = 0.0001, p = 0.0002), and a barometric pressure was higher than a mean value in the period of investigation (p = 0.0016). From the further analysis of these data by multiple regression analysis, it was suggested that the number of patients carried to hospitals by ambulance on asthma attacks was influenced by low temperature. In addition, it was suggested that the changes of meteorologic parameters on the passing over of typhoon, especially, the decrease of temperature and barometric pressure, were related to induce asthma attacks.

Inhibition of human immunodeficiency virus type 1 replication in myelomonocytic cells derived from retroviral vector-transduced peripheral blood progenitor cells.

Monocytes and macrophages (Mo/Mphi) contribute to the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. A successful hematopoietic stem/progenitor cell (HSPC)-based gene therapy strategy for HIV-1 disease must protect Mo/Mphi as well as T cells from HIV-1-related pathology. In this report, we demonstrate that RevM10-transduced HSPCs isolated from cytokine-mobilized peripheral blood give rise to Mo/Mphi suppressing replication of Mphi-tropic HIV-1 isolates. A Moloney murine leukemia virus (MoMLV)-based retroviral vector encoding a bicistronic mRNA co-expressing RevM10 and the murine CD8alpha' chain (Lyt2) was used to transduce HSPCs. Following transduction, these cells were expanded and differentiated by short-term culture in methylcellulose containing various cytokines. In vitro differentiated Mo/Mphi were enriched by fluorescence activated cell sorting (FACS) for the co-expressed transgene (Lyt2) and myelomonocytic (CD33, CD14) surface markers. HIV-1 replication of two Mphi-tropic isolates (JR-FL, BaL) was inhibited in Mo/Mphi expressing RevM10 and Lyt2 relative to control cells expressing only Lyt2 but no functional RevM10 gene product. Cell proliferation and expression of lineage-specific surface markers was not altered in transduced, in vitro differentiated Mo/Mphi cells. This study supports the feasibility of HSPC-based gene therapy as a future treatment for HIV-1 disease.

Comparative analyses of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication.

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3' long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the transdominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse.

The SCID-hu mouse implanted with human fetal tissue is a novel model for investigating human viral pathogenesis. Infection of human skin implants was used to investigate the basis for the clinical attenuation of the varicella-zoster virus (VZV) strain, V-Oka, from which the newly licensed vaccine is made. The pathogenicity of V-Oka was compared with that of its parent, P-Oka, another low-passage clinical isolate, strain Schenke (VZV-S), and VZV-Ellen, a standard laboratory strain. The role of glycoprotein C (gC) in infectivity for human skin was assessed by using gC-negative mutants of V-Oka and VZV-Ellen. Whereas all of these VZV strains replicated well in tissue culture, only low-passage clinical isolates were fully virulent in skin, as shown by infectious virus yields and analysis of implant tissues for VZV DNA and viral protein synthesis. The infectivity of V-Oka in skin was impaired compared to that of P-Oka, providing the first evidence of a virologic basis for the clinical attenuation of V-Oka. The infectivity of V-Oka was further diminished in the absence of gC expression. All strains except gC-Ellen retained some capacity to replicate in human skin, but cell-free virus was recovered only from implants infected with P-Oka or VZV-S. Although VZV is closely related to herpes simplex virus type 1 (HSV-1) genetically, experiments in the SCID-hu model revealed differences in tropism for human cells that correlated with differences in VZV and HSV-1 disease. VZV caused extensive infection of epidermal and dermal skin cells, while HSV-1 produced small, superficial lesions restricted to the epidermis. As in VZV, gC expression was a determinant for viral replication in skin. VZV infects human CD4+ and CD8+ T cells in thymus/liver implants, but HSV-1 was detected only in epithelial cells, with no evidence of lymphotropism. These SCID-hu mouse experiments show that the clinical attenuation of the varicella vaccine can be attributed to decreased replication of V-Oka in skin and that tissue culture passage alone reduces the ability of VZV to infect human skin in vivo. Furthermore, gC, which is dispensable for replication in tissue culture, plays a critical role in the virulence of the human alphaherpesviruses VZV and HSV-1 for human skin.

Antiviral potency of drug-gene therapy combinations against human immunodeficiency virus type 1.

Gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infection using intracellular immunization strategies is currently being tested in clinical trials. With the continuing development of potent antiretroviral drugs (e.g., reverse transcriptase [RT] and protease [PR] inhibitors), it is likely that HIV-1 gene therapy will be applied to humans concurrently receiving such antiretroviral medication. In this study, we assessed the in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10, Gag antisense RNA) in combination with clinically relevant RT (AZT, ddC) or PR (indinavir) inhibitors. Retrovirally transduced, human T cell lines expressing antiviral gene constructs were inoculated with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone. More importantly, we also addressed whether gene therapy strategies are effective against drug-resistant HIV-1 isolates. Both the RevM10 and Gag antisense RNA strategies showed antiviral efficacy against several RT inhibitor-resistant HIV-1 isolates equivalent to their inhibition of HIV-1HXB3 replication. In summary, our data demonstrate the greater than additive antiviral efficacy of gene therapy strategies and RT or PR inhibitors, and that gene therapy approaches are effective against drug-resistant HIV-1 viral isolates.

The SCID-hu mouse: an in vivo model for HIV-1 infection in humans.

The lack of suitable animal models for the in vivo study of HIV-1 infection has prompted investigators to take advantage of the graft-rejection deficit in severe combined immunodeficient (SCID) mice. Two separate approaches have been used to transplant human lymphoid and/or hemolymphoid tissues into SCID mice to generate chimeric animals in which distinct elements of the human immune system could be maintained and studied in vivo. The two models that arose from this work were the SCID-hu mouse and hu-PBL-SCID mouse. The goal of producing these distinct models is to provide an easily manipulable model for the in vivo analysis of HIV-1 infection and its ensuing pathophysiology. Both models support HIV-1 replication and display potential as models for studying antiviral strategies and mechanisms of viral pathogenesis. This review focuses on the SCID-hu mouse.

Hematopoietic potential and retroviral transduction of CD34+ Thy-1+ peripheral blood stem cells from asymptomatic human immunodeficiency virus type-1-infected individuals mobilized with granulocyte colony-stimulating factor.

The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)-infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+ Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1-infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 microg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+ Thy-1+ cell population of high purity (76% to 92% CD34+ Thy-1+ cells). This cell population showed no evidence of HIV-1-containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+ Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1-infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.

RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.

A key feature of the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection is the gradual loss of CD4-positive T cells. A number of gene therapy strategies have been designed with the intent of inhibiting HIV replication in mature T cells. As T cells are products of hematolymphoid differentiation, insertion of antiviral genes into hematopoietic stem cells could serve as a vehicle to confer long-term protection in progeny T cells derived from transduced stem cells. One such "cellular immunization" strategy utilizes the gene coding for the HIV-1 rev trans-dominant mutant protein RevM10 which has been demonstrated to inhibit HIV-1 replication in T-cell lines and in primary T cells. In this study, we used a Moloney murine leukemia virus-based retrovirus encoding a bicistronic message coexpressing RevM10 and the murine CD8-alpha' chain (Lyt2). This vector allows rapid selection of transgene-expressing cells as well as quantitation of transgene expression. We demonstrate that RevM10-transduced CD34-enriched hematopoietic progenitor-stem cells (HPSC) isolated from human umbilical cord blood or from granulocyte colony-stimulating factor-mobilized peripheral blood can give rise to mature thymocytes in the SCID-hu thymus/liver mouse model. The phenotypic distribution of HPSC-derived thymocytes is normal, and expression of the transgene can be detected by flow cytometric analysis. Moreover, we demonstrate that RevM10 can inhibit HIV replication in T cells derived from transduced HPSC after expansion in vitro. This is the first demonstration of anti-HIV efficacy in T cells derived from transduced human HPSC.

Hematopoietic stem cell-based gene therapy for acquired immunodeficiency syndrome: efficient transduction and expression of RevM10 in myeloid cells in vivo and in vitro.

Gene delivery via the hematopoietic stem cell (HSC) offers an attractive means to introduce antiviral genes into both T cells and macrophages for acquired immunodeficiency syndrome (AIDS) gene therapy. An amphotropic retroviral vector encoding a bicistronic gene coexpressing RevM10 and the murine CD8alpha' chain (lyt2) was developed to transduce HSC/progenitor cells. After transduction of CD34+ cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytometry was used to monitor the level of gene transduction and expression and to enrich RevM10-expressing cells by cell sorting without drug selection. Using this quantitative method, high levels of gene transduction and expression (around 20%) were achieved by high-speed centrifugation of CD34+ cells with the retroviral supernatant (spinoculation). After reconstitution of human bone marrow implanted in SCID mice (SCID-hu bone) with the transduced HSC/progenitor cells, a significant number of donor-derived CD14+ bone marrow cells were found to express the RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus-type 1 (HIV-1) isolate was greatly inhibited in the lyt2+/CD14+ cells differentiated from transduced CD34+ cells after the enrichment of lyt2+ population. Thus, the RevM10 gene did not appear to inhibit the differentiation of HSC/progneitor cells into monocytes/macrophages. The level of retrovirus-mediated RevM10 expression in monocytes/macrophages derived from transduced HSCs is sufficient to suppress HIV-1 replication.

The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells.

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication.

Identification of HIV-1 determinants for replication in vivo.

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.