Why does hormonal contraception and menopausal hormonal treatment have such a small effect on breast cancer risk?

Oestrogen is considered by many to be a major cause of breast cancer, and yet hormonal contraception and menopausal hormonal therapy have a paradoxically small effect on breast cancer risk. Also, in the oestrogen-only arm of the Women's Health Initiative, subjects given oestrogen had a reduced risk of breast cancer compared to controls. Initiation of breast cancer likely begins early in life, in the long-lived ER-PR- breast stem cell. The main mitogen of ER+PR+ breast cancers is oestrogen derived from local breast fat and the tumour itself, rather than circulating oestrogens. Progesterone is relatively breast neutral, but progestins in the laboratory have been shown to expand malignant breast stem cell number.

Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Fatal Human Neurologic Infection Caused by Pigeon Avian Paramyxovirus-1, Australia.

Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples.

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.

New Detection of Locally Acquired Japanese Encephalitis Virus Using Clinical Metagenomics, New South Wales, Australia.

In the context of an emerging Japanese encephalitis outbreak within Australia, we describe a novel locally acquired case in New South Wales. A man in his 70s had rapidly progressive, fatal meningoencephalitis, diagnosed as caused by Japanese encephalitis virus by RNA-based metagenomic next-generation sequencing performed on postmortem brain tissue.

Safety and effectiveness of a novel home-use therapeutic ultrasound device for the treatment of vaginal dryness in postmenopausal women: a pilot study.

To evaluate safety and effectiveness of therapeutic ultrasound for treatment of postmenopausal vaginal dryness. In a pilot study, postmenopausal women with self-reported vaginal dryness were randomized (1:1) to double-blind ultrasound treatment (n = 21) or sham (n = 21) for 12 weeks. Primary effectiveness endpoint was change from baseline to week 12 in Vaginal Assessment Scale symptoms (dryness, soreness, irritation, dyspareunia). Secondary effectiveness endpoint was scoring of clinician-reported Vaginal Health Index (elasticity, fluid, pH, mucosa, moisture). After 12 weeks, participants received open-label ultrasound treatment to 1 year. Safety endpoint was treatment-emergent adverse events. In the modified intent-to-treat population, women showed (mean ± standard error) reduction in Vaginal Assessment Scale with ultrasound treatment versus sham (n = 15, −0.5 ± 0.2 vs n = 15, −0.4 ± 0.3; P = 0.9) and improved Vaginal Health Index (n = 9, 2.7 ± 0.9 vs n = 9, 0.6 ± 1.4; P = 0.3). In the per-protocol analysis population, ultrasound treatment (n = 9) versus sham (n = 8) significantly reduced symptoms score (−0.6 ± 0.3 vs −0.0 ± 0.4; P = 0.05) and significantly improved Vaginal Health Index (2.7 ± 0.9 vs −0.4 ± 1.2; P = 0.03). Improvement in effectiveness endpoints were seen at 1 year compared with baseline. There were no differences in treatment-emergent adverse events between ultrasound treatment versus sham and no serious adverse events. Home-use ultrasound was safe and effective for treating vaginal dryness after 12 weeks. Effectiveness was maintained to 1 year. Therapeutic ultrasound could offer a new, nonhormonal treatment option for postmenopausal women with vulvovaginal atrophy.

Novel variant Hendra virus genotype 2 infection in a horse in the greater Newcastle region, New South Wales, Australia.

In October 2021, the first contemporary detection of Hendra virus genotype 2 (HeV-g2) was made by veterinary priority disease investigation in a horse near Newcastle, New South Wales, Australia, as part of routine veterinary priority disease surveillance. This discovery followed an update of Hendra virus diagnostic assays following retrospective identification of this variant from 2015 via sentinel emerging infectious disease research, enabling timely detection of this case. The sole infected horse was euthanized in moribund condition. As the southernmost recognised HeV spill-over detection to date, it extends the southern limit of known cases by approximately 95 km. The event occurred near a large urban centre, characterised by equine populations of diverse type, husbandry, and purpose, with low HeV vaccination rates. Urgent multi-agency outbreak response involved risk assessment and monitoring of 11 exposed people and biosecurity management of at-risk animals. No human or additional animal cases were recognised. This One Health investigation highlights need for research on risk perception and strategic engagement to support owners confronted with the death of companion animals and potential human exposure to a high consequence virus. The location and timing of this spill-over event diverging from that established for prototype HeV (HeV-g1), highlight benefit in proactive One Health surveillance and research activities that improve understanding of dynamic transmission and spill-over risks of both HeV genotypic lineages and related but divergent emerging pathogens.

Emerging Genotype IV Japanese Encephalitis Virus Outbreak in New South Wales, Australia.

The detection of a new and unexpected Japanese encephalitis virus (JEV) outbreak in March 2022 in Australia, where JEV is not endemic, demanded the rapid development of a robust diagnostic framework to facilitate the testing of suspected patients across the state of New South Wales (NSW). This nascent but comprehensive JEV diagnostic service encompassed serological, molecular and metagenomics testing within a centralised reference laboratory. Over the first three months of the outbreak (4 March 2022 to 31 May 2022), 1,061 prospective samples were received from 878 NSW residents for JEV testing. Twelve confirmed cases of Japanese encephalitis (JE) were identified, including ten cases diagnosed by serology alone, one case by metagenomic next generation sequencing and real-time polymerase chain reaction (RT-PCR) of brain tissue and serology, and one case by RT-PCR of cerebrospinal fluid, providing an incidence of JE over this period of 0.15/100,000 persons in NSW. As encephalitis manifests in <1% of cases of JEV infection, the population-wide prevalence of JEV infection is likely to be substantially higher. Close collaboration with referring laboratories and clinicians was pivotal to establishing successful JEV case ascertainment for this new outbreak. Sustained and coordinated animal, human and environmental surveillance within a OneHealth framework is critical to monitor the evolution of the current outbreak, understand its origins and optimise preparedness for future JEV and arbovirus outbreaks.

Emergence of Japanese encephalitis in Australia: a diagnostic perspective.

The unprecedented emergence of Japanese encephalitis (JE) in mainland Australia represents an outbreak of high clinical and public health significance. JE is a zoonosis spread by mosquitoes and is one of the most important causes of endemic viral encephalitis in South-East Asia and the Indian subcontinent. While occasional cases of human Japanese encephalitis virus (JEV) infection have occurred in far north Australia, its detection in pigs and the substantial number of locally acquired human cases across multiple jurisdictions in early 2022 prompted the declaration of this outbreak as a Communicable Disease Incident of National Significance. Laboratory testing for JEV is complex, and most cases are diagnosed by serology, for which interpretation is difficult. This review provides a comprehensive outline of currently available methods for JEV diagnosis including serology, nucleic acid amplification testing, virus isolation, sequencing and metagenomics. The relative advantages and disadvantages of the diagnostic tests are presented, as well as their value in clinical and public health contexts. This review also explores the role of mosquito, veterinary and human surveillance as part of the laboratory response to JEV. As JEV may become endemic in Australia, a collaborative and coordinated One Health approach involving animal, human and environmental health is required for optimal disease response and control.

Primary dysmenorrhoea in adolescents and young women: A twin family study of maternal transmission, genetic influence and associations.

AIMS: The extent to which maternal transmission of primary dysmenorrhoea is genetically determined in adolescents and young women has yet to be determined. We aimed to assess heritability and associations relevant to primary pain syndromes using a twin family study. METHODS: Participants were young menstruating female twins, and their oldest sisters and mothers, whose families were registered with Twins Research Australia and previously participated in a twin family study of primary paediatric pain disorders. Questionnaire packs were mailed, assessing current maximum and average menstrual pain intensity, current pain interference with activities and retrospective dysmenorrhea secondary symptoms. RESULTS: The sample comprised 206 twin individuals (57 monozygous (MZ) and 46 dizygous (DZ) pairs) aged 10-22 years, eldest siblings (n = 38) aged 13-28 years and mothers (n = 101) aged 32-61 years. The estimated regression coefficient of the relationship between mother-daughter and twin-sibling dyads indicated significant associations for the measures of dysmenorrhea and supported heritability. Adjusted for age, the within twin-pair correlation for MZ twins was generally more than twice that of DZ twins. Heritability estimates were maximal pain intensity 0.67 (P = 3.8 × 10-11 ), average pain intensity 0.63 (P = 3.7 × 10-10 ), pain interference 0.57 (P = 1.8 × 10-8 ) and retrospective symptoms 0.57 (P = 1.8 × 10-8 ). Twin individuals with a lifetime (three-month) history of iron deficiency and those with painless restless legs syndrome (RLS) were significantly more likely to have more intense pain associated with menstruation. CONCLUSION: Primary dysmenorrhea in adolescents and young women was shown to be relatively strongly genetically influenced and associated especially with a history of iron deficiency and painless RLS which have potential therapeutic implications.

Off-season RSV epidemics in Australia after easing of COVID-19 restrictions.

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.

Potent monoclonal antibody-mediated neutralization of a divergent Hendra virus variant.

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/F­mediated 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.

Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption.

The HIV-1 reservoir is composed of cells harboring latent proviruses that have the potential to contribute to viremia upon antiretroviral treatment (ART) interruption. While this reservoir is known to be maintained by clonal expansion of infected cells, the contribution of these cell clones to residual viremia and viral rebound remains underexplored. Here, we conducted an extensive analysis on four ART-treated individuals who underwent an analytical treatment interruption (ATI), characterizing the proviral genomes and associated integration sites of large infected clones and phylogenetically linking these to plasma viremia. We show discrepancies between different assays in their ability to assess clonal expansion. Furthermore, we demonstrate that proviruses could phylogenetically be linked to plasma virus obtained before or during an ATI. This study highlights a role for HIV-infected cell clones in the maintenance of the replication-competent reservoir and suggests that infected cell clones can directly contribute to rebound viremia upon ATI.

Novel Hendra Virus Variant Circulating in Black Flying Foxes and Grey-Headed Flying Foxes, Australia.

A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

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.

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes.

Genetically-characterizing full-length HIV-1 RNA is critical for identifying genetically-intact genomes and for comparing these RNA genomes to proviral DNA. We have developed a method for sequencing plasma-derived RNA using long-range sequencing (PRLS assay; ∼8.3 kb from gag to the 3' end or ∼5 kb from integrase to the 3' end). We employed the gag-3' PRLS assay to sequence HIV-1 RNA genomes from ART-naive participants during acute/early infection (n = 6) or chronic infection (n = 2). On average, only 65% of plasma-derived genomes were genetically-intact. Defects were found in all genomic regions but were concentrated in env and pol. We compared these genomes to near-full-length proviral sequences from paired peripheral blood mononuclear cell (PBMC) samples for the acute/early group and found that near-identical (>99.98% identical) sequences were identified only during acute infection. For three participants who initiated therapy during acute infection, we used the int-3' PRLS assay to sequence plasma-derived genomes from an analytical treatment interruption and identified 100% identical genomes between pretherapy and rebound time points. The PRLS assay provides a new level of sensitivity for understanding the genetic composition of plasma-derived HIV-1 RNA from viremic individuals either pretherapy or after treatment interruption, which will be invaluable in assessing possible HIV-1 curative strategies. IMPORTANCE We developed novel plasma-derived RNA using long-range sequencing assays (PRLS assay; 8.3 kb, gag-3', and 5.0 kb, int-3'). Employing the gag-3' PRLS assay, we found that 26% to 51% of plasma-derived genomes are genetically-defective, largely as a result of frameshift mutations and deletions. These genetic defects were concentrated in the env region compared to gag and pol, likely a reflection of viral immune escape in env during untreated HIV-1 infection. Employing the int-3' PRLS assay, we found that analytical treatment interruption (ATI) plasma-derived sequences were identical and genetically-intact. Several sequences from the ATI plasma samples were identical to viral sequences from pretherapy plasma and PBMC samples, indicating that HIV-1 reservoirs established prior to therapy contribute to viral rebound during an ATI. Therefore, near-full-length sequencing of HIV-1 particles is required to gain an accurate picture of the genetic landscape of plasma HIV-1 virions in studies of HIV-1 replication and persistence.

Cellular Activation, Differentiation, and Proliferation Influence the Dynamics of Genetically Intact Proviruses Over Time.

Human immunodeficiency virus (HIV) persists in cells despite antiretroviral therapy; however, the influence of cellular mechanisms such as activation, differentiation, and proliferation upon the distribution of proviruses over time is unclear. To address this, we used full-length sequencing to examine proviruses within memory CD4+ T-cell subsets longitudinally in 8 participants. Over time, the odds of identifying a provirus increased in effector and decreased in transitional memory cells. In all subsets, more activated (HLA-DR-expressing) cells contained a higher frequency of intact provirus, as did more differentiated cells such as transitional and effector memory subsets. The proportion of genetically identical proviruses increased over time, indicating that cellular proliferation was maintaining the persistent reservoir; however, the number of genetically identical proviral clusters in each subset was stable. As such, key biological processes of activation, differentiation, and proliferation influence the dynamics of the HIV reservoir and must be considered during the development of any immune intervention.

Metagenomic Identification of Viral Sequences in Laboratory Reagents.

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete 'infectome' (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple 'blank' negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

The spatial-temporal dynamics of respiratory syncytial virus infections across the east-west coasts of Australia during 2016-17.

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.