Virology-The next fifty years.

Virology has made enormous advances in the last 50 years but has never faced such scrutiny as it does today. Herein, we outline some of the major advances made in virology during this period, particularly in light of the COVID-19 pandemic, and suggest some areas that may be of research importance in the next 50 years. We focus on several linked themes: cataloging the genomic and phenotypic diversity of the virosphere; understanding disease emergence; future directions in viral disease therapies, vaccines, and interventions; host-virus interactions; the role of viruses in chronic diseases; and viruses as tools for cell biology. We highlight the challenges that virology will face moving forward-not just the scientific and technical but also the social and political. Although there are inherent limitations in trying to outline the virology of the future, we hope this article will help inspire the next generation of virologists.

Genetic tracing of market wildlife and viruses at the epicenter of the COVID-19 pandemic.

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Seafood Wholesale Market. Here, we analyze environmental qPCR and sequencing data collected in the Huanan market in early 2020. We demonstrate that market-linked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity is consistent with market emergence and find increased SARS-CoV-2 positivity near and within a wildlife stall. We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and raccoon dogs, previously identified as possible intermediate hosts. We also detect animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them with those from farms and other markets. This analysis provides the genetic basis for a shortlist of potential intermediate hosts of SARS-CoV-2 to prioritize for serological and viral sampling.

Zoonotic risk assessment among farmed mammals.

Farmed mammals may act as hosts for zoonotic viruses that can cause disease outbreaks in humans. This SnapShot shows which farmed mammals, and to what extent, are of particular risk of harboring and spreading viruses from viral families that are commonly associated with zoonotic disease. It also discusses genome surveillance methods and biosafety measures. To view this SnapShot, open or download the PDF.

Host traits shape virome composition and virus transmission in wild small mammals.

Bats, rodents, and shrews are the most important animal sources of human infectious diseases. However, the evolution and transmission of viruses among them remain largely unexplored. Through the meta-transcriptomic sequencing of internal organ and fecal samples from 2,443 wild bats, rodents, and shrews sampled from four Chinese habitats, we identified 669 viruses, including 534 novel viruses, thereby greatly expanding the mammalian virome. Our analysis revealed high levels of phylogenetic diversity, identified cross-species virus transmission events, elucidated virus origins, and identified cases of invertebrate viruses in mammalian hosts. Host order and sample size were the most important factors impacting virome composition and patterns of virus spillover. Shrews harbored a high richness of viruses, including many invertebrate-associated viruses with multi-organ distributions, whereas rodents carried viruses with a greater capacity for host jumping. These data highlight the remarkable diversity of mammalian viruses in local habitats and their ability to emerge in new hosts.

Virome characterization of game animals in China reveals a spectrum of emerging pathogens.

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one 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 inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we 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.

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

Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2-related and three SARS-CoV-related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2-related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV.

The origins of SARS-CoV-2: A critical review.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

A Genomic Perspective on the Origin and Emergence of SARS-CoV-2.

The ongoing pandemic of a new human coronavirus, SARS-CoV-2, has generated enormous global concern. We and others in China were involved in the initial genome sequencing of the virus. Herein, we describe what genomic data reveal about the emergence SARS-CoV-2 and discuss the gaps in our understanding of its origins.

Using Metagenomics to Characterize an Expanding Virosphere.

We know less about viruses than any other lifeform. Fortunately, metagenomics has led to a massive expansion in the known diversity of the virosphere. Here, we discuss how metagenomics has changed our understanding of RNA viruses and present some of the remaining challenges, including characterization of the "dark matter" of divergent viral genomes.

An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis.

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.

Human Adaptation of Ebola Virus during the West African Outbreak.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

Mapping glycoprotein structure reveals Flaviviridae evolutionary history.

Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis1. Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae2, a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized3. Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae.

Farmed fur animals harbour viruses with zoonotic spillover potential.

Animals such as raccoon dogs, mink and muskrats are farmed for fur and are sometimes used as food or medicinal products1,2, yet they are also potential reservoirs of emerging pathogens3. Here we performed single-sample metatranscriptomic sequencing of internal tissues from 461 individual fur animals that were found dead due to disease. We characterized 125 virus species, including 36 that were novel and 39 at potentially high risk of cross-species transmission, including zoonotic spillover. Notably, we identified seven species of coronaviruses, expanding their known host range, and documented the cross-species transmission of a novel canine respiratory coronavirus to raccoon dogs and of bat HKU5-like coronaviruses to mink, present at a high abundance in lung tissues. Three subtypes of influenza A virus-H1N2, H5N6 and H6N2-were detected in the lungs of guinea pig, mink and muskrat, respectively. Multiple known zoonotic viruses, such as Japanese encephalitis virus and mammalian orthoreovirus4,5, were detected in guinea pigs. Raccoon dogs and mink carried the highest number of potentially high-risk viruses, while viruses from the Coronaviridae, Paramyxoviridae and Sedoreoviridae families commonly infected multiple hosts. These data also reveal potential virus transmission between farmed animals and wild animals, and from humans to farmed animals, indicating that fur farming represents an important transmission hub for viral zoonoses.

AMPD2 regulates GTP synthesis and is mutated in a potentially treatable neurodegenerative brainstem disorder.

Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease.

A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here, we propose adding 2 sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present an assignment tool to show that the proposed lineages are useful for regional, national, and subnational discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

After the pandemic: perspectives on the future trajectory of COVID-19.

There is a realistic expectation that the global effort in vaccination will bring the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under control. Nonetheless, uncertainties remain about the type of long-term association that the virus will establish with the human population and, in particular, whether coronavirus disease 2019 (COVID-19) will become an endemic disease. Although the trajectory is difficult to predict, the conditions, concepts and variables that influence this transition can be anticipated. Persistence of SARS-CoV-2 as an endemic virus, perhaps with seasonal epidemic peaks, may be fuelled by pockets of susceptible individuals and waning immunity after infection or vaccination, changes in the virus through antigenic drift that diminish protection and re-entries from zoonotic reservoirs. Here we review relevant observations from previous epidemics and discuss the potential evolution of SARS-CoV-2 as it adapts during persistent transmission in the presence of a level of population immunity. Lack of effective surveillance or adequate response could enable the emergence of new epidemic or pandemic patterns from an endemic infection of SARS-CoV-2. There are key pieces of data that are urgently needed in order to make good decisions; we outline these and propose a way forward.

A ~40-kb flavi-like virus does not encode a known error-correcting mechanism.

It is commonly held that there is a fundamental relationship between genome size and error rate, manifest as a notional "error threshold" that sets an upper limit on genome sizes. The genome sizes of RNA viruses, which have intrinsically high mutation rates due to a lack of mechanisms for error correction, must therefore be small to avoid accumulating an excessive number of deleterious mutations that will ultimately lead to population extinction. The proposed exceptions to this evolutionary rule are RNA viruses from the order Nidovirales (such as coronaviruses) that encode error-correcting exonucleases, enabling them to reach genome lengths greater than 40 kb. The recent discovery of large-genome flavi-like viruses (Flaviviridae), which comprise genomes up to 27 kb in length yet seemingly do not encode exonuclease domains, has led to the proposal that a proofreading mechanism is required to facilitate the expansion of nonsegmented RNA virus genomes above 30 kb. Herein, we describe a ~40 kb flavi-like virus identified in a Haliclona sponge metatranscriptome that does not encode a known exonuclease. Structural analysis revealed that this virus may have instead captured cellular domains associated with nucleic acid metabolism that have not been previously found in RNA viruses. Phylogenetic inference placed this virus as a divergent pesti-like lineage, such that we have provisionally termed it "Maximus pesti-like virus." This virus represents an instance of a flavi-like virus achieving a genome size comparable to that of the Nidovirales and demonstrates that RNA viruses have evolved multiple solutions to overcome the error threshold.

Author Correction: A new coronavirus associated with human respiratory disease in China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new coronavirus associated with human respiratory disease in China.

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health1-3. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China5. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.