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.

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.

Defective Severe Acute Respiratory Syndrome Coronavirus 2 Immune Responses in an Immunocompromised Individual With Prolonged Viral Replication.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2-specific antibody responses were absent and SARS-CoV-2-specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.

Virus survey in populations of two subspecies of bent-winged bats (Miniopterus orianae bassanii and oceanensis) in south-eastern Australia reveals a high prevalence of diverse herpesviruses.

While bats are often viewed as carriers of infectious disease agents, little research has been conducted on the effects these potential pathogens may have on the bat populations themselves. The southern bent-winged bat (Miniopterus orianae bassanii) is a critically endangered subspecies endemic to south-eastern Australia. Population numbers of this bat have been declining for the past 50 years, but the reasons for this are unclear. As part of a larger study to determine if disease could be a contributing factor to this decline, 351 southern bent-winged bats from four locations were captured, and oral swabs were collected and tested for the presence of potentially pathogenic viruses. Results were compared with those obtained from 116 eastern bent-winged bats (Miniopterus orianae oceanensis) from three different locations. The eastern bent-winged bat is a related but more common and widespread subspecies whose geographical range overlaps partly with southern bent-winged bats. Herpesviruses were detected in bent-winged bats from all seven locations. At least six novel herpesviruses (five betaherpesviruses and one gammaherpesvirus) were identified. The prevalence of herpesvirus infection was higher in eastern bent-winged bats (44%, 51/116), compared to southern bent-winged bats (27%, 95/351), although this varied across the locations and sampling periods. Adenoviruses and a range of different RNA viruses (lyssaviruses, filoviruses, coronaviruses and henipaviruses) were also tested for but not detected. The detected herpesviruses did not appear to be associated with obvious ill health, and may thus not be playing a role in the population decline of the southern bent-winged bat. The detection of multiple novel herpesviruses at a high prevalence of infection is consistent with our understanding of bats as hosts to a rich diversity of viruses.

Herpes simplex virus-2 transmission following solid organ transplantation: Donor-derived infection and transplantation from prior organ recipients.

BACKGROUND: Owing to limited availability of donor organs, previous solid organ transplant (SOT) recipients are increasingly considered as potential organ donors. We report donor-derived transmission of herpes simplex virus type-2 (HSV-2) to two clusters of SOT recipients with transmission from the original donor and an HSV-2-infected recipient who subsequently became a donor. METHODS: We reviewed medical records of the donors and recipients in both clusters. Pre-transplant serology and virological features of HSV-2 were characterized. Genotyping of HSV-2 isolates to determine potential for donor transmission of HSV-2 through transplantation of organs from prior organ recipients was performed. RESULTS: A kidney-pancreas recipient died day 9 post transplant. Following confirmation of brain death, the lungs and recently transplanted kidney were donated to two further recipients. The liver was not retrieved, but biopsy confirmed HSV-2 infection. Testing on the original donor showed negative HSV-2 polymerase chain reaction and HSV immunoglobulin (Ig)M, but positive HSV-2 IgG. The liver recipient from the original donor developed HSV-2 hepatitis and cutaneous infection that responded to treatment with intravenous acyclovir. In the second cluster, lung and kidney recipients both developed HSV-2 viremia that was successfully treated with antiviral therapy. Genotyping of all HSV-2-positive samples showed 100% sequence homology for three recipients. CONCLUSIONS: Donor-derived HSV infection affected two clusters of recipients because of transplantation of organs from a prior organ recipient. HSV should be considered as a possible cause of illness in febrile SOT recipients in the immediate post-transplant period and may cause disseminated disease and re-infection in HSV-2-seropositive recipients. Testing of HSV serology and prophylaxis may be considered in SOT recipients not receiving cytomegalovirus prophylaxis.

Characteristics of adenovirus urethritis among heterosexual men and men who have sex with men: a review of clinical cases.

BACKGROUND: The aim of this study was to characterise the clinical features of adenovirus urethritis in men and to compare the frequency of these between heterosexual men and men who have sex with men (MSM). METHODS: This was a review of the clinical and laboratory information from men diagnosed with PCR-confirmed adenovirus urethritis at the Melbourne Sexual Health Centre between January 2006 and April 2014. RESULTS: 102 adenovirus urethritis cases were reported, among which 61 were heterosexual men and 41 MSM. Eighty-nine per cent (n=91) had signs of meatitis or conjunctivitis: 51% had meatitis only; 32% meatitis together with conjunctivitis and 6% with conjunctivitis only. The distribution of symptoms and signs was similar among heterosexual men and MSM (p values >0.1). Adenovirus was the sole pathogen found in 93% of cases, excluding gonorrhoea, chlamydia, Mycoplasma genitalium and herpes simplex virus. Only 37% had ≥5 polymorphs per high-power field from a urethral smear. Where samples were still available for adenoviral sequencing (n=20), all were subgroup D. CONCLUSIONS: The clinical features of adenovirus urethritis in men can be distinctive and aid diagnosis, distinguishing it from other treatable causes of male urethritis.

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by PCR.

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.

Respiratory virus laboratory pandemic planning and surveillance in central Viet Nam, 2008-2010.

INTRODUCTION: Laboratory capacity is needed in central Viet Nam to provide early warning to public health authorities of respiratory outbreaks of importance to human health, for example the outbreak of influenza A(H1N1) pandemic in 2009. Polymerase chain reaction (PCR) procedures established as part of a capacity-building process were used to conduct prospective respiratory surveillance in a region where few previous studies have been undertaken. METHODS: Between October 2008 and September 2010, nose and throat swabs from adults and children (approximately 20 per week) presenting with an acute respiratory illness to the Ninh Hoa General Hospital were collected. Same-day PCR testing and result reporting for 13 respiratory viruses were carried out by locally trained scientists. RESULTS: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0-5 year age group. Viral co-infections were identified in 148 (6.9%) cases. DISCUSSION: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory's pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

Community epidemiology of human metapneumovirus, human coronavirus NL63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens.

OBJECTIVES: The purpose of this work was to assess the impact of recently described human metapneumovirus and human coronavirus NL63 compared with other respiratory viruses by using sensitive molecular techniques in a cohort of healthy preschool-aged children. We also aimed to assess the use of parent collection to obtain an adequate respiratory specimen from acutely unwell children in the community. PATIENTS AND METHODS: The community epidemiology and burden of human metapneumovirus and other respiratory viruses (influenza A, influenza B, respiratory syncytial virus, parainfluenza viruses, adenoviruses, and picornaviruses) were examined in a cohort of 234 preschool-aged children from Melbourne, Australia, over a 12-month period by using polymerase chain reaction testing. Parents collected a daily symptom diary for the duration of the study and were taught to collect a combined nose-throat swab and complete an impact diary when the study child had an acute respiratory illness. RESULTS: The average incidence of acute respiratory illness was 0.48 per child-month for the duration of the study, with a winter peak. Of 543 illnesses with > or = 1 specimen returned, 33 were positive for human metapneumovirus (6.1%) and 18 for human coronavirus NL63 (3.3%). Of all of the viruses for which we tested, human metapneumovirus and human coronavirus NL63 were most strongly linked to child care attendance, occurring in 82% and 78% of infected children, respectively. Picornaviruses were the most commonly identified virus group (269 [49.5%]). Influenza virus and adenovirus illnesses had the greatest impact, with fever in more than three quarters and requiring, on average, > 1 local doctor visit per illness. CONCLUSIONS: Recently identified human metapneumovirus and human coronavirus NL63 are important pathogens in community-based illness in children, particularly in those who attend child care. Picornaviruses were detected in half of the nose-throat swabs collected during acute respiratory illness in children but resulted in milder illnesses; influenza and adenovirus caused the highest-impact illnesses. The use of parent-collected specimens should be considered for additional community-based epidemiologic studies and vaccine trials.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.