Re-emergence of Severe Acute Diarrhea Syndrome Coronavirus (SADS-CoV) in Guangxi, China, 2021 (preprint)

Severe acute diarrhea syndrome coronavirus (SADS-CoV) has had a major impact on the swine industry in China, but has not been detected since 2019. Using real-time qPCR and metagenomic surveillance we identified SADS-CoV in a pig farm experiencing diarrheal disease. Genomic analysis supported the undetected circulation of SADS-CoV since 2019.

Faecal virome of the Australian grey-headed flying fox from urban/suburban environments contains novel coronaviruses, retroviruses and sapoviruses (preprint)

ABSTRACT Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae , with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting ( Coronaviridae, Caliciviridae, Retroviridae ) and one possible mammalian-infecting ( Birnaviridae ) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus ) and a novel bat sapovirus ( Caliciviridae ), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1,375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

The enteric virome of cats with feline panleukopenia differs in abundance and diversity from healthy cats (preprint)

Feline panleukopenia (FPL) is a severe, often fatal disease caused by feline parvovirus (FPV). How infection with FPV might impact the composition of the entire eukaryotic enteric virome in cats has not been characterized. We used metatranscriptomic and viral particle enrichment metagenomic approaches to characterize the enteric viromes of 23 cats naturally infected with FPV (FPV-cases) and 36 age-matched healthy shelter cats (healthy controls). Sequencing reads were detected from 11 mammalian infecting viral families mostly belonging to Coronaviridae, Parvoviridae and Astroviridae . Among the healthy control cats the most abundant viruses were Feline coronavirus, Mamastrovirus 2 and Carnivore bocaparvovirus 3 (Feline bocavirus 1) with frequent co-infections of all three. Feline chaphamaparvovirus was only detected in healthy controls (6/36, 16.7%). Among the FPV-cases, in addition to FPV, the most abundant viruses were Mamastrovirus 2 , Feline coronavirus and Carnivore bocaparvovirus 4 (Feline bocaparvovirus 2). The latter and Feline bocaparvovirus 3 were detected significantly more frequently in FPV-cases than in healthy controls. Feline calicivirus was present in a high proportion of FPV-cases (11/23, 47.8%) compared to healthy controls (5/36, 13.9%, p=0.0067). Feline kobuvirus infections were also common among FPV-cases (9/23, 39.1%) and were not detected in any healthy control cats (p<0.0001). While abundant in both groups, astroviruses were more frequently present in FPV-cases (19/23, 82.6%) than in healthy controls (18/36, p=0.0142). The differences in eukaryotic virome composition found in this study indicate that further investigations to determine associations between enteric viral co-infections on clinical disease severity in cats with FPL are warranted.

Total Infectomes Characterization of Respiratory Infections in pre-COVID-19 Wuhan, China (preprint)

At the end of 2019 Wuhan witnessed an outbreak of “atypical pneumonia” that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus - SARS-CoV-2. Herein, to provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their “total infectome”, including viruses, bacteria and fungi. Consequently, we identified 37 pathogen species, comprising 15 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (12.8%). However, SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen – Chlamydia psittaci . Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016-2017.

Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing (preprint)

Encephalitis is most often caused by a variety of infectious agents, the identity of which is commonly determined through diagnostic tests utilising cerebrospinal fluid (CSF). Immune-mediated disorders are also a differential in encephalitis cases. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic next-generation sequencing of residual clinical samples of multiple tissue types and independent clinical review. A total of 43 specimens, from both sterile and non-sterile sites, were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing to determine the presence and abundance of potential pathogens, to reveal mixed infections, pathogen genotypes, and epidemiological origins, and to describe the possible aetiologies of unexplained encephalitis. From this, we identified five RNA and two DNA viruses associated with human infection from both non-sterile (nasopharyngeal aspirates, nose/throat swabs, urine, stool rectal swab) and sterile (cerebrospinal fluid, blood) sites. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. With the exception of picobirnavirus all have been previously associated with respiratory disease. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases reported here. Immune-mediated encephalitis was considered clinically likely in six cases and RNA sequencing did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasises the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that the routine inclusion of non-CNS sampling in encephalitis clinical guidelines/protocols could improve the diagnostic yield. Author Summary Encephalitis is caused by both infectious agents and auto-immune disorders. However, the aetiological agents, including viruses, remain unknown in around half the cases of encephalitis in many cohorts. Importantly, diagnostic tests are usually based on the analysis of cerebrospinal fluid which may limit their utility. We used a combination of meta-transcriptomic sequencing and independent clinical review to identify the potential causative pathogens in cases of unexplained childhood encephalitis. Accordingly, we identified seven viruses associated with both sterile and non-sterile sampling sites. Human rhinovirus and seasonal coronaviruses were considered as most likely responsible for five of the 18 encephalitis cases studied, while immune-mediated encephalitis was considered the cause in six cases, and we were unable to determine the aetiology in nine cases. Overall, we demonstrate the role of respiratory viruses as a cause of unexplained encephalitis and that sampling sites other than cerebrospinal fluid is of diagnostic value.

Emergence and spread of SARS-CoV-2 lineages B.1.1.7 and P.1 in Italy (preprint)

Italy’s second wave of SARS-CoV-2 has hit hard, with more than 3 million cases and over 100,000 deaths, representing an almost ten-fold increase on the numbers reported by August 2020. Herein, we present the analysis of 6,515 SARS-CoV-2 sequences sampled in Italy between 29 th January 2020 and 1 st March 2021 and show how different lineages emerged multiple times independently despite lockdown restrictions. Virus lineage B.1.177 became the dominant variant in November 2020, when cases peaked at 40,000 a day, but since January 2021 this is being replaced by the B.1.1.7 ‘variant of concern’. In addition, we report a sudden increase in another documented variant of concern – lineage P.1 – from December 2020 onwards, most likely caused by a single introduction into Italy. We again highlight how international importations drive the emergence of new lineages and that genome sequencing should remain a top priority for ongoing surveillance in Italy.

Slippery when wet: cross-species transmission of divergent coronaviruses in bony and jawless fish and the evolutionary history of the Coronaviridae (preprint)

The Nidovirales comprise a genetically diverse group of positive-sense single-stranded RNA virus families that infect a range of invertebrate and vertebrate hosts. Recent metagenomic studies have identified nido-like virus sequences, particularly those related to the Coronaviridae , in a range of aquatic hosts including fish, amphibians and reptiles. We sought to identify additional members of the Coronaviridae in both bony and jawless fish through a combination of total RNA sequencing (meta-transcriptomics) and data mining of published RNA sequencing data, and from this reveal more of the long-term patterns and processes of coronavirus evolution. Accordingly, we identified a number of divergent viruses that fell within the Letovirinae subfamily of the Coronaviridae , including those in a jawless fish – the pouched lamprey. By mining fish transcriptome data we identified additional virus transcripts matching these viruses in bony fish from both marine and freshwater environments. These new viruses retained sequence conservation in the RNA-dependant RNA polymerase across the Coronaviridae , but formed a distinct and diverse phylogenetic group. Although there are broad-scale topological similarities between the phylogenies of the major groups of coronaviruses and their vertebrate hosts, the evolutionary relationships of viruses within the Letovirinae does not mirror that of their hosts. For example, the coronavirus found in the pouched lamprey fell within the phylogenetic diversity of bony fish letoviruses, indicative of past host switching events. Hence, despite possessing a phylogenetic history that likely spans the entire history of the vertebrates, coronavirus evolution has been characterised by relatively frequent cross-species transmission, particularly in hosts that reside in aquatic habitats.

Epidemiological and Genomic analysis of a Sydney Hospital COVID-19 Outbreak (preprint)

Australia’s early COVID-19 experience involved clusters in northern Sydney, including hospital and aged-care facility (ACF) outbreaks. We explore transmission dynamics, drivers and outcomes of a metropolitan hospital COVID-19 outbreak that occurred in the context of established local community transmission. A retrospective cohort analysis is presented, with integration of viral genome sequencing, clinical and epidemiological data. We demonstrate using genomic epidemiology that the hospital outbreak (n=23) was linked to a concurrent outbreak at a local aged care facility, but was phylogenetically distinct from other community clusters. Thirty day survival was 50% for hospitalised patients (an elderly cohort with significant comorbidities) and 100% for staff. Staff who acquired infection were unable to attend work for a median of 26.5 days (range 14-191); an additional 140 staff were furloughed for quarantine. Transmission from index cases showed a wide dispersion (mean 3.5 persons infected for every patient case and 0.6 persons infected for every staff case). One patient, who received regular nebulised medication prior to their diagnosis being known, acted as an apparent superspreader. No secondary transmissions occurred from isolated cases or contacts who were quarantined prior to becoming infectious. This analysis elaborates the wide-ranging impacts on patients and staff of nosocomial COVID-19 transmission and highlights the utility of genomic analysis as an adjunct to traditional epidemiological investigations. Delayed case recognition resulted in nosocomial transmission but once recognised, prompt action by the outbreak management team and isolation with contact and droplet (without airborne) precautions were sufficient to prevent transmission within this cohort. Our findings support current PPE recommendations in Australia but demonstrate the risk of administering nebulised medications when COVID-19 is circulating locally.

Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia (preprint)

Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp ( Cyprinus carpio ). This invasive species now occupies up to 90% of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 ( CyHV-3 ) has been proposed as a potentially effective biological control. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3 , we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of 36 RNA libraries representing 10 species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified 18 vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further 27 that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fishes sampled here, suggesting that there is little virus transmission from common carp to native fish species. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories. Author Summary The ongoing invasion of the common carp in the Murray-Darling Basin, Australia, has wreaked havoc on native freshwater ecosystems. This has stimulated research into the possible biological control of invasive carp through the deliberate release of the virus cyprinid herpesvirus 3 ( CyHV-3 ). However, little is known on the diversity of viruses that naturally circulate in wild freshwater fauna, whether these viruses are transmitted between invasive and native species, nor if CyHV-3 or other cyprinid herpesviruses are already present in the basin. To address these fundamental questions we employed meta-transcriptomic next-generation sequencing to characterise the total assemblage of viruses (i.e. the viromes) in three invasive and seven native fish species cohabiting at 10 sites across 2,200 km of the river system. From this analysis we identified 18 vertebrate-associated viruses across 14 viral families, yet a marked lack of virus transmission between invasive and native species. Importantly, no CyHV-3 was detected. This study shows that freshwater fish harbour a high diversity and abundance of viruses, that viruses have likely been associated with fish for millennia, and that there is likely little direct virus transmission between introduced and native species.

Epidemiological and evolutionary considerations of SARS-CoV-2 vaccine dosing regimes (preprint)

As the threat of Covid-19 continues and in the face of vaccine dose shortages and logistical challenges, various deployment strategies are being proposed to increase population immunity levels. How timing of delivery of the second dose affects infection burden but also prospects for the evolution of viral immune escape are critical questions. Both hinge on the strength and duration (i.e. robustness) of the immune response elicited by a single dose, compared to natural and two-dose immunity. Building on an existing immuno-epidemiological model, we find that in the short-term, focusing on one dose generally decreases infections, but longer-term outcomes depend on this relative immune robustness. We then explore three scenarios of selection, evaluating how different second dose delays might drive immune escape via a build-up of partially immune individuals. Under certain scenarios, we find that a one-dose policy may increase the potential for antigenic evolution. We highlight the critical need to test viral loads and quantify immune responses after one vaccine dose, and to ramp up vaccination efforts throughout the world.

Co-Infecting Pathogens Can Contribute to Inflammatory Responses and Severe Symptoms in COVID-19 (preprint)

Background: The current pandemic of COVID-19 is posing a major challenge to public health on a global scale. While it is generally believed severe COVID-19 results from over-expression of inflammatory mediators (i.e. a “cytokine storm”), it is still unclear whether and how co-infecting pathogens contribute to disease pathogenesis. To address this, we followed the entire course of disease in severe COVID-19 cases to reveal the presence and abundance of all potential pathogens present - the total “infectome” - and how they interact with the host immune system in the context of severe COVID-19 disease.Methods: We considered one severe and three critical cases of COVID-19, as well as a set of healthy controls, with longitudinal samples (throat swab, whole blood and serum) taken in each case. Total RNA sequencing (meta-transcriptomics) was performed to simultaneously reveal pathogen diversity and abundance, as well as host immune responses, within each sample. A Bio-Plex method was used to measure serum cytokine and chemokine levels.Findings: Eight pathogens were identified in these COVID-19 patients - Aspergillus fumigatus, Mycoplasma orale, Myroides odorantus, Acinetobacter baumannii, Candida tropicalis, herpes simplex virus and human cytomegalovirus - that appeared at different stages of disease course. Notably, the dynamics of inflammatory mediators in the serum as well as respiratory tract were better associated with the dynamics of the infectome as a whole rather than SARS-CoV-2 alone. Correlation analysis revealed that pulmonary injury was directly associated with cytokine levels, which in turn was associated with the proliferation of SARS-CoV-2 and the co-infecting pathogens.Interpretation: The cytokine storm that resulted in aggravated acute lung injury and death involved the highly complex and dynamic entire infectome of each patient, of which SARS-CoV-2 was a component. These results call for a precision-medicine approach to investigating both the infection and the host response on a daily basis as a standard means of infectious disease characterization.Funding: Guangzhou Institute of Respiratory Health Open Project (Funds provided by China Evergrande Group) - Project No. (2020GIRHHMS01), Guangdong Province “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project (2019ZT08Y464), Macao Science and Technology Development Fund (0042/2020/A), Science research project of the Guangdong Province (2019B030316028), Special Project for Scientific and Technological Development and Emergency Response in COVID-19 Prevention and Control of Guangdong Province (2020A111129028), Special Project for Research and Promotion of Prevention and Control Techniques of COVID-19 and Emergency Response in Dongguan City (202071715001114), Jack Ma Foundation (2020-CMKYGG-02), Guangzhou Medical University High-level University Clinical Research and Cultivation Program ([2017] 159 and 160) and ARC Australian Laureate Fellowship (FL170100022).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The ethics committee of the FAHGMU (Ethics No. 2020-85) and Dongguan’s People’s Hospital (KYKT2020-005-A1) approved the sampling procedure and the use of patient samples for this study. Informed consent was obtained from each patient.