Initial interactions between HIV-1 and the immune system at mucosal exposure sites play a critical role in determining whether the virus is eliminated or progresses to establish systemic infection. The virus that successfully crosses the mucosal barrier to establish infection in the new host is referred to as the transmitted/founder (TF) virus. Following mucosal HIV-1 transmission, type 1 interferons (IFN-I) are rapidly induced at sites of initial virus replication. The resistance of TF variants to these antiviral effects of the IFN-I has been studied among HIV-1 subtypes B and C. However, their role in restricting HIV-1 replication among subtypes D and AD recombinant remains unexplored. This study assessed the sensitivity of HIV-1 subtype D and AD recombinant TF viruses to IFN-I by infecting peripheral blood mononuclear cells in vitro with infectious molecular clones of these viruses. Cells were exposed to varying concentrations of interferon-α and interferon-β, and viral replicative capacity was measured using HIV-1 p24 antigen ELISA from culture supernatants. Sensitivity to IFN-I was quantified based on viral replication levels. The results showed that interferon-α was more effective in inhibiting viral replication than interferon-β, regardless of the varying amounts of IFN-I used. However, recombinant AD viruses were found to be more resistant to the antiviral effects of IFN-I compared to subtype D viruses. These findings highlight the differential sensitivity of HIV-1 subtypes AD recombinant and D TF viruses to IFN-I and underscore the potential of IFN-I as a therapeutic strategy to target TF viruses and reduce HIV-1 transmission, particularly in populations where subtype D is prevalent.

Summary: Viral genome sequencing and analysis are crucial for understanding the diversity and evolution of viruses. Traditional Sanger sequencing is limited by low sequence depth and is labor intensive. Next-Generation Sequencing (NGS) methods, such as Illumina, offer improved sequencing depth and throughput but face challenges with accurate reconstruction of viral genomes due to genome fragmentation. Third-generation sequencing platforms, such as PacBio and Oxford Nanopore Technologies (ONT), generate long reads with high throughput. However, PacBio is constrained by substantial resource requirements, while ONT suffers from inherently high error rates. Moreover, standardized pipelines for ONT sequencing encompassing basecalling to genome assembly remain limited. Results: Here, we introduce BonoboFlow, a standardized Nextflow pipeline designed to streamline ONT-based viral genome assembly/haplotype reconstruction. BonoboFlow integrates key processing steps, including basecalling, read filtering, chimeric read removal, error correction, draft genome assembly/haplotype reconstruction, and genome polishing. The pipeline accepts raw POD5 or basecalled FASTQ files as input, produces FASTA consensus files as output, and uses a reference genome (in FASTA format) for contaminant read filtering. BonoboFlow’s containerized implementation via Docker and Singularity ensures seamless deployment across diverse computing environments. While BonoboFlow excels in assembling small and medium viral genomes, it showed challenges when reconstructing large viral genomes. Availability and implementation: BonoboFlow and corresponding containerized images are publicly available at https://github.com/nchis09/BonoboFlow and https://hub.docker.com/r/nchis09/bonobo_image. The test dataset is available at SRA repository Accession number: PRJNA1137155, http://www.ncbi.nlm.nih.gov/bioproject/1137155.

Introduction: The envelope glycoprotein (Env) of HIV-1 Transmitted/Founder (T/F) viruses in subtypes B and C carries distinct genetic signatures that enhance transmission fitness, augment infectivity and immune evasion. However, there is limited data on such signatures in T/F subtypes A1, D and A1D recombinants that predominate East Africa’s HIV epidemic.Methods: We used phylogenetically corrected approaches to detect distinct genetic signatures by comparing 44 contemporary HIV-1 T/F Envs with 229 historical Envs of the same subtype in East Africa.Results and Discussion: Subtype analysis based on the full-length Env gene of contemporary T/F viruses revealed a high proportion of subtype A1, followed by A1D recombinants, and fewer subtype D. Signature analysis revealed that the contemporary subtype A1 T/Fs were more likely to select distinct amino acids, including M22 in the signal peptide, R82 in gp120, A172 in the V2 loop, E230 in the glycosite 230, K275 in the D loop, Y317 in the V3 loop, K476 and N477 in the CD4 contact site, when compared with the historical Envs (q-value < 0.2). Conversely, the contemporary subtype A1 T/F Envs were less likely to carry the amino acids Q432 in the CD4 contact site, and the L784 signature within the LLP-2 (q-value < 0.2). The A1D recombinant T/Fs were more likely to select the D620 in the C-helix, but under selected the L34 in gp120, P299 in the V3 loop and Y643 in the Heptad repeat-2, compared to the historical Envs (q-value < 0.2). The distinct signature sites reported in this study may contribute to the successful establishment of acute infection as well as the persistence of long-term infection. Therefore, effective therapeutics and vaccines may target these distinct amino acid signatures especially for the East African region as it may be necessary to employ subtype-specific vaccines according to the subtype distribution.

HIV-1 Transmitted/Founder (T/F) viruses cause 80-90% of heterosexual transmissions, making their rapid identification vital for vaccine and cure development. Single-genome amplification (SGA) followed by Sanger sequencing is the gold standard for T/F virus detection, but low throughput and high cost limit its scalability. Here, we evaluated the Oxford Nanopore Technology (ONT) as a high-throughput alternative. We sequenced 195 archived HIV-1 single genome amplicons (SGAs) from 20 acutely infected participants, encompassing both 3' and 5' genome halves. Libraries were prepared via end repair, native barcoding, and adapter ligation, and then sequenced on a MinION MK1C device with R9.4 flow cells. Data processing included read filtering, error correction, and haplotype reconstruction. T/F viruses were identified using Highlighter plots and by applying a criterion of intra-patient mean pairwise diversity <0.60%, together with phylogenetic clustering. The sequence most closely related to the most recent common ancestor (MRCA) was designated as the T/F virus. Phylogenetic analysis showed strong concordance between ONT and Sanger sequences, with 100% bootstrap support. ONT identified 35 of 39 T/F viruses detected by Sanger, achieving 89.70% sensitivity. Sequence similarity between ONT and Sanger - derived T/Fs averaged 99.81% (95% CI: 99.76-99.87%), ranging from 99.45-99.96%. These findings demonstrate ONT's promise as a reliable, high-throughput alternative for HIV-1 T/F identification. Advances such as the Dorado basecaller and Q20+ chemistry are expected to further improve ONT's accuracy, supporting its use in large-scale, resource-limited settings.

Background: Adenoviral vectors are widely used in vaccine development; however, pre-existing immunity to common human adenovirus serotypes can limit their effectiveness. Canine adenovirus type 2 (CAV-2) is a non-human adenovirus with low seroprevalence in humans, making it a suitable alternative vector. Despite its promise, comprehensive preclinical safety data for CAV-2 vector-based vaccine platforms remain limited. In this study, we evaluated the safety profile of a replication-defective CAV-2 vector expressing the Omicron BA.4 SARS-CoV-2 spike immunogen in BALB/c mouse models. Methods: The SARS-CoV-2 CAV-2 vector-based vaccine was expressed and propagated in AD293 cells. The mice received intramuscular prime-boost immunisations with low (1 × 10⁶ PFU), moderate (0.5 × 10¹⁰ PFU), or high (1 × 10¹⁰ PFU) vaccine doses, alongside empty CAV-2 vector and physiological buffer control groups, and were monitored longitudinally up to Day 72. The mice were clinically assessed at days 0, 7, 21, 42, and 72 for any deviations from normal conditions in comparison to the control groups. Biochemical analyses were performed to evaluate liver and kidney function, as well as any tissue injury due to the vaccine candidate. Hematological parameters were assessed by conducting complete blood counts. Body temperature and weight changes were also monitored as an indicator of systemic toxicity. Results: The biochemical and haematological parameters remained within physiological reference ranges across all dose groups and timepoints, with no dose-related deviations, indicating that the vaccine candidate did not show evidence of hepatotoxicity, nephrotoxicity, tissue or haematological toxicity. Body temperatures remained within normal physiological ranges following both prime and booster immunisations, and body weights increased normally across all groups as the animals grew throughout the study period without any abnormal weight gain or loss. Conclusions: These results suggest that the replication-defective CAV-2-vectored SARS-CoV-2 vaccine candidate was well tolerated and did not demonstrate evidence of systemic toxicity under the conditions tested. These findings demonstrate that the CAV-2 vector exhibits a favourable safety profile in murine models when used as a vaccine delivery platform, supporting its translational potential as an alternative adenoviral vector-based vaccine platform. Further studies incorporating additional safety endpoints, including histopathological, vector persistence and shedding evaluation, are warranted to support continued development of the platform.

Understanding the genetic evolution of HIV-1 Transmitted/Founder (T/F) virus is crucial for developing effective treatment and prevention strategies due to