Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infection amongst all ages, causing a significant global health burden. Preventative and therapeutic options for RSV infection have long been under development, and recently, several widely-publicised vaccines targeting older adult and maternal populations have become available. Promising monoclonal antibody (mAb) and antiviral (AV) therapies are also progressing in clinical trials, with the prophylactic mAb nirsevimab recently approved for clinical use in infant populations. A systematic review on current progress in this area is lacking. We performed a systematic literature search (PubMed, Embase, Web of Science, ClinicalTrials.gov, EudraCT, ANZCTR-searched Nov 29th, 2023) to identify studies on all RSV-specific mAbs and AV therapies that has undergone human clinical trials since year 2000. Data extraction focused on outcomes related to the therapeutic efficacy and safety of the intervention on trial, and all studies were graded against the OCEBM Levels of Evidence Table. Results from 59 studies were extracted, covering efficacy and safety data on six mAbs (motavizumab, motavizumab-YTE, nirsevimab, ALX-0171, suptavumab, clesrovimab) and 12 AV therapies (ALN-RSV01, RSV604, presatovir, MDT-637, lumicitabine, IFN-α1b, rilematovir, enzaplatovir, AK0529, sisunatovir, PC786, EDP-938). Of the mAbs reviewed, nirsevimab and clesrovimab hold considerable promise. The timeline for RSV-specific AV availability is less advanced, although EDP-938 and AK0529 have reported promising phase 2 efficacy and safety data. Moving forward, passive immunisation and treatment options for RSV infection will play a significant role in reducing the health burden of RSV, complementing recent advancements in vaccine development. TRIAL REGISTRATION: PROSPERO registration: CRD42022376633.

OBJECTIVES: The 2022 seasonal respiratory syncytial virus (RSV) epidemic in Sydney, Australia saw an unprecedented number of RSV detections. We aimed to characterize genomic and immunologic factors associated with the surge in RSV cases. METHODS: Whole genome sequences of RSV were generated from 264 RSV-infected infants and linked to case-matched clinical data from the 2022 southern hemisphere RSV season. We then performed an immunologic analysis of baseline RSV-specific humoral immunity in women of childbearing age before and throughout the coronavirus disease 2019 pandemic. RESULTS: Clinical analysis revealed a high burden of disease across patients of all health backgrounds. More than one-half of RSV-related health care visits by infants resulted in hospitalization, and one-quarter required high-flow respiratory support or a higher level of care. Viral phylogenetic analyses revealed that 2022 Sydney RSV sequences were closely related to viruses that had been circulating globally since 2017, including those detected in recent US outbreaks. Nonsynonymous mutations within the palivizumab and nirsevimab binding sites were detected at low frequencies. There was no difference in baseline RSV-neutralizing antibody titers between 2020 and 2022. CONCLUSIONS: Collectively, these findings suggest that neither the emergence of a novel RSV genotype nor hypothesized immune debt was associated with the surge of RSV cases and hospitalizations in 2022. Continued genomic and immunologic surveillance is required to further understand the factors driving outbreaks of RSV globally, and to inform guidelines for the rollout and ongoing use of recently developed immunotherapeutics and vaccines.

The World Health Organisation has set targets of reducing the transmission of new hepatitis C (HCV) infections by 90%, and ending human immunodeficiency virus-1 (HIV) as a public health threat, by 2030. To achieve this, efficient and timely viral surveillance, and effective public health interventions, are required. Traditional epidemiological methods are largely dependent on the recognition of incident cases with symptomatic illness; acute HIV and HCV infections are commonly asymptomatic, which may lead to delays in the recognition of such new infections. Instead, for these viruses, molecular epidemiology may improve the detection of, and response to, clusters of viral transmission. Molecular epidemiology using historical datasets has highlighted key populations that may have benefitted from a timely intervention. Similar analyses performed on contemporary samples are needed to underpin the 2030 targets, but this requires the generation of a cohesive dataset of viral genome sequences in near-real-time. To generate such data, methodologies harnessing next-generation sequencing (NGS) should be utilised. Here we discuss the opportunity presented by NGS for public health surveillance of HIV and HCV, and discuss three methods that can generate sequences for such analysis. These include full-length genome amplification, utilised for analysis of HCV in the research space; tiling PCR, which was the method of choice for many diagnostic laboratories in the SARS-CoV-2 pandemic; and bait-capture hybridisation, which has been utilised in local HIV outbreaks. These techniques could be applied for near-real-time HIV and HCV surveillance, informing public health strategies that will be key to achieving 2030 targets.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. A 17-nucleotide frameshift-inducing deletion in ORF7a rapidly became represented at the consensus level (Delta-ORF7aΔ17del) in most Australian outbreak cases. Studies from early in the COVID-19 pandemic suggest that frameshift-inducing deletions in ORF7a do not persist for long in the population; therefore, Delta-ORF7aΔ17del genomes should have disappeared early in the Australian outbreak. In this study, we conducted a retrospective analysis of global Delta genomes to characterise the dynamics of Delta-ORF7aΔ17del over time, determined the frequency of all ORF7a deletions worldwide, and compared global trends with those of the Australian Delta outbreak. We downloaded all GISAID clade GK Delta genomes and scanned them for deletions in ORF7a. For each deletion we identified, we characterised its frequency, the number of countries it was found in, and how long it persisted. Of the 4,018,216 Delta genomes identified globally, 134,751 (~3.35%) possessed an ORF7a deletion, and ORF7aΔ17del was the most common. ORF7aΔ17del was the sole deletion in 28,014 genomes, of which 27,912 (~99.6%) originated from the Australian outbreak. During the outbreak, ~87% of genomes were Delta-ORF7aΔ17del, and genomes with this deletion were sampled until the outbreak’s end. These data demonstrate that, contrary to suggestions early in the COVID-19 pandemic, genomes with frameshifting deletions in ORF7a can persist over long time periods. We suggest that the proliferation of Delta-ORF7aΔ17del genomes was likely a chance founder effect. Nonetheless, the frequency of ORF7a deletions in SARS-CoV-2 genomes worldwide suggests they might have some benefit for virus transmission.

Plasmonic nanoparticles are finding applications within the single molecule sensing field in a "dimer" format, where interaction of the target with hairpin DNA causes a decrease in the interparticle distance, leading to a localized surface plasmon resonance shift. While this shift may be detected using spectroscopy, achieving statistical relevance requires the measurement of thousands of nanoparticle dimers and the timescales required for spectroscopic analysis are incompatible with point-of-care devices. However, using dark-field imaging of the dimer structures, simultaneous digital analysis of the plasmonic resonance shift after target interaction of thousands of dimer structures may be achieved in minutes. The main challenge of this digital analysis on the single-molecule scale was the occurrence of false signals caused by non-specifically bound clusters of nanoparticles. This effect may be reduced by digitally separating dimers from other nanoconjugate types. Variation in image intensity was observed to have a discernible impact on the color analysis of the nanoconjugate constructs and thus the accuracy of the digital separation. Color spaces wherein intensity may be uncoupled from the color information (hue, saturation, and value (HSV) and luminance, a* vector, and b* vector (LAB) were contrasted to a color space which cannot uncouple intensity (RGB) to train a classifier algorithm. Each classifier algorithm was validated to determine which color space produced the most accurate digital separation of the nanoconjugate types. The LAB-based learning classifier demonstrated the highest accuracy for digitally separating nanoparticles. Using this classifier, nanoparticle conjugates were monitored for their plasmonic color shift after interaction with a synthetic RNA target, resulting in a platform with a highly accurate yes/no response with a true positive rate of 88% and a true negative rate of 100%. The sensor response of tested single stranded RNA (ssRNA) samples was well above control responses for target concentrations in the range of 10 aM-1 pM.

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

Measures to control the COVID-19 pandemic such as antiviral therapy and vaccination have been challenged by ongoing virus evolution under antiviral and immune pressures. Understanding viral evolutionary dynamics is crucial for responding to SARS-CoV-2, and preparing for the next pandemic, by informing prediction of virus adaptation, public health strategies, and design of broadly effective therapies. Whole-genome sequencing (WGS) of SARS-CoV-2 during the pandemic enabled fine-grained studies of virus evolution in the human population. Serial passaging in vitro offers a controlled environment to investigate the emergence and persistence of genetic variants that may confer selective advantage. Nine virus lineages, including four “variants of concern” and three former “variants under investigation” as designated by the World Health Organisation, were chosen to investigate intra- and inter-lineage evolution through long-term serial passaging in Vero E6 cells. Viruses were sampled over at least 33 passages (range 33-100) and analysed using WGS to examine evolutionary dynamics and identify key mutations with implications for virus fitness, transmissibility, and immune evasion. All passages continued to replicate in culture, despite regular accumulation of mutations. There was evidence of convergent acquisition of mutations both across passage lines and compared with contemporaneous SARS CoV-2 clinical sequences from population studies. Some of these convergent mutations are hypothesised to be important in proliferation of SARS-CoV-2 lineages, such as by evading host immune responses (e.g. S:A67V, S:H655Y). Given these mutations arose in vitro, in the absence of a multicellular host immune response, this suggests virus genome mutation resulted from stochastic events, rather than immune-driven mutation. There was a regular gain and loss of low-frequency variants during serial passaging, but some became fixed in subsequent multiple passages, suggesting either a benefit of the mutation in vitro , or at least a lack of deleterious effect. Our findings reveal valuable insights into the evolution of SARS-CoV-2 by quantitatively investigating evolutionary dynamics of the virus over the greatest number of serial passages to date. Knowledge of these evolutionary trends will be useful for public health and the development of antiviral and vaccine measures to reduce the effects of SARS CoV-2 infection on the human population.

Acute respiratory infections (ARIs) are a major cause of morbidity among children. Respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. The rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. Children otherwise healthy aged 1 to 8 years were prospectively enrolled in the study during two consecutive winters, when ARIs peak in New Zealand. Parents completed a daily symptom diary for 8 weeks, during which time they collected a nasal swab from the child for each clinical ARI episode. A further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. All samples were tested for 15 respiratory viruses commonly causing ARI using molecular multiplex polymerase chain reaction assays. There were 575 ARIs identified from 301 children completing the study, at a rate of 1.04 per child-month. Swabs collected during an ARI were positive for a respiratory virus in 76.8% (307 of 400), compared with 37.3% (79 of 212) of swabs collected during asymptomatic periods. The most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. All of these were significantly more likely to be detected during ARIs than asymptomatic periods. Parent-administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. Interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses.

Journal of Medical VirologyVolume 94, Issue 9 p. 4043-4046 LETTER TO THE EDITOR Persistent high-level shedding of cultivable SARS-CoV-2 Delta virus 33 days after onset of COVID-19 in a hospitalized patient with pneumonia Ki Wook Kim, Corresponding Author Ki Wook Kim [email protected] orcid.org/0000-0001-9579-6408 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Correspondence Ki Wook Kim, Virology Research Laboratory, Serology and Virology Division (SAViD), Level 3 Clinical Sciences Bldg, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia. Email: [email protected]Search for more papers by this authorXinye Wang, Xinye Wang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAnurag Adhikari, Anurag Adhikari Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMalinna Yeang, Malinna Yeang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorFrances Jenkins, Frances Jenkins Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorZin Naing, Zin Naing Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorGregory J. Walker, Gregory J. Walker Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorCharles S. P. Foster, Charles S. P. Foster Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSacha Stelzer-Braid, Sacha Stelzer-Braid orcid.org/0000-0001-6037-9305 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorIra Deveson, Ira Deveson Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia Faculty of Medicine, St Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMaria E. Craig, Maria E. Craig orcid.org/0000-0001-6004-576X Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, AustraliaSearch for more papers by this authorNicodemus Tedla, Nicodemus Tedla Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorRowena A. Bull, Rowena A. Bull Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMarianne Martinello, Marianne Martinello Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAngie N. Pinto, Angie N. Pinto Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorRaymond Chan, Raymond Chan Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorStuart Turville, Stuart Turville Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorWilliam D. Rawlinson, William D. Rawlinson orcid.org/0000-0003-0988-7827 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia Faculty of Science, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSebastiaan van Hal, Sebastiaan van Hal Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, AustraliaSearch for more papers by this author Ki Wook Kim, Corresponding Author Ki Wook Kim [email protected] orcid.org/0000-0001-9579-6408 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Correspondence Ki Wook Kim, Virology Research Laboratory, Serology and Virology Division (SAViD), Level 3 Clinical Sciences Bldg, Prince of Wales Hospital, Randwick, Sydney, NSW 2031, Australia. Email: [email protected]Search for more papers by this authorXinye Wang, Xinye Wang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAnurag Adhikari, Anurag Adhikari Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMalinna Yeang, Malinna Yeang Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorFrances Jenkins, Frances Jenkins Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorZin Naing, Zin Naing Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorGregory J. Walker, Gregory J. Walker Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorCharles S. P. Foster, Charles S. P. Foster Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSacha Stelzer-Braid, Sacha Stelzer-Braid orcid.org/0000-0001-6037-9305 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorIra Deveson, Ira Deveson Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, New South Wales, Australia Faculty of Medicine, St Vincent's Clinical School, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMaria E. Craig, Maria E. Craig orcid.org/0000-0001-6004-576X Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, AustraliaSearch for more papers by this authorNicodemus Tedla, Nicodemus Tedla Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorRowena A. Bull, Rowena A. Bull Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorMarianne Martinello, Marianne Martinello Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorAngie N. Pinto, Angie N. Pinto Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorRaymond Chan, Raymond Chan Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, AustraliaSearch for more papers by this authorStuart Turville, Stuart Turville Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia The Kirby Institute for Infection and Immunity, The University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorWilliam D. Rawlinson, William D. Rawlinson orcid.org/0000-0003-0988-7827 Virology Research and Diagnostics Laboratories, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital, Sydney, New South Wales, Australia Discipline of Paediatrics and Child Health, Faculty of Medicine and Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia Faculty of Medicine and Health, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia Faculty of Science, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, AustraliaSearch for more papers by this authorSebastiaan van Hal, Sebastiaan van Hal Department of Microbiology and Infectious Diseases, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, AustraliaSearch for more papers by this author First published: 04 May 2022 https://doi.org/10.1002/jmv.27832Citations: 1 William D. Rawlinson and Sebastiaan van Hal are Joint Senior authors. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Supporting Information Filename Description jmv27832-sup-0001-JMV_Supplementary_Material_final.docx17.9 KB Supplementary information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 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Diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has primarily been achieved using reverse transcriptase polymerase chain reaction (RT-PCR) for acute infection, and serology for prior infection. Assay with RT-PCR provides data on presence or absence of viral RNA, with no information on virus replication competence, infectivity, or virus characterisation. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) is typically not used in clinical virology, despite its potential to provide supplemental data about the presence of viral proteins and thus the potential for replication-competent, transmissible virus. Using the SARS-CoV-2 as a model virus, we developed a fast ‘bottom-up’ proteomics workflow for discovery of target virus peptides using ‘serum-free’ culture conditions, providing high coverage of viral proteins without the need for protein or peptide fractionation techniques. This workflow was then applied to Coronaviruses OC43 and 229E, Influenza A/H1N1 and H3N2, Influenza B, and Respiratory Syncytial Viruses A and B. Finally, we created an LC–MS/MS method for targeted detection of the eight-virus panel in clinical specimens, successfully detecting peptides from the SARS-CoV-2 ORF9B and nucleoprotein in RT-PCR positive samples. The method provides specific detection of respiratory viruses from clinical samples containing moderate viral loads and is an important further step to the use of LC–MS/MS in diagnosis of viral infection.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. Whole-genome sequencing of virus from an early case revealed a sub-consensus level of sequencing reads supporting a 17-nucleotide frameshift-inducing deletion in ORF7a that truncated the peptide sequence. The variant rapidly became represented at the consensus level (Delta-ORF7a{Delta}17del) in most of the outbreak cases in Australia. Retrospective analysis of ORF7a deletions in all GISAID clade GK Delta genomes showed that of 4,018,216 genomes, 134,751 (~3.35%) possessed a deletion in ORF7a, with the ORF7a{Delta}17del mutation by far the most common. Approximately 99.05% of Delta-ORF7a{Delta}17del genomes on GISAID originated from the Australian Delta outbreak, and comprised 87% of genomes in the outbreak. In vitro comparison of lineages in cell culture showed a significantly greater proportion of cells were infected with Delta-ORF7a{Delta}17del than with a contemporaneous Delta variant without ORF7a{Delta}17del (Delta-ORF7aintact), and the proportion was also measurably higher than an early SARS-CoV-2 strain (A.2.2). These results showed that Delta-ORF7a{Delta}17del potentially has a slight growth advantage compared to Delta-ORF7aintact. Delta-ORF7a{Delta}17del viruses still produced ORF7a protein, but significantly less than A.2.2, in a different cellular distribution with a more diffuse expression throughout the cytoplasm of infected cells. These data suggest that the proliferation of Delta-ORF7a{Delta}17del genomes during the Australian Delta outbreak was likely not a result of an intrinsic benefit of the ORF7a{Delta}17del mutation, but rather a chance founder effect. Nonetheless, the abundance of different ORF7a deletions in genomes worldwide suggests these have some benefit to virus transmission. IMPORTANCE Deletions in the ORF7a region of SARS-CoV-2 have been noted since early in the COVID-19 pandemic, but are generally reported as transient mutations that are quickly lost in the population. Consequently, ORF7a deletions are considered disadvantageous to the virus through possible loss-of-function effects. In constrast to these earlier reports, we present the first report of a SARS-CoV-2 variant with an ORF7a deletion that dominated for the entirety of a protracted outbreak, and found no associated fitness disadvantage or advantage in cell culture. The relatively common rise and fall of ORF7a deletion variants over time likely represent chance founder events followed by proliferation until a more fit variant(s) is introduced to the population. Our global clade-level survey of ORF7a deletions will be a useful resource for future studies into this gene region.

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.

Serological testing for SARS-CoV-2 antibodies provides important research and diagnostic information relating to COVID-19 immune response and surveillance. A major challenge when addressing protection post- infection or vaccination is the difficulty of specimen collection from infants and children. Dried blood spots (DBS) collected by finger prick or heel prick are a minimally invasive sample collection alternative previously used to detect antibodies to other viruses. In this study we evaluated DBS for the detection of SARS-CoV-2 antibodies on three commercially available enzyme (EIA) and chemiluminescent (CLIA) immunoassays by analysing paired DBS and serum samples collected from 54 subjects. We demonstrate that testing of DBS samples was highly sensitive and specific, and quantitative results strongly correlated with those of paired serum. These results suggest that DBS derived blood is a viable alternative to plasma or serum for use in EIAs and CLIAs, and has particular utility as a minimally invasive collection tool for COVID-19 serological testing of infants and children.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

AimAs the COVID‐19 pandemic evolves, human milk banks world‐wide continue to provide donor human milk to vulnerable infants who lack access to mother's own milk. Under these circumstances, ensuring the safety of donor human milk is paramount, as the risk of vertical transmission of SARS‐CoV‐2 is not fully understood. Here, we investigate the inactivation of SARS‐CoV‐2 in human milk by pasteurisation and the stability of SARS‐CoV‐2 in human milk under cold storage.MethodsSARS‐CoV‐2 was experimentally inoculated into human milk samples from healthy donors or into a control medium. Triplicates of each sample were layered onto uninfected cells after Holder pasteurisation (63°C for 30 min), heating to 56°C for 30 min, or after 48 h of storage at 4°C or −30°C. Infectious titres of virus were determined at 72 h post‐infection by endpoint titration.ResultsFollowing heating to 63°C or 56°C for 30 min, replication competent (i.e. live) SARS‐CoV‐2 was undetected in both human milk and the control medium. Cold storage of SARS‐CoV‐2 in human milk (either at 4°C or −30°C) did not significantly impact infectious viral load over a 48 h period.ConclusionSARS‐CoV‐2 is effectively inactivated by Holder pasteurisation, suggesting that existing milk bank processes will effectively mitigate the risk of transmission of SARS‐COV‐2 to vulnerable infants through pasteurised donor human milk. The demonstrated stability of SARS‐CoV‐2 in refrigerated or frozen human milk may assist in the development of guidelines around safe expressing and storing of milk from COVID‐19 infected mothers.

There is increasing demand for organ and tissue donations to cater for a growing waiting list of recipients. Serological screening of donors remains the initial assessment upon which many decisions are made, particularly if donors are found to be seropositive. Multiple different platforms are now available, although the Abbott ARCHITECT platform assays are currently licensed globally for testing of blood collected at less than 15 h post-mortem. Compliance with the specified maximum collection times drastically decreases the number of eligible deceased donors, with ~ 70% more donations available if screened at up to 24 h post mortem. A large scale study on deceased donors was performed where blood was collected between 12 and 25 h post-mortem. A total of 194 cadaveric serological specimens were tested using the Abbott ARCHITECT analyser for Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human T Lymphotropic Virus type I/II, and syphilis infection. The specificity, sensitivity, accuracy, reproducibility and influence of storage conditions were assessed for testing with Abbott ARCHITECT platform for HIV antigen/antibody Combo, HCV antibody, HBV surface antigen (HBsAg), HBV core antibody (HBcAb), HTLVI/II antibody (rHTLV-I/II), and Syphilis TP assays. There was no significant difference between testing of sera from living and cadaveric individuals in terms of assay specificity, sensitivity and accuracy. The findings show testing of human serum and plasma specimens collected up to 24 h post-mortem with these assays is acceptable and reflects host status accurately.

Isolation of the new pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for diagnostic and research purposes including assessment of novel therapeutics. Several primary and continuous cell lines are currently used, and new organoid and engineered cell lines are being developed for improved investigation and understanding of the human immune response to this virus. Here we review the growth of SARS-CoV-2 in reference standard cell lines, engineered cell lines and new developments in this field.

The first reported case of Middle East respiratory syndrome coronavirus (MERS‐CoV) infection was identified in Saudi Arabia in September 2012, since which time there have been over 2000 laboratory‐confirmed cases, including 750 deaths in 27 countries. Nucleic acid testing (NAT) is the preferred method for the detection of MERS‐CoV. A single round of a Proficiency Testing Program (PTP) was used to assess the capability of laboratories globally to accurately detect the presence of MERS‐CoV using NAT. A panel of eleven lyophilized specimens containing different viral loads of MERS‐CoV, common coronaviruses, and in vitro RNA transcripts was distributed to laboratories in all six World Health Organization regions. A total of 96 laboratories from 79 countries participating in the PTP, with 76 of 96 (79.2%) reporting correct MERS‐CoV results for all nine scored specimens. A further 10 laboratories (10.4%) scored correctly in eight of nine specimens of the PTP. The majority of laboratories demonstrated satisfactory performance in detecting the presence of MERS‐CoV using NAT. However, some laboratories require improved assay sensitivity, reduced cross contamination of samples, and improved speciation of coronavirus subtypes for potentially complex clinical specimens. Further PTP and enhanced links with expert laboratories globally may improve the laboratory performance.

Acute respiratory tract infections are a major cause of morbidity and mortality and represent a significant burden on the health care system. Laboratory testing is required to definitively distinguish infecting influenza virus from other pathogens, resulting in prolonged emergency department (ED) visits and unnecessary antibiotic use. Recently available rapid point-of-care tests (POCT) may allow for appropriate use of antiviral and antibiotic treatments and decrease patient lengths of stay. We undertook a systematic review to assess the effect of POCT for influenza on three outcomes: (1) antiviral prescription, (2) antibiotic prescription, and (3) patient length of stay in the ED. The databases Medline and Embase were searched using MeSH terms and keywords for influenza, POCT, antivirals, antibiotics, and length of stay. Amongst 245 studies screened, 30 were included. The majority of papers reporting on antiviral prescription found that a positive POCT result significantly increased use of antivirals for influenza compared with negative POCT results and standard supportive care. A positive POCT result also led to decreased antibiotic use. The results of studies assessing the effect of POCT on ED length of stay were not definitive. The studies assessed in this systematic review support the use of POCT for diagnosis of influenza in patients suffering an acute respiratory infection. Diagnosis using POCT may lead to more appropriate prescription of treatments for infectious agents. Further studies are needed to assess the effect of POCT on the length of stay in ED.