Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4+ T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12–20 mo, viruses exhibited concentrated mutations at 17–34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.

Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 ؋ 10 ؊5 . Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 ؋ 10 ؊5 substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of moreconventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.

Lack of effective therapy of primary brain tumors has promoted the development of novel experimental approaches utilizing oncolytic viruses combined with gene therapy. Towards this end, we have assessed a conditionally replication-competent, g 1 34.5-deleted herpes simplex virus type 1 (HSV-1) expressing cytosine deaminase (CD) for treatment of malignant brain tumors. Our results are summarized as follows: (i) a recombinant HSV (M012) was constructed in which both copies of the g 1 34.5 gene were replaced with the bacterial CD gene, under the control of the cellular promoter Egr-1; (ii) M012-infected cells in vitro efficiently convert 5-fluorocytosine (5-FC) to 5-fluorouracil, thereby enhancing cytotoxicity of neighboring, uninfected cells; (iii) both direct and bystander cytotoxicity of murine neuroblastoma and human glioma cell lines after infection with M012 were demonstrated; (iv) direct intracerebral inoculation of A/J mice demonstrated lack of neurotoxicity at doses similar to G207, a g 1 34.5-deleted HSV with demonstrated safety in human patient trials and (v) intratumoral injection of M012 into Neuro-2a flank tumors in combination with 5-FC administration significantly reduced tumor growth versus tumors treated with R3659 combined with 5-FC, or treated with M012 alone. Thus, M012 is a promising new oncolytic HSV vector with an enhanced prodrug-mediated, antineoplastic effect that is safe for intracranial administration.

Summary OBJECTIVES To describe specific causes of the high rates of stillbirth, neonatal death and early child childhood death in Zambia. METHODS We conducted a household-based survey in rural Zambia. Socio-demographic and delivery characteristics were recorded, alongside a maternal HIV test. Verbal autopsy questionnaires were administered to elicit mortality-related information and independently reviewed by three experienced paediatricians who assigned a cause and contributing factor to death. For this secondary analysis, deaths were categorized into: stillbirths (foetal death ≥28 weeks of gestation), neonatal deaths (≤28 days) and early childhood deaths (>28 days to <2 years). RESULTS Among 1679 households, information was collected on 148 deaths: 34% stillbirths, 26% neonatal and 40% early childhood deaths. Leading identifiable causes of stillbirth were intrauterine infection (26%) and birth asphyxia (18%). Of 32 neonatal deaths, 38 (84%) occurred within the first week of life, primarily because of infections (37%) and prematurity (34%). The majority of early childhood deaths were caused by suspected bacterial infections (82%). HIV prevalence was significantly higher in mothers who reported an early childhood death (44%) than mothers who did not (17%; P < 0.01). Factors significantly associated with mortality were lower socio-economic status (P < 0.01), inadequate water or sanitation facilities (P < 0.01), home delivery (P = 0.04) and absence of a trained delivery attendant (P < 0.01). CONCLUSION We provide community-level data about the causes of death among children under 2 years of age. Infectious etiologies for mortality ranked highest. At a public health level, such information may have an important role in guiding prevention and treatment strategies to address perinatal and early childhood mortality.

We observed a higher point estimate for VE against increased severity of illness compared with milder disease, but were not powered to detect a low level of VE against milder disease.