Mutational signature dynamics indicate SARS-CoV-2's evolutionary capacity is driven by host antiviral molecules.

The COVID-19 pandemic has been characterised by sequential variant-specific waves shaped by viral, individual human and population factors. SARS-CoV-2 variants are defined by their unique combinations of mutations and there has been a clear adaptation to more efficient human infection since the emergence of this new human coronavirus in late 2019. Here, we use machine learning models to identify shared signatures, i.e., common underlying mutational processes and link these to the subset of mutations that define the variants of concern (VOCs). First, we examined the global SARS-CoV-2 genomes and associated metadata to determine how viral properties and public health measures have influenced the magnitude of waves, as measured by the number of infection cases, in different geographic locations using regression models. This analysis showed that, as expected, both public health measures and virus properties were associated with the waves of regional SARS-CoV-2 reported infection numbers and this impact varies geographically. We attribute this to intrinsic differences such as vaccine coverage, testing and sequencing capacity and the effectiveness of government stringency. To assess underlying evolutionary change, we used non-negative matrix factorisation and observed three distinct mutational signatures, unique in their substitution patterns and exposures from the SARS-CoV-2 genomes. Signatures 1, 2 and 3 were biased to C→T, T→C/A→G and G→T point mutations. We hypothesise assignments of these mutational signatures to the host antiviral molecules APOBEC, ADAR and ROS respectively. We observe a shift amidst the pandemic in relative mutational signature activity from predominantly Signature 1 changes to an increasingly high proportion of changes consistent with Signature 2. This could represent changes in how the virus and the host immune response interact and indicates how SARS-CoV-2 may continue to generate variation in the future. Linkage of the detected mutational signatures to the VOC-defining amino acids substitutions indicates the majority of SARS-CoV-2's evolutionary capacity is likely to be associated with the action of host antiviral molecules rather than virus replication errors.

SARS-CoV-2 in Domestic UK Cats from Alpha to Omicron: Swab Surveillance and Case Reports.

Although domestic cats are susceptible to infection with SARS-CoV-2, the role of the virus in causing feline disease is less well defined. We conducted a large-scale study to identify SARS-CoV-2 infections in UK pet cats, using active and passive surveillance. Remnant feline respiratory swab samples, submitted for other pathogen testing between May 2021 and February 2023, were screened using RT-qPCR. In addition, we appealed to veterinarians for swab samples from cats suspected of having clinical SARS-CoV-2 infections. Bespoke testing for SARS-CoV-2 neutralising antibodies was also performed, on request, in suspected cases. One RT-qPCR-positive cat was identified by active surveillance (1/549, 0.18%), during the Delta wave (1/175, 0.57%). Passive surveillance detected one cat infected with the Alpha variant, and two of ten cats tested RT-qPCR-positive during the Delta wave. No cats tested RT-qPCR-positive after the emergence of Omicron BA.1 and its descendants although 374 were tested by active and eleven by passive surveillance. We describe four cases of SARS-CoV-2 infection in pet cats, identified by RT-qPCR and/or serology, that presented with a range of clinical signs, as well as their SARS-CoV-2 genome sequences. These cases demonstrate that, although uncommon in cats, a variety of clinical signs can occur.

Direct Nanopore Sequencing of Human Cytomegalovirus Genomes from High-Viral-Load Clinical Samples.

Nanopore sequencing is becoming increasingly commonplace in clinical settings, particularly for diagnostic assessments and outbreak investigations, due to its portability, low cost, and ability to operate in near real-time. Although high sequencing error rates initially hampered the wider implementation of this technology, improvements have been made continually with each iteration of the sequencing hardware and base-calling software. Here, we assess the feasibility of using nanopore sequencing to determine the complete genomes of human cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a hybrid bioinformatic approach that involved assembling the reads de novo, improving the consensus sequence by aligning reads to the best-matching genome from a collated set of published sequences, and polishing the improved consensus sequence. The final genomes from a urine sample and a lung sample, the former with an HCMV to human DNA load approximately 50 times greater than the latter, achieved 99.97 and 99.93% identity, respectively, to the benchmark genomes obtained independently by Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of determining HCMV genomes directly from high-viral-load clinical samples with a high accuracy.

Evaluating the potential of whole-genome sequencing for tracing transmission routes in experimental infections and natural outbreaks of bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle. Genomic sequencing can resolve phylogenetic relationships between virus populations, which can be used to infer transmission routes and potentially inform the design of biosecurity measures. Sequencing of short (<2000 nt) segments of the 15 000-nt BRSV genome has revealed geographic and temporal clustering of BRSV populations, but insufficient variation to distinguish viruses collected from herds infected close together in space and time. This study investigated the potential for whole-genome sequencing to reveal sufficient genomic variation for inferring transmission routes between herds. Next-generation sequencing (NGS) data were generated from experimental infections and from natural outbreaks in Jämtland and Uppsala counties in Sweden. Sufficient depth of coverage for analysis of consensus and sub-consensus sequence diversity was obtained from 47 to 20 samples respectively. Few (range: 0-6 polymorphisms across the six experiments) consensus-level polymorphisms were observed along experimental transmissions. A much higher level of diversity (146 polymorphic sites) was found among the consensus sequences from the outbreak samples. The majority (144/146) of polymorphisms were between rather than within counties, suggesting that consensus whole-genome sequences show insufficient spatial resolution for inferring direct transmission routes, but might allow identification of outbreak sources at the regional scale. By contrast, within-sample diversity was generally higher in the experimental than the outbreak samples. Analyses to infer known (experimental) and suspected (outbreak) transmission links from within-sample diversity data were uninformative. In conclusion, analysis of the whole-genome sequence of BRSV from experimental samples discriminated between circulating isolates from distant areas, but insufficient diversity was observed between closely related isolates to aid local transmission route inference.

Recent changes to virus taxonomy ratified by the International Committee on Taxonomy of Viruses (2022).

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2022. The entire ICTV was invited to vote on 174 taxonomic proposals approved by the ICTV Executive Committee at its annual meeting in July 2021. All proposals were ratified by an absolute majority of the ICTV members. Of note, the Study Groups have started to implement the new rule for uniform virus species naming that became effective in 2021 and mandates the binomial 'Genus_name species_epithet' format with or without Latinization. As a result of this ratification, the names of 6,481 virus species (more than 60 percent of all species names currently recognized by ICTV) now follow this format.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Quantifying and Cataloguing Unknown Sequences within Human Microbiomes.

Advances in genome sequencing technologies and lower costs have enabled the exploration of a multitude of known and novel environments and microbiomes. This has led to an exponential growth in the raw sequence data that are deposited in online repositories. Metagenomic and metatranscriptomic data sets are typically analysed with regard to a specific biological question. However, it is widely acknowledged that these data sets are comprised of a proportion of sequences that bear no similarity to any currently known biological sequence, and this so-called "dark matter" is often excluded from downstream analyses. In this study, a systematic framework was developed to assemble, identify, and measure the proportion of unknown sequences present in distinct human microbiomes. This framework was applied to 40 distinct studies, comprising 963 samples, and covering 10 different human microbiomes including fecal, oral, lung, skin, and circulatory system microbiomes. We found that while the human microbiome is one of the most extensively studied, on average 2% of assembled sequences have not yet been taxonomically defined. However, this proportion varied extensively among different microbiomes and was as high as 25% for skin and oral microbiomes that have more interactions with the environment. A rate of taxonomic characterization of 1.64% of unknown sequences being characterized per month was calculated from these taxonomically unknown sequences discovered in this study. A cross-study comparison led to the identification of similar unknown sequences in different samples and/or microbiomes. Both our computational framework and the novel unknown sequences produced are publicly available for future cross-referencing. Our approach led to the discovery of several novel viral genomes that bear no similarity to sequences in the public databases. Some of these are widespread as they have been found in different microbiomes and studies. Hence, our study illustrates how the systematic characterization of unknown sequences can help the discovery of novel microbes, and we call on the research community to systematically collate and share the unknown sequences from metagenomic studies to increase the rate at which the unknown sequence space can be classified.

Selection analysis identifies unusual clustered mutational changes in Omicron lineage BA.1 that likely impact Spike function.

Among the 30 non-synonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (i) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (ii) interactions of Spike with ACE2 receptors, and (iii) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any genomes within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron over all previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

The antiviral state has shaped the CpG composition of the vertebrate interferome to avoid self-targeting.

Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2.

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.

Changes to virus taxonomy and to the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2021).

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2021. The entire ICTV was invited to vote on 290 taxonomic proposals approved by the ICTV Executive Committee at its meeting in October 2020, as well as on the proposed revision of the International Code of Virus Classification and Nomenclature (ICVCN). All proposals and the revision were ratified by an absolute majority of the ICTV members. Of note, ICTV mandated a uniform rule for virus species naming, which will follow the binomial 'genus-species' format with or without Latinized species epithets. The Study Groups are requested to convert all previously established species names to the new format. ICTV has also abolished the notion of a type species, i.e., a species chosen to serve as a name-bearing type of a virus genus. The remit of ICTV has been clarified through an official definition of 'virus' and several other types of mobile genetic elements. The ICVCN and ICTV Statutes have been amended to reflect these changes.

Detection of SARS-CoV-2 in respiratory samples from cats in the UK associated with human-to-cat transmission.

OBJECTIVES: The aim of the study was to find evidence of SARS-CoV-2 infection in UK cats. DESIGN: Tissue samples were tested for SARS-CoV-2 antigen using immunofluorescence and for viral RNA by in situ hybridisation. A set of 387 oropharyngeal swabs that had been submitted for routine respiratory pathogen testing was tested for SARS-CoV-2 RNA using reverse transcriptase quantitative PCR. RESULTS: Lung tissue collected post-mortem from cat 1 tested positive for both SARS-CoV-2 nucleocapsid antigen and RNA. SARS-CoV-2 RNA was detected in an oropharyngeal swab collected from cat 2 that presented with rhinitis and conjunctivitis. High throughput sequencing of the viral genome revealed five single nucleotide polymorphisms (SNPs) compared to the nearest UK human SARS-CoV-2 sequence, and this human virus contained eight SNPs compared to the original Wuhan-Hu-1 reference sequence. An analysis of the viral genome of cat 2 together with nine other feline-derived SARS-CoV-2 sequences from around the world revealed no shared cat-specific mutations. CONCLUSIONS: These findings indicate that human-to-cat transmission of SARS-CoV-2 occurred during the COVID-19 pandemic in the UK, with the infected cats developing mild or severe respiratory disease. Given the ability of the new coronavirus to infect different species, it will be important to monitor for human-to-cat, cat-to-cat and cat-to-human transmission.

Characterizing and Evaluating the Zoonotic Potential of Novel Viruses Discovered in Vampire Bats.

The contemporary surge in metagenomic sequencing has transformed knowledge of viral diversity in wildlife. However, evaluating which newly discovered viruses pose sufficient risk of infecting humans to merit detailed laboratory characterization and surveillance remains largely speculative. Machine learning algorithms have been developed to address this imbalance by ranking the relative likelihood of human infection based on viral genome sequences, but are not yet routinely applied to viruses at the time of their discovery. Here, we characterized viral genomes detected through metagenomic sequencing of feces and saliva from common vampire bats (Desmodus rotundus) and used these data as a case study in evaluating zoonotic potential using molecular sequencing data. Of 58 detected viral families, including 17 which infect mammals, the only known zoonosis detected was rabies virus; however, additional genomes were detected from the families Hepeviridae, Coronaviridae, Reoviridae, Astroviridae and Picornaviridae, all of which contain human-infecting species. In phylogenetic analyses, novel vampire bat viruses most frequently grouped with other bat viruses that are not currently known to infect humans. In agreement, machine learning models built from only phylogenetic information ranked all novel viruses similarly, yielding little insight into zoonotic potential. In contrast, genome composition-based machine learning models estimated different levels of zoonotic potential, even for closely related viruses, categorizing one out of four detected hepeviruses and two out of three picornaviruses as having high priority for further research. We highlight the value of evaluating zoonotic potential beyond ad hoc consideration of phylogeny and provide surveillance recommendations for novel viruses in a wildlife host which has frequent contact with humans and domestic animals.

Diversification of mammalian deltaviruses by host shifting.

Hepatitis delta virus (HDV) is an unusual RNA agent that replicates using host machinery but exploits hepatitis B virus (HBV) to mobilize its spread within and between hosts. In doing so, HDV enhances the virulence of HBV. How this seemingly improbable hyperparasitic lifestyle emerged is unknown, but it underpins the likelihood that HDV and related deltaviruses may alter other host-virus interactions. Here, we show that deltaviruses diversify by transmitting between mammalian species. Among 96,695 RNA sequence datasets, deltaviruses infected bats, rodents, and an artiodactyl from the Americas but were absent from geographically overrepresented Old World representatives of each mammalian order, suggesting a relatively recent diversification within the Americas. Consistent with diversification by host shifting, both bat and rodent-infecting deltaviruses were paraphyletic, and coevolutionary modeling rejected cospeciation with mammalian hosts. In addition, a 2-y field study showed common vampire bats in Peru were infected by two divergent deltaviruses, indicating multiple introductions to a single host species. One vampire bat-associated deltavirus was detected in the saliva of up to 35% of individuals, formed phylogeographically compartmentalized clades, and infected a sympatric bat, illustrating horizontal transmission within and between species on ecological timescales. Consistent absence of HBV-like viruses in two deltavirus-infected bat species indicated acquisitions of novel viral associations during the divergence of bat and human-infecting deltaviruses. Our analyses support an American zoonotic origin of HDV and reveal prospects for future cross-species emergence of deltaviruses. Given their peculiar life history, deltavirus host shifts will have different constraints and disease outcomes compared to ordinary animal pathogens.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.

Population genomics of louping ill virus provide new insights into the evolution of tick-borne flaviviruses.

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors.

No evidence for distinct types in the evolution of SARS-CoV-2.

A recent study by Tang et al. claimed that two major types of severe acute respiratory syndrome-coronavirus-2 (CoV-2) had evolved in the ongoing CoV disease-2019 pandemic and that one of these types was more 'aggressive' than the other. Given the repercussions of these claims and the intense media coverage of these types of articles, we have examined in detail the data presented by Tang et al., and show that the major conclusions of that paper cannot be substantiated. Using examples from other viral outbreaks, we discuss the difficulty in demonstrating the existence or nature of a functional effect of a viral mutation, and we advise against overinterpretation of genomic data during the pandemic.