SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes.

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 - Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

Improved Neutralisation of the SARS-CoV-2 Omicron Variant following a Booster Dose of Pfizer-BioNTech (BNT162b2) COVID-19 Vaccine.

In late November 2021, the World Health Organization declared the SARS-CoV-2 lineage B.1.1.529 the fifth variant of concern, Omicron. This variant has acquired over 30 mutations in the spike protein (with 15 in the receptor-binding domain), raising concerns that Omicron could evade naturally acquired and vaccine-derived immunity. We utilized an authentic virus, multicycle neutralisation assay to demonstrate that sera collected one, three, and six months post-two doses of Pfizer-BioNTech BNT162b2 had a limited ability to neutralise SARS-CoV-2. However, four weeks after a third dose, neutralising antibody titres were boosted. Despite this increase, neutralising antibody titres were reduced fourfold for Omicron compared to lineage A.2.2 SARS-CoV-2.

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture.

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts. Discussion and Conclusion: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

Experimental infection of Asian house geckos with Enterococcus lacertideformus demonstrates multiple disease transmission routes and the in-vivo efficacy of antibiotics.

The disease caused by Enterococcus lacertideformus is multisystemic and ultimately fatal. Since its emergence, the bacterium has significantly impacted the captive breeding programs of the extinct in the wild Christmas Island Lister's gecko (Lepidodactylus listeri) and blue-tailed skink (Cryptoblepharus egeriae). The bacterium's pathogenicity, inability to grow in-vitro, and occurrence beyond the confines of Christmas Island necessitated the development of an experimental infection and treatment model. Asian house geckos (Hemidactylus frenatus) were challenged with a single dose of E. lacertideformus inoculum either by mouth, application to mucosal abrasion or skin laceration, subcutaneous injection, coelomic injection, or via co-housing with an infected gecko. Five healthy geckos acted as controls. Each transmission route resulted in disease in at least 40% (n = 2) geckos, expanding to 100% (n = 5) when E. lacertideformus was applied to skin laceration and mucosal abrasion groups. Incubation periods post-infection ranged between 54 and 102 days. To determine the efficacy of antibiotic treatment, infected geckos were divided into six groups (enrofloxacin 10 mg/kg, per os (PO), every 24 h (q24), amoxicillin-clavulanic acid 10 mg/kg, PO, q24, enrofloxacin 10 mg/kg combined with amoxicillin-clavulanic acid 10 mg/kg, PO, q24, rifampicin 15 mg/kg, PO, q24, clarithromycin 15 mg/kg, PO, q24, and untreated controls) for 21 days. Response to treatment was assessed by the change in lesion size, bacterial dissemination, and histological evidence of a host immune response. Irrespective of the antibiotic given, histology revealed that geckos inoculated by skin laceration were observed to have more extensive disease spread throughout the animal's body compared to other inoculation routes. The reduction in the average surface area of gross lesions was 83.6% for geckos treated with enrofloxacin, followed by the combination therapy amoxicillin-clavulanic acid and enrofloxacin (62.4%), amoxicillin-clavulanic acid (58.2%), rifampicin (45.5%), and clarithromycin (26.5%). Lesions in geckos untreated with antibiotics increased in size between 100 and 300%. In summary, enrofloxacin and amoxicillin-clavulanic acid show promising properties for the treatment of E. lacertideformus infection in geckos. The Asian house gecko E. lacertideformus infection model therefore provides foundational findings for the development of effective therapeutic treatment protocols aimed at conserving the health of infected and at-risk reptiles.

Pharmacokinetic profile of enrofloxacin and its metabolite ciprofloxacin in Asian house geckos (Hemidactylus frenatus) after single-dose oral administration of enrofloxacin.

The pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were determined following oral administration in 21 Asian house geckos (Hemidactylus frenatus) at a dose of 10 mg/kg. Changes in enrofloxacin and ciprofloxacin plasma concentrations were quantified at regular intervals over 72 h (1, 2, 6, 12, 24, 48, and 72 h). Samples were analysed by high-pressure liquid chromatography (HPLC) and the enrofloxacin pharmacokinetic data underwent a two-compartment analysis. Due to the limited ciprofloxacin plasma concentrations above the lower limit of quantification (LLOQ), the ciprofloxacin data underwent non-compartment analysis and the half-life was determined by the Lineweaver-Burke plot and analysis. The enrofloxacin and ciprofloxacin mean half-lives (t ½) were 0.95 h (α) / 24.36 h (ß), and 11.06 h respectively, area under the curve (AUC0-24h) were 60.56 and 3.14 µg/mL*h, respectively, maximum concentrations (C max) were 12.31 and 0.24 µg/mL, respectively, and time required to reach the C max (T max) were 1 and 2 h respectively. Enrofloxacin was minimally converted to the active metabolite ciprofloxacin, with ciprofloxacin concentrations contributing only 4.91% of the total fluoroquinolone concentrations (AUC0-24h). Based on the pharmacokinetic indices when using susceptibility breakpoints when determined at mammalian body temperature it is predicted that single oral administration of enrofloxacin (10 mg/kg) would result in plasma concentrations effective against susceptible bacterial species inhibited by an enrofloxacin MIC ≤ 0.5 µg/mL in vitro, but additional studies will be required to determine its efficacy in vivo.

Opportunistic sampling of wild native and invasive birds reveals a rich diversity of adenoviruses in Australia.

Little is known about the diversity of adenoviruses in wild birds and how they have evolved and are maintained in complex ecosystems. In this study, 409 samples were collected from woodland birds caught for banding (droppings), birds submitted to a wildlife hospital (droppings and tissues), silver gulls (droppings or tissues), and feral pigeons (Columbia livia; oral, cloacal swabs, or tissues) from the Greater Sydney area in NSW, Australia. Additional samples were from native pigeons and doves (swabs) presented to the Healesville Sanctuary, VIC, Australia. Samples were screened for adenovirus DNA using degenerate primers and polymerase chain reaction. Adenovirus sequences were detected in eighty-three samples representing thirty-five novel amino acid sequences. Fourteen novel sequences were atadenoviruses, seven were aviadenoviruses, twelve were siadenoviruses, and one was a mastadenovirus. Sequences from passerine birds were predominately found to form a single lineage within the atadenoviruses, a second lineage in the siadenoviruses, and a third smaller aviadenovirus lineage. These viruses appeared to have co-evolved with a diverse group of woodland birds that share similar habitat. Evidence for host/virus co-evolution in some viruses and a wide host range in others was observed. A high prevalence of adenovirus infection was found in rainbow lorikeets (Trichoglossus haematodus), galahs (Eolophus roseicapilla), and sulphur-crested cockatoos (Cacatua galerita). Sequences were either identical to or mapped to already established lineages in the Aviadenovirus, Siadenovirus, and Atadenovirus genera, suggesting a possible origin of the psittacine adenoviruses in ancestral Australian psittacine birds. The sequences of passerine and psittacine origin provided insight into diversity and structure of the Atadenovirus genus and demonstrated for the first-time viruses of passerine origin in the Aviadenovirus genus. Four unrelated adenovirus sequences were found in silver gull samples (Chroicocephalus novaehollandiae), including one of pigeon origin, suggesting environmental virus exposure. Three pigeon adenovirus types were detected in feral pigeons and infection prevalence was high. Evidence for host switching between invasive species and native species and native species and invasive species was documented. A variant of a murine adenovirus was detected in kidney tissue from two bird species suggesting mouse to bird transmission.

New insights into Sauropsid Papillomaviridae evolution and epizootiology: discovery of two novel papillomaviruses in native and invasive Island geckos.

Papillomaviruses cause persistent infections in skin and mucosal membranes and, in at least one species, are also be able to infect a tissue of mesenchymal origin. Infections may either be subclinical or induce proliferative lesions. Of the known papillomaviruses, the majority that have been characterized are from humans and other mammals. Currently, only fifteen complete bird and reptile papillomavirus genomes have been described, and they have been found in birds (n = 11), turtles (n = 2), and snakes (n = 2). Using next-generation sequencing technologies and virus-specific PCR, we have identified two novel papillomavirus genomes, Hemidactylus frenatus Papillomavirus 1 and 2 (HfrePV1, HfrePV2), in the widely distributed and highly invasive Asian house gecko (H.frenatus) and mute gecko (Gehyra mutilata) on Christmas Island and Cocos (Keeling) Islands. HfrePV1 was also detected in critically endangered Lister's geckos (Lepidodactylus listeri) in their captive breeding colony on Christmas Island. Tissue-containing virus included epidermis, oral mucosa, and liver (HfrePV1) and epidermis, liver, and colon (HfrePV2). Concurrent infections were found in both H.frenatus and G.mutilata. Invasive mourning geckos (Lepidodactylus lugubris) (n = 4), Sri Lankan house geckos (Hemidactylus parvimaculatus) (n = 3), flat-tailed house geckos (Hemidactylus platyurus) (n = 4) from the Cocos Islands, and blue-tailed skinks (Cryptoblepharus egeriae) (n = 10) from Christmas Island were also screened but were not found to be infected. The novel HfrePV1 and HfrePV2 genomes were 7,378 bp and 7,380 bp in length, respectively, and each contained the early (E1, E2, and E7), and late (L1 and L2) open-reading frames. Phylogenetic analysis of the concatenated E1, E2, and L1 proteins from both papillomaviruses revealed that they clustered with, but were basal to, the Sauropsida clade containing bird and reptile viruses. This study sheds light on the evolution of papillomaviruses and the distribution of pathogens in a highly invasive species impacting endangered populations of geckos.