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.

The global H5N1 influenza panzootic in mammals.

Influenza A viruses (IAV) have caused more documented global pandemics in human history than any other pathogen1,2. High pathogenicity avian influenza (HPAI) viruses belonging to the H5N1 subtype are a leading pandemic risk. Two decades after H5N1 "bird flu" became established in poultry in Southeast Asia, its descendants have resurged3, setting off an H5N1 panzootic in wild birds that is fueled by (a) rapid intercontinental spread, reaching South America and Antarctica for the first time4,5; (b) fast evolution via genomic reassortment6; and (c) frequent spillover into terrestrial7,8 and marine mammals9. The virus has sustained mammal-to-mammal transmission in multiple settings, including European fur farms10,11, South American marine mammals12-15, and US dairy cattle16-19, raising questions about whether humans are next. Historically, swine are considered optimal intermediary hosts that help avian influenza viruses (AIV) adapt to mammals before jumping to humans20. However, the altered ecology of H5N1 has opened the door to new evolutionary pathways. Could dairy cattle, farmed mink, or South American sea lions serve as new mammalian gateways to humans? Here we explore the molecular and ecological factors driving H5N1's sudden expansion in host range and assess the likelihood of different zoonotic pathways leading to an H5N1 pandemic.

H5N1 influenza: Urgent questions and directions.

H5N1 is an avian influenza virus that causes respiratory disease in birds and several land and sea mammals. The recent outbreak in the United States, including infection of dairy workers, has increased the concern around potential transmission and spread. We asked virologists, epidemiologists, and public health experts what the most urgent questions and action points are at this stage of the outbreak.

APOBEC3 deaminase editing in mpox virus as evidence for sustained human transmission since at least 2016.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

Determinants of de novo B cell responses to drifted epitopes in post-vaccination SARS-CoV-2 infections.

Vaccine-induced immunity may impact subsequent de novo responses to drifted epitopes in SARS-CoV-2 variants, but this has been difficult to quantify due to the challenges in recruiting unvaccinated control groups whose first exposure to SARS-CoV-2 is a primary infection. Through local, statewide, and national SARS-CoV-2 testing programs, we were able to recruit cohorts of individuals who had recovered from either primary or post-vaccination infections by either the Delta or Omicron BA.1 variants. Regardless of variant, we observed greater Spike-specific and neutralizing antibody responses in post-vaccination infections than in those who were infected without prior vaccination. Through analysis of variant-specific memory B cells as markers of de novo responses, we observed that Delta and Omicron BA.1 infections led to a marked shift in immunodominance in which some drifted epitopes elicited minimal responses, even in primary infections. Prior immunity through vaccination had a small negative impact on these de novo responses, but this did not correlate with cross-reactive memory B cells, arguing against competitive inhibition of naïve B cells. We conclude that dampened de novo B cell responses against drifted epitopes are mostly a function of altered immunodominance hierarchies that are apparent even in primary infections, with a more modest contribution from pre-existing immunity, perhaps due to accelerated antigen clearance.

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 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.

The recency and geographical origins of the bat viruses ancestral to SARS-CoV and SARS-CoV-2.

The emergence of SARS-CoV in 2002 and SARS-CoV-2 in 2019 has led to increased sampling of related sarbecoviruses circulating primarily in horseshoe bats. These viruses undergo frequent recombination and exhibit spatial structuring across Asia. Employing recombination-aware phylogenetic inference on bat sarbecoviruses, we find that the closest-inferred bat virus ancestors of SARS-CoV and SARS-CoV-2 existed just ~1-3 years prior to their emergence in humans. Phylogeographic analyses examining the movement of related sarbecoviruses demonstrate that they traveled at similar rates to their horseshoe bat hosts and have been circulating for thousands of years in Asia. The closest-inferred bat virus ancestor of SARS-CoV likely circulated in western China, and that of SARS-CoV-2 likely circulated in a region comprising southwest China and northern Laos, both a substantial distance from where they emerged. This distance and recency indicate that the direct ancestors of SARS-CoV and SARS-CoV-2 could not have reached their respective sites of emergence via the bat reservoir alone. Our recombination-aware dating and phylogeographic analyses reveal a more accurate inference of evolutionary history than performing only whole-genome or single gene analyses. These results can guide future sampling efforts and demonstrate that viral genomic fragments extremely closely related to SARS-CoV and SARS-CoV-2 were circulating in horseshoe bats, confirming their importance as the reservoir species for SARS viruses.

Using archived and biocollection samples towards deciphering the DNA virus diversity associated with rodent species in the families cricetidae and heteromyidae.

Rodentia is the most speciose order of mammals, and they are known to harbor a wide range of viruses. Although there has been significant research on zoonotic viruses in rodents, research on the diversity of other viruses has been limited, especially for rodents in the families Cricetidae and Heteromyidae. In fecal and liver samples of nine species of rodents, we identify 346 distinct circular DNA viral genomes. Of these, a large portion are circular, single-stranded DNA viruses in the families Anelloviridae (n = 3), Circoviridae (n = 5), Genomoviridae (n = 7), Microviridae (n = 297), Naryaviridae (n = 4), Vilyaviridae (n = 15) and in the phylum Cressdnaviricota (n = 13) that cannot be assigned established families. We also identified two large bacteriophages of 36 and 50 kb that are part of the class Caudoviricetes. Some of these viruses are clearly those that infect rodents, however, most of these likely infect various organisms associated with rodents, their environment or their diet.

Sensitive SARS-CoV-2 salivary antibody assays for clinical saline gargle samples using smartphone-based competitive particle immunoassay platforms.

Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1-5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.

Diverse DNA virus genomes identified in fecal samples of Mexican free-tailed bats (Tadarida brasiliensis) captured in Chiricahua Mountains of southeast Arizona (USA).

Bats (order Chiroptera) are some of the most abundant mammals on earth and their species ecology strongly influences zoonotic potential. While substantial research has been conducted on bat-associated viruses, particularly on those that can cause disease in humans and/or livestock, globally, limited research has focused on endemic bats in the USA. The southwest region of the US is of particular interest because of its high diversity of bat species. We identified 39 single-stranded DNA virus genomes in the feces of Mexican free-tailed bats (Tadarida brasiliensis) sampled in the Rucker Canyon (Chiricahua Mountains) of southeast Arizona (USA). Twenty-eight of these belong to the virus families Circoviridae (n = 6), Genomoviridae (n = 17), and Microviridae (n = 5). Eleven viruses cluster with other unclassified cressdnaviruses. Most of the viruses identified represent new species. Further research on identification of novel bat-associated cressdnaviruses and microviruses is needed to provide greater insights regarding their co-evolution and ecology relative to bats.

Discovery of three cycloviruses in fecal samples from silver-haired bats (Lasionycteris noctivagans) in Arizona (USA).

Bats harbour a diverse array of viruses, some of which are zoonotic, and are one of the most speciose groups of mammals on earth. As part of an ongoing bat-associated viral diversity research project, we identified three cycloviruses (family Circoviridae) in fecal samples of silver-haired bats (Lasionycteris noctivagans) caught in Cave Creek Canyon of Arizona (USA). Two of the three identified genomes represent two new species in the genus Cyclovirus. Cycloviruses have been found in a wide range of environments and hosts; however, little is known about their biology. These new genomes of cycloviruses are the first from silver-haired bats, adding to the broader knowledge of cyclovirus diversity. With continuing studies, it is likely that additional viruses of the family Circoviridae will be identified in Arizona bat populations.

Genomic epidemiology of the first two waves of SARS-CoV-2 in Canada.

Tracking the emergence and spread of SARS-CoV-2 lineages using phylogenetics has proven critical to inform the timing and stringency of COVID-19 public health interventions. We investigated the effectiveness of international travel restrictions at reducing SARS-CoV-2 importations and transmission in Canada in the first two waves of 2020 and early 2021. Maximum likelihood phylogenetic trees were used to infer viruses' geographic origins, enabling identification of 2263 (95% confidence interval: 2159-2366) introductions, including 680 (658-703) Canadian sublineages, which are international introductions resulting in sampled Canadian descendants, and 1582 (1501-1663) singletons, introductions with no sampled descendants. Of the sublineages seeded during the first wave, 49% (46-52%) originated from the USA and were primarily introduced into Quebec (39%) and Ontario (36%), while in the second wave, the USA was still the predominant source (43%), alongside a larger contribution from India (16%) and the UK (7%). Following implementation of restrictions on the entry of foreign nationals on 21 March 2020, importations declined from 58.5 (50.4-66.5) sublineages per week to 10.3-fold (8.3-15.0) lower within 4 weeks. Despite the drastic reduction in viral importations following travel restrictions, newly seeded sublineages in summer and fall 2020 contributed to the persistence of COVID-19 cases in the second wave, highlighting the importance of sustained interventions to reduce transmission. Importations rebounded further in November, bringing newly emergent variants of concern (VOCs). By the end of February 2021, there had been an estimated 30 (19-41) B.1.1.7 sublineages imported into Canada, which increasingly displaced previously circulating sublineages by the end of the second wave.Although viral importations are nearly inevitable when global prevalence is high, with fewer importations there are fewer opportunities for novel variants to spark outbreaks or outcompete previously circulating lineages.

The molecular epidemiology of multiple zoonotic origins of SARS-CoV-2.

Understanding the circumstances that lead to pandemics is important for their prevention. We analyzed the genomic diversity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) early in the coronavirus disease 2019 (COVID-19) pandemic. We show that SARS-CoV-2 genomic diversity before February 2020 likely comprised only two distinct viral lineages, denoted "A" and "B." Phylodynamic rooting methods, coupled with epidemic simulations, reveal that these lineages were the result of at least two separate cross-species transmission events into humans. The first zoonotic transmission likely involved lineage B viruses around 18 November 2019 (23 October to 8 December), and the separate introduction of lineage A likely occurred within weeks of this event. These findings indicate that it is unlikely that SARS-CoV-2 circulated widely in humans before November 2019 and define the narrow window between when SARS-CoV-2 first jumped into humans and when the first cases of COVID-19 were reported. As with other coronaviruses, SARS-CoV-2 emergence likely resulted from multiple zoonotic events.

The Huanan Seafood Wholesale Market in Wuhan was the early epicenter of the COVID-19 pandemic.

Understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 is critical to preventing future zoonotic outbreaks before they become the next pandemic. The Huanan Seafood Wholesale Market in Wuhan, China, was identified as a likely source of cases in early reports, but later this conclusion became controversial. We show here that the earliest known COVID-19 cases from December 2019, including those without reported direct links, were geographically centered on this market. We report that live SARS-CoV-2-susceptible mammals were sold at the market in late 2019 and that within the market, SARS-CoV-2-positive environmental samples were spatially associated with vendors selling live mammals. Although there is insufficient evidence to define upstream events, and exact circumstances remain obscure, our analyses indicate that the emergence of SARS-CoV-2 occurred through the live wildlife trade in China and show that the Huanan market was the epicenter of the COVID-19 pandemic.

Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic.

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment.

Competent immune responses to SARS-CoV-2 variants in older adults following two doses of mRNA vaccination.

Aging is associated with a reduced magnitude of primary immune responses to vaccination. mRNA-based SARS-CoV-2 vaccines have shown efficacy in older adults but virus variant escape is still unclear. Here we analyze humoral and cellular immunity against an early-pandemic viral isolate and compare that to the P.1 (Gamma) and B.1.617.2 (Delta) variants in two cohorts (<50 and >55 age) of mRNA vaccine recipients. We further measure neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595, with the latter SARS-CoV-2 isolate bearing the spike mutation E484Q. Robust humoral immunity is measured following second vaccination, and older vaccinees manifest cellular immunity comparable to the adult group against early-pandemic SARS-CoV-2 and more recent variants. More specifically, the older cohort has lower neutralizing capacity at 7-14 days following the second dose but equilibrates with the younger cohort after 2-3 months. While long-term vaccination responses remain to be determined, our results implicate vaccine-induced protection in older adults against SARS-CoV-2 variants and inform thinking about boost vaccination.

Sensitive, smartphone-based SARS-CoV-2 detection from clinical saline gargle samples.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/µL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

Coevolutionary Analysis Implicates Toll-Like Receptor 9 in Papillomavirus Restriction.

Upon infection, DNA viruses can be sensed by pattern recognition receptors (PRRs), leading to the activation of type I and III interferons to block infection. Therefore, viruses must inhibit these signaling pathways, avoid being detected, or both. Papillomavirus virions are trafficked from early endosomes to the Golgi apparatus and wait for the onset of mitosis to complete nuclear entry. This unique subcellular trafficking strategy avoids detection by cytoplasmic PRRs, a property that may contribute to the establishment of infection. However, as the capsid uncoats within acidic endosomal compartments, the viral DNA may be exposed to detection by Toll-like receptor 9 (TLR9). In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we showed that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts. Finally, we demonstrated that the cancer-associated human papillomaviruses show a reduction in CpG dinucleotides within a TLR9 recognition complex. IMPORTANCE Viruses must avoid detection by the innate immune system. In this study, we characterized two new papillomaviruses from bats and used molecular archeology to demonstrate that their genomes altered their nucleotide compositions to avoid detection by TLR9, providing evidence that TLR9 acts as a PRR during papillomavirus infection. Furthermore, we demonstrated that TLR9, like other components of the innate immune system, is under evolutionary selection in bats, providing the first direct evidence for coevolution between papillomaviruses and their hosts.

Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip.

Respiratory viruses, especially coronaviruses, have resulted in worldwide pandemics in the past couple of decades. Saliva-based paper microfluidic assays represent an opportunity for noninvasive and rapid screening, yet both the sample matrix and test method come with unique challenges. In this work, we demonstrated the rapid and sensitive detection of SARS-CoV-2 from saliva samples, which could be simpler and more comfortable for patients than existing methods. Furthermore, we systematically investigated the components of saliva samples that affected assay performance. Using only a smartphone, an antibody-conjugated particle suspension, and a paper microfluidic chip, we made the assay user-friendly with minimal processing. Unlike the previously established flow rate assays that depended solely on the flow rate or distance, this unique assay analyzes the flow profile to determine infection status. Particle-target immunoagglutination changed the surface tension and subsequently the capillary flow velocity profile. A smartphone camera automatically measured the flow profile using a Python script, which was not affected by ambient light variations. The limit of detection (LOD) was 1 fg/µL SARS-CoV-2 from 1% saliva samples and 10 fg/µL from simulated saline gargle samples (15% saliva and 0.9% saline). This method was highly specific as demonstrated using influenza A/H1N1. The sample-to-answer assay time was <15 min, including <1-min capillary flow time. The overall accuracy was 89% with relatively clean clinical saline gargle samples. Despite some limitations with turbid clinical samples, this method presents a potential solution for rapid mass testing techniques during any infectious disease outbreak as soon as the antibodies become available.

Evolution and Diversity of Bat and Rodent Paramyxoviruses from North America.

Paramyxoviruses are a diverse group of negative-sense, single-stranded RNA viruses of which several species cause significant mortality and morbidity. In recent years the collection of paramyxovirus sequences detected in wild mammals has substantially grown; however, little is known about paramyxovirus diversity in North American mammals. To better understand natural paramyxovirus diversity, host range, and host specificity, we sought to comprehensively characterize paramyxoviruses across a range of diverse cooccurring wild small mammals in southern Arizona. We used highly degenerate primers to screen fecal and urine samples and obtained a total of 55 paramyxovirus sequences from 12 rodent species and 6 bat species. We also performed Illumina transcriptome sequencing (RNA-seq) and de novo assembly on 14 of the positive samples to recover a total of 5 near-full-length viral genomes. We show there are at least two clades of rodent-borne paramyxoviruses in Arizona, while bat-associated paramyxoviruses formed a putative single clade. Using structural homology modeling of the viral attachment protein, we infer that three of the five novel viruses likely bind sialic acid in a manner similar to other respiroviruses, while the other two viruses from heteromyid rodents likely bind a novel host receptor. We find no evidence for cross-species transmission, even among closely related sympatric host species. Taken together, these data suggest paramyxoviruses are a common viral infection in some bat and rodent species present in North America and illuminate the evolution of these viruses. IMPORTANCE There are a number of viral lineages that are potential zoonotic threats to humans. One of these, paramyxoviruses have jumped into humans multiple times from wild and domestic animals. We conducted one of the largest viral surveys of wild mammals in the United States to better understand paramyxovirus diversity and evolution.