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.

The Updated Mouse Universal Genotyping Array Bioinformatic Pipeline Improves Genetic QC in Laboratory Mice.

The MiniMUGA genotyping array is a popular tool for genetic QC of laboratory mice and genotyping of samples from most types of experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve array's performance and the associated report's usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA in 2020. Here we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for inbred strains and increase the number of constructs that can reliably be detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have made changes to the layout of the report, to simplify the interpretation and completeness of the analysis and added a table summarizing the ideogram. We believe that these changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research.

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.

Ictogenesis proceeds through discrete phases in hippocampal CA1 seizures in mice.

Epilepsy is characterized by spontaneous non-provoked seizures, yet the mechanisms that trigger a seizure and allow its evolution remain underexplored. To dissect out phases of ictogenesis, we evoked hypersynchronous activity with optogenetic stimulation. Focal optogenetic activation of putative excitatory neurons in the mouse hippocampal CA1 reliably evoked convulsive seizures in awake mice. A time-vs-time pulsogram plot characterized the evolution of the EEG pulse response from a light evoked response to induced seizure activity. Our results depict ictogenesis as a stepwise process comprised of three distinctive phases demarcated by two transition points. The induction phase undergoes the first transition to reverberant phase activity, followed by the second transition into the paroxysmal phase or a seizure. Non-seizure responses are confined to either induction or reverberant phases. The pulsogram was then constructed in seizures recorded from a murine model of temporal lobe epilepsy and it depicted a similar reverberance preceding spontaneous seizures. The discovery of these distinct phases of ictogenesis may offer means to abort a seizure before it develops.

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.

Understanding the Combined Effects of High Glucose Induced Hyper-Osmotic Stress and Oxygen Tension in the Progression of Tumourigenesis: From Mechanism to Anti-Cancer Therapeutics.

High glucose (HG), a hallmark of the tumour microenvironment, is also a biomechanical stressor, as it exerts hyper-osmotic stress (HG-HO), but not much is known regarding how tumour cells mechanoadapt to HG-HO. Therefore, this study aimed to delineate the novel molecular mechanisms by which tumour cells mechanoadapt to HG/HG-HO and whether phytochemical-based interference in these mechanisms can generate tumour-cell-selective vulnerability to cell death. Mannitol and L-glucose were used as hyper-osmotic equivalents of high glucose. The results revealed that the tumour cells can efficiently mechanoadapt to HG-HO only in the normoxic microenvironment. Under normoxic HG/HG-HO stress, tumour cells polySUMOylate a higher pool of mitotic driver pH3(Ser10), which translocates to the nucleus and promotes faster cell divisions. On the contrary, acute hypoxia dampens HG/HG-HO-associated excessive proliferation by upregulating sentrin protease SENP7. SENP7 promotes abnormal SUMOylation of pH3(Ser10), thereby restricting its nuclear entry and promoting the M-phase arrest and cell loss. However, the hypoxia-arrested cells that managed to survive showed relapse upon reversal to normoxia as well as upregulation of pro-survival-associated SENP1, and players in tumour growth signalling, autophagy, glycolytic pathways etc. Depletion of SENP1 in both normoxia and hypoxia caused significant loss of tumour cells vs undepleted controls. SENP1 was ascertained to restrict the abnormal SUMOylation of pH3(Ser10) in both normoxia and hypoxia, although not so efficiently in hypoxia, due to the opposing activity of SENP7. Co-treatment with Momordin Ic (MC), a natural SENP1 inhibitor, and Gallic Acid (GA), an inhibitor of identified major pro-tumourigenic signalling (both enriched in Momordica charantia), eliminated surviving tumour cells in normal glucose, HG and HG-HO normoxic and hypoxic microenvironments, suggesting that appropriate and enhanced polySUMOylation of pH3(Ser10) in response to HG/HG-HO stress was attenuated by this treatment along with further dampening of other key tumourigenic signalling, due to which tumour cells could no longer proliferate and grow.

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.

Hyperkalemia and Plant-Based Diets in Chronic Kidney Disease.

Traditional dietary guidelines for patients with kidney disease recommend restriction of plant foods due to concerns about hyperkalemia and associated adverse events. Studies conducted over several decades have shown that the basis for these guidelines does not match the evidence. Serum potassium levels can be elevated in patients with reduced kidney function after consumption of foods with potassium-based additives or with highly concentrated potassium content such as juices, dried fruit, or purees. However, plant foods may have certain qualities that may blunt potassium retention including their alkalinizing effects, the lack of bioavailable potassium, and the impact of dietary fiber in organic plant foods on colonic potassium excretion. Furthermore, there are many benefits of plant foods that patients with kidney disease may be missing by excluding them from their diets by recommending the historical low-potassium "renal diet." Revised dietary recommendations for kidney health may encourage patient-centered kidney recipes that revolve around plant foods and do not restrict them.

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.

Calidad de vida y sarcopenia en pacientes adultos con insuficiencia renal crónica / Quality of life and sarcopenia in adult patients with chronic renal failure

RESUMEN Introducción: la calidad de vida puede alterarse en los pacientes con insuficiencia renal crónica, sobre todo si sufren concomitantemente de sarcopenia. Objetivo: determinar la relación entre sarcopenia y calidad de vida en pacientes adultos con insuficiencia renal crónica del Hospital Nacional (Itauguá) y Hospital Militar (Asunción) entre abril y noviembre del 2021. Metodología: se utilizó un diseño observacional, descriptivo, transversal. Se incluyó a sujetos adultos de ambos sexos, portadores de insuficiencia renal crónica. Se midieron variables antropométricas, clínicas y laboratoriales. La calidad de vida se evaluó con el cuestionario EQ-5D y la sarcopenia con la fuerza de prensión palmar y el índice de masa muscular. Los datos se sometieron a estadística descriptiva y analítica con el programa Epi Info 7™. El estudio contó con la aprobación del Comité de Ética de la Universidad Privada del Este, Paraguay. Resultados: ingresaron al estudio 62 varones con edad media 56 ± 15 años y 57 mujeres con edad media 51 ± 16 años. La más frecuente de la insuficiencia renal fue la hipertensión arterial asociada a la diabetes mellitus (45,4 %). La sarcopenia se confirmó en 38,6 % de los pacientes. La calidad de vida fue buena en 30,25 %, regular en 39,5 % y mala en 30,25 %. Conclusión: en pacientes adultos con insuficiencia renal crónica, la sarcopenia predominó en los sujetos con regular y mala calidad de vida.

ABSTRACT Introduction: the quality of life can be altered in patients with chronic renal failure, especially if they suffer concomitantly from sarcopenia. Objective: to determine the relationship between sarcopenia and quality of life in adult patients with chronic renal failure at the Hospital Nacional (Itauguá) and the Hospital Militar (Asunción) between April and November 2021. Methodology: an observational, descriptive, cross-sectional design was used. Adult subjects of both sexes, carriers of chronic renal failure were included. Anthropometric, clinical and laboratory variables were measured. Quality of life was evaluated with the EQ-5D questionnaire and sarcopenia with palm grip strength and muscle mass index. The data was submitted to descriptive and analytical statistics with the Epi Info 7™ program. The study was approved by the Ethics Committee of the Universidad Privada del Este, Paraguay. Results: 62 men with a mean age of 56 ± 15 years and 57 women with a mean age of 51 ± 16 years entered the study. The most frequent renal failure was arterial hypertension associated with diabetes mellitus (45.4 %). Sarcopenia was confirmed in 38.6 % of the patients. The quality of life was good in 30.25 %, regular in 39.5 % and bad in 30.25 %. Conclusion: in adult patients with chronic renal failure, sarcopenia prevailed in subjects with fair and poor quality of life.

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.

DREADDs in Epilepsy Research: Network-Based Review.

Epilepsy can be interpreted as altered brain rhythms from overexcitation or insufficient inhibition. Chemogenetic tools have revolutionized neuroscience research because they allow "on demand" excitation or inhibition of neurons with high cellular specificity. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are the most frequently used chemogenetic techniques in epilepsy research. These engineered muscarinic receptors allow researchers to excite or inhibit targeted neurons with exogenous ligands. As a result, DREADDs have been applied to investigate the underlying cellular and network mechanisms of epilepsy. Here, we review the existing literature that has applied DREADDs to understand the pathophysiology of epilepsy. The aim of this review is to provide a general introduction to DREADDs with a focus on summarizing the current main findings in experimental epilepsy research using these techniques. Furthermore, we explore how DREADDs may be applied therapeutically as highly innovative treatments for epilepsy.

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.