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Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

New HIV-1 infections are genotyped as part of standard of care testing to ensure that antiretroviral treatment will be efficacious against the virus. Historically this has been performed by sequencing the pol region of the HIV-1 genome only. The popularity of next-generation sequencing (NGS) methods during the SARS-CoV-2 pandemic has resulted in a shift towards using NGS in diagnostic sequencing, but there remain limited methodologies utilising the strengths of NGS for robust diagnostic sequencing of longer regions of the HIV-1 genome. Given the acceptance and success of tiling PCR methodologies during the SARS-CoV-2 pandemic, we aimed to design and verify a novel tiling PCR method for routine HIV-1 sequencing. A set of tiling PCR primers was designed to amplify the 5’ half of HIV-1 in six overlapping segments of 1,000 bp in only two PCR reactions. The assay can move from sample to sequencer in under a day. The tiling PCR was able to generate HIV-1 sequences from 90 (100%) samples in a comparison panel, and complete protease-reverse transcriptase and integrase regions were amplified in > 90% of samples with a viral load > 5000 copies/mL. Seven additional drug resistance mutations were identified when using this novel method. As such, this novel designer tiling PCR is a promising method for the routine NGS-based diagnostic sequencing of HIV-1.

The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Future HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4 + T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1. IMPORTANCE The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically-intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Our investigations into the possible cellular mechanisms maintaining the HIV-1 reservoir in different T-cell subsets have revealed a link between the half-lives of T-cells and the level of proviruses they contain. Taken together, we believe our study shows that more differentiated and proliferative cells, such as transitional and effector memory T-cells, contain the highest levels of genetically-intact proviruses, and the rapid turnover rate of these cells contributes to the expansion of genetically-intact proviruses within them. Therefore, our study delivers an in-depth assessment of the cellular mechanisms, such as cellular proliferation and half-life, that contribute to and maintain the latent HIV-1 reservoir.

Human respiratory syncytial virus (RSV) is an important cause of acute respiratory infection with the most severe disease in the young and elderly. Non-pharmaceutical interventions and travel restrictions for controlling COVID-19 have impacted the circulation of most respiratory viruses including RSV globally, particularly in Australia, where during 2020 the normal winter epidemics were notably absent. However, in late 2020, unprecedented widespread RSV outbreaks occurred, beginning in spring, and extending into summer across two widely separated regions of the Australian continent, New South Wales (NSW) and Australian Capital Territory (ACT) in the east, and Western Australia. Through genomic sequencing we reveal a major reduction in RSV genetic diversity following COVID-19 emergence with two genetically distinct RSV-A clades circulating cryptically, likely localised for several months prior to an epidemic surge in cases upon relaxation of COVID-19 control measures. The NSW/ACT clade subsequently spread to the neighbouring state of Victoria and to cause extensive outbreaks and hospitalisations in early 2021. These findings highlight the need for continued surveillance and sequencing of RSV and other respiratory viruses during and after the COVID-19 pandemic, as mitigation measures may disrupt seasonal patterns, causing larger or more severe outbreaks. Non-pharmaceutical interventions for COVID-19 also reduced incidence of respiratory pathogens such as respiratory syncytial virus (RSV). Here, the authors report the resurgence of RSV in Australia following lifting of some of the restrictions and describe reduction in genetic diversity in circulating clades.

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic Henipaviruses (HNVs) responsible for recurrent outbreaks in humans and domestic species of highly fatal (50 to 95%) disease. A HeV variant (HeV-g2) of unprecedented genetic divergence has been identified in two fatally diseased horses, and in two flying fox species in regions of Australia not previously considered at risk for HeV spillover. Given the HeV-g2 divergence from HeV while retaining equivalent pathogenicity and spillover potential, understanding receptor usage and antigenic properties is urgently required to guide One Health biosecurity. Here, we show that the HeV-g2 G glycoprotein shares a conserved receptor tropism with prototypic HeV and that a panel of monoclonal antibodies recognizing the G and F glycoproteins potently neutralizes HeV-g2– and HeV G/F–mediated entry into cells. We determined a crystal structure of the Fab fragment of the hAH1.3 antibody bound to the HeV G head domain, revealing an antigenic site associated with potent cross-neutralization of both HeV-g2 and HeV. Structure-guided formulation of a tetravalent monoclonal antibody (mAb) mixture, targeting four distinct G head antigenic sites, results in potent neutralization of HeV and HeV-g2 and delineates a path forward for implementing multivalent mAb combinations for postexposure treatment of HNV infections.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Abstract Respiratory syncytial virus (RSV) is an important human respiratory pathogen. In temperate regions, a distinct seasonality is observed, where peaks of infections typically occur in early winter, often preceding the annual influenza season. Infections are associated with high rates of morbidity and mortality and in some populations exceed that of influenza. Two subtypes, RSV-A and RSV-B, have been described, and molecular epidemiological studies have shown that both viruses mostly co-circulate. This trend also appears to be the case for Australia; however, previous genomic studies have been limited to cases from one Eastern state—New South Wales. As such, the broader spatial patterns and viral traffic networks across the continent are not known. Here, we conducted a whole-genome study of RSV comparing strains across eastern and Western Australia during the period January 2016 to June 2017. In total, 96 new RSV genomes were sequenced, compiled with previously generated data, and examined using a phylodynamic approach. This analysis revealed that both RSV-A and RSV-B strains were circulating, and each subtype was dominated by a single genotype, RSV-A ON1-like and RSV-B BA10-like viruses. Some geographical clustering was evident in strains from both states with multiple distinct sub-lineages observed and relatively low mixing across jurisdictions, suggesting that endemic transmission was likely seeded from imported, unsampled locations. Overall, the RSV phylogenies reflected a complex pattern of interactions across multiple epidemiological scales from fluid virus traffic across global and regional networks to fine-scale local transmission events.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of [CD4.sup.+] T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory [CD4.sup.+] T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory [CD4.sup.+] T cells when compared with naive, central, and transitional memory [CD4.sup.+] T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

A novel Hendra virus (HeV) variant was identified from a horse that suffered acute fatal disease consistent with HeV infection through multidisciplinary and interagency syndromic sentinel surveillance research. Novel molecular assays for HeV detection are described in the light of routine testing failure. In silico analysis of the variant receptor-binding protein in comparison with prototypic HeV supported that the monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the equine vaccine, should be effective also against this novel variant. Similarity of this virus (99%) to a partial sequence detected from a South Australian grey-headed flying fox, along with case exposure to this species in Queensland, suggests the variant circulates at least across the range of this flying fox species. Investigation into HeV diversity, comparative kinetics and pathogenicity, reservoir-species associations, viral-host co-evolution and spillover dynamics should be prioritized. Biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes regardless of species. Competing Interest Statement The authors have declared no competing interest.