Mpox diagnostics: Review of current and emerging technologies.

Mpox is a zoonotic disease caused by monkeypox virus (MPXV) from the Orthopoxvirus genus. Unprecedented transmission events have led to more than 70 000 cases reported worldwide by October 2022. The change in mpox epidemiology has raised concerns of its ability to establish endemicity beyond its traditional geographical locations. In this review, we discuss the current understanding of mpox virology and viral dynamics that are relevant to mpox diagnostics. A synopsis of the traditional and emerging laboratory technologies useful for MPXV detection and in guiding "elimination" strategies is outlined in this review. Importantly, development in MPXV genomics has rapidly advanced our understanding of the role of viral evolution and adaptation in the current outbreak.

Intra- and interhost genomic diversity of monkeypox virus.

The impact and frequency of infectious disease outbreaks demonstrate the need for timely genomic surveillance to inform public health responses. In the largest known outbreak of mpox, genomic surveillance efforts have primarily focused on high-incidence nations in Europe and the Americas, with a paucity of data from South-East Asia and the Western Pacific. Here we analyzed 102 monkeypox virus (MPXV) genomes sampled from 56 individuals in Melbourne, Australia. All genomes fell within the 2022 MPXV outbreak lineage (B.1), with likely onward local transmission detected. We observed within-host diversity and instances of co-infection, and highlight further examples of structural variation and apolipoprotein B editing complex-driven micro-evolution in the current MPXV outbreak. Updating our understanding of MPXV emergence and diversification will inform public health measures and enable monitoring of the virus' evolutionary trajectory throughout the mpox outbreak.

Lymphocytic Choriomeningitis Virus Infection, Australia.

During a mouse plague in early 2021, a farmer from New South Wales, Australia, sought treatment for aseptic meningitis and was subsequently diagnosed with locally acquired lymphocytic choriomeningitis virus infection. Whole-genome sequencing identified a divergent and geographically distinct lymphocytic choriomeningitis virus strain compared with other published sequences.

Monkeypox infection presenting as genital rash, Australia, May 2022.

Rapid diagnosis and whole genome sequencing confirmed a case of monkeypox in an HIV-positive individual receiving antiretroviral therapy. The patient had a normal CD4+ T-cell count and suppressed HIV viral load and presented with a genital rash in Melbourne, Australia after return from Europe in May 2022. He subsequently developed systemic illness and disseminated rash and 11 days after symptom onset, he was hospitalised to manage painful bacterial cellulitis of the genital area.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology.

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia.

OBJECTIVES: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. SETTING: SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

Divergent Barmah Forest Virus from Papua New Guinea.

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia.

Increasing proportion of herpes simplex virus type 1 among women and men diagnosed with first-episode anogenital herpes: a retrospective observational study over 14 years in Melbourne, Australia.

OBJECTIVES: Reports of rising herpes simplex virus type 1 (HSV-1) genital infections relative to HSV-2 have been published up to 2006 in Australia. These changes have been attributed to declining childhood immunity to HSV-1. We described the temporal trends of HSV-1 and HSV-2 up to 2017 in Melbourne, Australia, to determine if the earlier trend is continuing. METHODS: We conducted a retrospective review of the medical records of 4517 patients who were diagnosed with first episode of anogenital HSV infection at the Melbourne Sexual Health Centre, Australia, between January 2004 and December 2017. HSV-1 and HSV-2 were calculated as a proportion of all first episode of anogenital HSV infections. The change in the proportions of HSV-1 and HSV-2 over time was assessed by a χ2 trend test. Risk factors associated with HSV-1 were examined using a multivariable logistic regression model. RESULTS: The proportion of first episode of anogenital herpes due to HSV-1 increased significantly over time in women (from 45% to 61%; ptrend<0.001) and heterosexual men (from 38% to 41%; ptrend=0.01) but not in men who have sex with men (MSM) (ptrend=0.21). After adjusting for condom use, partner number and age, the annual increase remained significant only in women (OR 1.08, 95% CI 1.03 to 1.13, p<0.001). In MSM, HSV-1 caused up to two-thirds of anogenital herpes in most years and HSV-1 was more likely to be diagnosed at an anal site than genital site (OR 1.69, 95% CI 1.23 to 2.32, p<0.001). Younger age (<28 years) was an independent risk factor for HSV-1 in all groups. CONCLUSIONS: The proportion of first-episode anogenital herpes due to HSV-1 has been rising in women since 2004. HSV-1 has become the leading cause of anogenital herpes in younger populations, women and MSM.

c-Jun N-terminal kinase activity is required for efficient respiratory syncytial virus production.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly, leading to more deaths than influenza each year worldwide. With no RSV antiviral or efficacious vaccine currently available, improved understanding of the host-RSV interaction is urgently required. Here we examine the contribution to RSV infection of the host stress-regulated c-Jun N-terminal kinase (JNK), for the first time. Peak JNK1/2 phosphoactivation is observed at ∼24 h post-infection, correlating with the time of virus assembly. The release of infectious RSV virions from infected cells was significantly reduced by either JNK1/2 siRNA knockdown or treatment with the JNK-specific inhibitor, JNK-IN-VIII. High resolution microscopy confirmed RSV accumulation in the host cell cytoplasm. The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics.

New host factors important for respiratory syncytial virus (RSV) replication revealed by a novel microfluidics screen for interactors of matrix (M) protein.

Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.

Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate.

Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications.

Uncovering strain- and age-dependent innate immune responses to SARS-CoV-2 infection in air-liquid-interface cultured nasal epithelia.

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Non-SARS-CoV-2 respiratory viral detection and whole genome sequencing from COVID-19 rapid antigen test devices: a laboratory evaluation study.

BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche devices; rhinovirus in 24 of 28 from Panbio and 27 of 28 from Roche devices; influenza B in four of 15 in both devices; and RSV in 16 of 18 in both devices. Of the 31 COVID-19 devices collected from The Royal Melbourne Hospital emergency department, 11 tested positive for a respiratory virus on rtPCR, including one device positive for influenza A virus, one positive for RSV, four positive for rhinovirus, and five positive for SARS-CoV-2. Sequences of target respiratory viruses from archival samples were detected in 55 (98·2%) of 56 samples from Panbio and 48 (85·7%) of 56 from Roche rapid antigen test devices. 98 (87·5%) of 112 viral genomes were completely assembled from these data, enabling subtyping for RSV and influenza viruses. All 11 samples collected from the emergency department had viral sequences detected, with near-complete genomes assembled for influenza A and RSV. INTERPRETATION: Non-SARS-CoV-2 respiratory viruses can be detected and sequenced from COVID-19 rapid antigen devices. Recovery of near full-length viral sequences from these devices provides a valuable opportunity to expand genomic surveillance programmes for public health monitoring of circulating respiratory viruses. FUNDING: Australian Government Medical Research Future Fund and Australian National Health and Medical Research Council.

Characterising HIV-1 transmission in Victoria, Australia: a molecular epidemiological study.

Background: In Australia the incidence of HIV has declined steadily, yet sustained reduction of HIV transmission in this setting requires improved public health responses. As enhanced public health responses and prioritisation of resources may be guided by molecular epidemiological data, here we aimed to assess the applicability of these approaches in Victoria, Australia. Methods: A comprehensive collection of HIV-1 pol sequences from individuals diagnosed with HIV in Victoria, Australia, between January 1st 2000 and December 31st 2020 were deidentified and used as the basis of our assessment. These sequences were subtyped and surveillance drug resistance mutations (SDRMs) identified, before definition of transmission groups was performed using HIV-TRACE (0.4.4). Phylodynamic methods were applied using BEAST (2.6.6), assessing effective reproductive numbers for large groups, and additional demographic data were integrated to provide a high resolution view of HIV transmission in Victoria on a decadal time scale. Findings: Based on standard settings for HIV-TRACE, 70% (2438/3507) of analysed HIV-1 pol sequences were readily assigned to a transmission group. Individuals in transmission groups were more commonly males (aOR 1.50), those born in Australia (aOR 2.13), those with probable place of acquisition as Victoria (aOR 6.73), and/or those reporting injectable drug use (aOR 2.13). SDRMs were identified in 375 patients (10.7%), with sustained transmission of these limited to a subset of smaller groups. Informative patterns of epidemic growth, stabilisation, and decline were observed; many transmission groups showed effective reproductive numbers (R e ) values reaching greater than 4.0, representing considerable epidemic growth, while others maintained low R e values. Interpretation: This study provides a high resolution view of HIV transmission in Victoria, Australia, and highlights the potential of molecular epidemiology to guide and enhance public health responses in this setting. This informs ongoing discussions with community groups on the acceptability and place of molecular epidemiological approaches in Australia. Funding: National Health and Medical Research Council, Australian Research Council.

Comparative Longitudinal Serological Study of Anti-SARS-CoV-2 Antibody Profiles in People with COVID-19.

Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.

Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.

BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology. METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands. FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per µL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples. INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing. FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.

At Least Three Doses of Leading Vaccines Essential for Neutralisation of SARS-CoV-2 Omicron Variant.

Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT50) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.