Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic (preprint)

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Wholesale Seafood Market, the site with the most reported wildlife vendors in the city of Wuhan, China. Here, we analyze publicly available qPCR and sequencing data from environmental samples collected in the Huanan market in early 2020. We demonstrate that the SARS-CoV-2 genetic diversity linked to this market is consistent with market emergence, and find increased SARS-CoV-2 positivity near and within a particular wildlife stall. We identify wildlife DNA in all SARS-CoV-2 positive samples from this stall. This includes species such as civets, bamboo rats, porcupines, hedgehogs, and one species, raccoon dogs, known to be capable of SARS-CoV-2 transmission. We also detect other animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them to those from other markets. This analysis provides the genetic basis for a short list of potential intermediate hosts of SARS-CoV-2 to prioritize for retrospective serological testing and viral sampling.

Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses (preprint)

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

Co-infection with SARS-COV-2 Omicron and Delta Variants Revealed by Genomic Surveillance (preprint)

We identified the co-infection of the SARS-CoV-2 Omicron and Delta variants in two epidemiologically unrelated patients with chronic kidney disease requiring haemodialysis. Both SARS-CoV-2 variants were co-circulating locally at the time of detection. Amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified Omicron and Delta subpopulations in respiratory samples from the two patients. These findings highlight the importance of genomic surveillance in vulnerable populations.

Replacement of the Gamma by the Delta variant in Brazil: impact of lineage displacement on the ongoing pandemic (preprint)

The COVID-19 epidemic in Brazil was driven mainly by the spread of Gamma (P.1), a locally emerged Variant of Concern (VOC) that was first detected in early January 2021. This variant was estimated to be responsible for more than 96% of cases reported between January and June 2021, being associated with increased transmissibility and disease severity, a reduction in neutralization antibodies and effectiveness of treatments or vaccines, as well as diagnostic detection failure. Here we show that, following several importations predominantly from the USA, the Delta variant rapidly replaced Gamma after July 2021. However, in contrast to what was seen in other countries, the rapid spread of Delta did not lead to a large increase in the number of cases and deaths reported in Brazil. We suggest that this was likely due to the relatively successful early vaccination campaign coupled with natural immunity acquired following prior infection with Gamma. Our data reinforces reports of the increased transmissibility of the Delta variant and, considering the increasing concern due to the recently identified Omicron variant, argues for the necessity to strengthen genomic monitoring on a national level to quickly detect and curb the emergence and spread of other VOCs that might threaten global health.

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens (preprint)

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

Genomic epidemiology reveals how restriction measures shaped the SARS-CoV-2 epidemic in Brazil (preprint)

Brazil has experienced some of the highest numbers of COVID-19 infections and deaths globally and made Latin America a pandemic epicenter from May 2021. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of local virus transmission dynamics. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and an adjacent country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed, the absence of effective restriction measures led to the local emergence and international spread of Variants of Concern (VOC) and under monitoring (VUM), including the Gamma (P.1) and Zeta (P.2) variants. In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring and providing a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

Assessment of COVID-19 intervention strategies in the Nordic countries using genomic epidemiology (preprint)

The Nordic countries, defined here as Norway, Sweden, Denmark, Finland and Iceland, are known for their comparable demographics and political systems. Since these countries implemented different COVID-19 intervention strategies, they provide a natural laboratory for examining how COVID-19 policies and mitigation strategies affected the propagation, evolution and spread of the SARS-CoV-2 virus. We explored how the duration, the size and number of transmission clusters, defined as country-specific monophyletic groups in a SARS-CoV-2 phylogenetic tree, differed between the Nordic countries. We found that Sweden had the largest number of COVID-19 transmission clusters followed by Denmark, Norway, Finland and Iceland. Moreover, Sweden and Denmark had the largest, and most enduring, transmission clusters followed by Norway, Finland and Iceland. In addition, there was a significant positive association between transmission cluster size and duration, suggesting that the size of transmission clusters could be reduced by rapid and effective contact tracing. Thus, these data indicate that to reduce the general burden of COVID-19 there should be a focus on limiting dense gatherings and their subsequent contacts to keep the number, size and duration of transmission clusters to a minimum. Our results further suggest that although geographical connectivity, population density and openness influence the spread and the size of SARS-CoV-2 transmission clusters, country-specific intervention strategies had the largest single impact.

Off-season RSV epidemics in Australia after easing of COVID-19 restrictions (preprint)

Human respiratory syncytial virus (RSV) is an important cause of acute respiratory infection (ARI) with the most severe disease in the young and elderly. Non-pharmaceutical interventions (NPIs) and travel restrictions for controlling COVID-19 have impacted the circulation of most respiratory viruses including RSV globally, particularly in Australia, where during 2020 the normal winter epidemics were notably absent. However, in late 2020, unprecedented widespread RSV outbreaks occurred, beginning in spring, and extending into summer across two widely separated states of Australia, Western Australia (WA) and New South Wales (NSW) including the Australian Capital Territory (ACT). Genome sequencing revealed a significant reduction in RSV genetic diversity following COVID-19 emergence except for two genetically distinct RSV-A clades. These clades circulated cryptically, likely localized for several months prior to an epidemic surge in cases upon relaxation of COVID-19 control measures. The NSW/ACT clade subsequently spread to the neighbouring state of Victoria (VIC) and caused extensive outbreaks and hospitalisations in early 2021. These findings highlight the need for continued surveillance and sequencing of RSV and other respiratory viruses during and after the COVID-19 pandemic as mitigation measures introduced may result in unusual seasonality, along with larger or more severe outbreaks in the future.

Vaccine nationalism and the dynamics and control of SARS-CoV-2 (preprint)

Vaccines provide powerful tools to mitigate the enormous public health and economic costs that the ongoing SARS-CoV-2 pandemic continues to exert globally, yet vaccine distribution remains unequal between countries. To examine the potential epidemiological and evolutionary impacts of 'vaccine nationalism', we extend previous models to include simple scenarios of stockpiling. In general, we find that stockpiling vaccines by countries with high availability leads to large increases in infections in countries with low vaccine availability, the magnitude of which depends on the strength and duration of natural and vaccinal immunity. Additionally, a number of subtleties arise when the populations and transmission rates in each country differ depending on evolutionary assumptions and vaccine availability. Furthermore, the movement of infected individuals between countries combined with the possibility of increases in viral transmissibility may greatly magnify local and combined infection numbers, suggesting that countries with high vaccine availability must invest in surveillance strategies to prevent case importation. Dose-sharing is likely a high-return strategy because equitable allocation brings non-linear benefits and also alleviates costs of surveillance (e.g. border testing, genomic surveillance) in settings where doses are sufficient to maintain cases at low numbers. Across a range of immunological scenarios, we find that vaccine sharing is also a powerful tool to decrease the potential for antigenic evolution, especially if infections after the waning of natural immunity contribute most to evolutionary potential. Overall, our results stress the importance of equitable global vaccine distribution.

Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses (preprint)

Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.

The impact of early public health interventions on SARS-CoV-2 transmission and evolution (preprint)

Many countries have attempted to control COVID-19 through the implementation of non-pharmaceutical interventions. However, it remains unclear how different control strategies have impacted SARS-CoV-2 virus transmission dynamics at the local level. Using complete SARS-CoV-2 genomes, we inferred the relative frequencies of virus importation and exportation, as well as virus transmission chain dynamics in Nordic countries - Denmark, Finland, Iceland, Norway and Sweden - during the first months of the pandemic. Our analyses revealed that Sweden experienced more numerous transmission chains, which tended to have more cases, and were of longer duration, a set of features that increased with time. Together with Denmark, Sweden was also a net exporter of SARS-CoV-2. Hence, Sweden effectively constituted an epidemiological and evolutionary refugia that enabled the virus to maintain active transmission and spread to other geographic localities. This analysis highlights the utility of genomic surveillance where active transmission chain monitoring is a key metric.

A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners. (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modeling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 2020, the cat- owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred. Article Summary LineSARS-CoV-2 in naturally infected cats present a shorter shedding pattern compared to their owners.

The power and limitations of genomics to track COVID-19 outbreaks: a case study from New Zealand (preprint)

Background. Real-time genomic sequencing has played a major role in tracking the global spread and local transmission of SARS-CoV-2, contributing greatly to disease mitigation strategies. After effectively eliminating the virus, New Zealand experienced a second outbreak of SARS-CoV-2 in August 2020. During this August outbreak, New Zealand utilised genomic sequencing in a primary role to support its track and trace efforts for the first time, leading to a second successful elimination of the virus. Methods. We generated the genomes of 80% of the laboratory-confirmed samples of SARS-CoV-2 from New Zealand's August 2020 outbreak and compared these genomes to the available global genomic data. Findings. Genomic sequencing was able to rapidly identify that the new COVID-19 cases in New Zealand belonged to a single cluster and hence resulted from a single introduction. However, successful identification of the origin of this outbreak was impeded by substantial biases and gaps in global sequencing data. Interpretation. Access to a broader and more heterogenous sample of global genomic data would strengthen efforts to locate the source of any new outbreaks.

In Silico Modeling of Virus Particle Propagation and Infectivity along the Respiratory Tract: A Case Study for SARS-COV-2 (preprint)

Respiratory viruses including Respiratory syncytial virus (RSV), influenza virus and cornaviruses such as Middle Eastern respiratory virus (MERS) and SARS-CoV-2 infect and cause serious and sometimes fatal disease in thousands of people annually. It is critical to understand virus propagation dynamics within the respiratory system because new insights will increase our understanding of virus pathogenesis and enable infection patterns to be more predictable in vivo, which will enhance targeting of vaccines and drug delivery. This study presents a computational model of virus propagation within the respiratory tract network. The model includes the generation network branch structure of the respiratory tract, biophysical and infectivity properties of the virus, as well as air flow models that aid the circulation of the virus particles. The model can also consider the impact of the immune response aim to inhibit virus replication and spread. The model was applied to the SARS-CoV-2 virus by integrating data on its life-cycle, as well as density of Angiotensin Converting Enzyme (ACE2) expressing cells along the respiratory tract network. Using physiological data associated with the respiratory rate and virus load that is inhaled, the model can improve our understanding of the concentration and spatiotemporal dynamics of virus.

SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier (preprint)

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

Covidex: an ultrafast and accurate tool for virus subtyping (preprint)

Covidex is an open-source, alignment-free machine learning subtyping tool for viral species. It is a shiny app that allows a fast and accurate classification in pre-defined clusters for SARS-CoV-2 and FMDV genome sequences. The user can also build its own classification models with the Covidex model generator. AvailabilityCovidex is open-source, cross-platform compatible, and is available under the terms of the GNU General Public License v3 (http://www.gnu.org/licenses/gpl.txt). Covidex is available via SourceForge https://sourceforge.net/projects/covidex or the web application https://cacciabue.shinyapps.io/shiny2/ Contactcacciabue.marco@inta.gob.ar; marcocacciabue@yahoo.com

Characterization of cells susceptible to SARS-COV-2 and methods for detection of neutralizing antibody by focus forming assay (preprint)

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

Ubiquitous Forbidden Order in R-group classified protein sequence of SARS-CoV-2 and other viruses (preprint)

Each amino acid in a polypeptide chain has a distinctive R-group associated with it. We report here a novel method of species characterization based upon the order of these R-group classified amino acids in the linear sequence of the side chains associated with the codon triplets. In an otherwise pseudo-random sequence, we search for forbidden combinations of kth order. We applied this method to analyze the available protein sequences of various viruses including SARS-CoV-2. We found that these ubiquitous forbidden orders (UFO) are unique to each of the viruses we analyzed. This unique structure of the viruses may provide an insight into viruses chemical behavior and the folding patterns of the proteins. This finding may have a broad significance for the analysis of coding sequences of species in general.

Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom (preprint)

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.

Targeting pentose phosphate pathway for SARS-CoV-2 therapy (preprint)

It becomes more and more obvious that deregulation of host metabolism play an important role in SARS-CoV-2 pathogenesis with implication for increased risk of severe course of COVID-19. Furthermore, it is expected that COVID-19 patients recovered from severe disease may experience long-term metabolic disorders. Thereby understanding the consequences of SARS-CoV-2 infection on host metabolism can facilitate efforts for effective treatment option. We have previously shown that SARS-CoV-2-infected cells undergo a shift towards glycolysis and that 2-deoxy-D-glucose (2DG) inhibits SARS-CoV-2 replication. Here, we show that also pentose phosphate pathway (PPP) is remarkably deregulated. Since PPP supplies ribonucleotides for SARS-CoV-2 replication, this could represent an attractive target for an intervention. On that account, we employed the transketolase inhibitor benfooxythiamine and showed dose-dependent inhibition of SARS-CoV-2 in non-toxic concentrations. Importantly, the antiviral efficacy of benfooxythiamine was further increased in combination with 2DG.