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.

Aquaculture mediates global transmission of a viral pathogen to wild salmon.

Global expansion of aquaculture and agriculture facilitates disease emergence and catalyzes transmission to sympatric wildlife populations. The health of wild salmon stocks critically concerns Indigenous peoples, commercial and recreational fishers, and the general public. Despite potential impact of viral pathogens such as Piscine orthoreovirus-1 (PRV-1) on endangered wild salmon populations, their epidemiology in wild fish populations remains obscure, as does the role of aquaculture in global and local spread. Our phylogeographic analyses of PRV-1 suggest that development of Atlantic salmon aquaculture facilitated spread from Europe to the North and South East Pacific. Phylogenetic analysis and reverse transcription polymerase chain reaction surveillance further illuminate the circumstances of emergence of PRV-1 in the North East Pacific and provide strong evidence for Atlantic salmon aquaculture as a source of infection in wild Pacific salmon. PRV-1 is now an important infectious agent in critically endangered wild Pacific salmon populations, fueled by aquacultural transmission.

Variable routes to genomic and host adaptation among coronaviruses.

Natural selection operating on the genomes of viral pathogens in different host species strongly contributes to adaptation facilitating host colonization. Here, we analyse, quantify and compare viral adaptation in genomic sequence data derived from seven zoonotic events in the Coronaviridae family among primary, intermediate and human hosts. Rates of nonsynonymous (dN ) and synonymous (dS ) changes on specific amino acid positions were quantified for each open reading frame (ORF). Purifying selection accounted for 77% of all sites under selection. Diversifying selection was most frequently observed in viruses infecting the primary hosts of each virus and predominantly occurred in the orf1ab genomic region. Within all four intermediate hosts, diversifying selection on the spike gene was observed either solitarily or in combination with orf1ab and other genes. Consistent with previous evidence, pervasive diversifying selection on coronavirus spike genes corroborates the role this protein plays in host cellular entry, adaptation to new hosts and evasion of host cellular immune responses. Structural modelling of spike proteins identified a significantly higher proportion of sites for SARS-CoV-2 under positive selection in close proximity to sites of glycosylation relative to the other coronaviruses. Among human coronaviruses, there was a significant inverse correlation between the number of sites under positive selection and the estimated years since the virus was introduced into the human population. Abundant diversifying selection observed in SARS-CoV-2 suggests the virus remains in the adaptive phase of the host switch, typical of recent host switches. A mechanistic understanding of where, when and how genomic adaptation occurs in coronaviruses following a host shift is crucial for vaccine design, public health responses and predicting future pandemics.

Discovery and surveillance of viruses from salmon in British Columbia using viral immune-response biomarkers, metatranscriptomics, and high-throughput RT-PCR.

The emergence of infectious agents poses a continual economic and environmental challenge to aquaculture production, yet the diversity, abundance, and epidemiology of aquatic viruses are poorly characterised. In this study, we applied salmon host transcriptional biomarkers to identify and select fish in a viral disease state, but only those that were negative for known viruses based on RT-PCR screening. These fish were selected for metatranscriptomic sequencing to discover potential viral pathogens of dead and dying farmed Atlantic (Salmo salar) and Chinook (Oncorhynchus tshawytscha) salmon in British Columbia (BC). We found that the application of the biomarker panel increased the probability of discovering viruses in aquaculture populations. We discovered two viruses that have not previously been characterised in Atlantic salmon farms in BC (Atlantic salmon calicivirus and Cutthroat trout virus-2), as well as partially sequenced three putative novel viruses. To determine the epidemiology of the newly discovered or emerging viruses, we conducted high-throughput reverse transcription polymerase chain reaction (RT-PCR) and screened over 9,000 farmed and wild salmon sampled over one decade. Atlantic salmon calicivirus and Cutthroat trout virus-2 were in more than half of the farmed Atlantic salmon we tested. Importantly we detected some of the viruses we first discovered in farmed Atlantic salmon in Chinook salmon, suggesting a broad host range. Finally, we applied in situ hybridisation to determine infection and found differing cell tropism for each virus tested. Our study demonstrates that continual discovery and surveillance of emerging viruses in these ecologically important salmon will be vital for management of both aquaculture and wild resources in the future.

Coronaviruses in the Sea.

Interest in coronaviruses because of the 2019 novel coronavirus (SARS-CoV-2) pandemic has generated concern about their occurrence and persistence in aquatic habitats. Coronaviruses are not quantitatively significant constituents of marine virioplankton. Members of the Nidovirales (to which human coronaviruses belong) infect marine mammals, teleosts and possibly invertebrates, and human coronaviruses may persist in marine plankton receiving wastewater effluent. However, virions likely experience significant particle and infectivity decay rates in surface seawater, similar to other enveloped RNA viruses.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

Distribution and Phylogeny of Erythrocytic Necrosis Virus (ENV) in Salmon Suggests Marine Origin.

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10-8). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely.

ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees.

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006-2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

Novel RNA Virus Genome Discovered in Ghost Ants (Tapinoma melanocephalum) from Hawaii.

Here, we report the full-genome sequence of Milolii virus, a novel single-stranded (positive-sense) RNA virus discovered from Tapinoma melanocephalum ants in Hawaii. The genome is 10,475 nucleotides long, encoding a polyprotein of 3,304 amino acids.

A Comparison of Deformed Wing Virus in Deformed and Asymptomatic Honey Bees.

Deformed wing virus (DWV) in association with Varroa destructor is currently attributed to being responsible for colony collapse in the western honey bee (Apis mellifera). The appearance of deformed individuals within an infested colony has long been associated with colony losses. However, it is unknown why only a fraction of DWV positive bees develop deformed wings. This study concerns two small studies comparing deformed and non-deformed bees. In Brazil, asymptomatic bees (no wing deformity) that had been parasitised by Varroa as pupae had higher DWV loads than non-parasitised bees. However, we found no greater bilateral asymmetry in wing morphology due to DWV titres or parasitisation. As expected, using RT-qPCR, deformed bees were found to contain the highest viral loads. In a separate study, next generation sequencing (NGS) was applied to compare the entire DWV genomes from paired symptomatic and asymptomatic bees from three colonies on two different Hawaiian islands. This revealed no consistent differences between DWV genomes from deformed or asymptomatic bees, with the greatest variation seen between locations, not phenotypes. All samples, except one, were dominated by DWV type A. This small-scale study suggests that there is no unique genetic variant associated with wing deformity; but that many DWV variants have the potential to cause deformity.

Schrödinger's Cheshire Cat: Are Haploid Emiliania huxleyi Cells Resistant to Viral Infection or Not?

Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the "Cheshire Cat" escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger's cat; of being simultaneously both dead and alive.

Moku virus; a new Iflavirus found in wasps, honey bees and Varroa.

There is an increasing global trend of emerging infectious diseases (EIDs) affecting a wide range of species, including honey bees. The global epidemic of the single stranded RNA Deformed wing virus (DWV), driven by the spread of Varroa destructor has been well documented. However, DWV is just one of many insect RNA viruses which infect a wide range of hosts. Here we report the full genome sequence of a novel Iflavirus named Moku virus (MV), discovered in the social wasp Vespula pensylvanica collected in Hawaii. The novel genome is 10,056 nucleotides long and encodes a polyprotein of 3050 amino acids. Phylogenetic analysis showed that MV is most closely related to Slow bee paralysis virus (SBPV), which is highly virulent in honey bees but rarely detected. Worryingly, MV sequences were also detected in honey bees and Varroa from the same location, suggesting that MV can also infect other hymenopteran and Acari hosts.

Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies.

Treatment of emerging RNA viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies. Declining honey bee populations have been attributed to the ectoparasitic mite Varroa destructor and its affiliation with Deformed Wing Virus (DWV). In the current study we use next-generation sequencing to investigate the DWV quasispecies in an apiary known to suffer from overwintering colony losses. We show that the DWV species complex is made up of three master variants. Our results indicate that a new DWV Type C variant is distinct from the previously described types A and B, but together they form a distinct clade compared with other members of the Iflaviridae. The molecular clock estimation predicts that Type C diverged from the other variants ∼319 years ago. The discovery of a new master variant of DWV has important implications for the positive identification of the true pathogen within global honey bee populations.

Superinfection exclusion and the long-term survival of honey bees in Varroa-infested colonies.

Over the past 50 years, many millions of European honey bee (Apis mellifera) colonies have died as the ectoparasitic mite, Varroa destructor, has spread around the world. Subsequent studies have indicated that the mite's association with a group of RNA viral pathogens (Deformed Wing Virus, DWV) correlates with colony death. Here, we propose a phenomenon known as superinfection exclusion that provides an explanation of how certain A. mellifera populations have survived, despite Varroa infestation and high DWV loads. Next-generation sequencing has shown that a non-lethal DWV variant 'type B' has become established in these colonies and that the lethal 'type A' DWV variant fails to persist in the bee population. We propose that this novel stable host-pathogen relationship prevents the accumulation of lethal variants, suggesting that this interaction could be exploited for the development of an effective treatment that minimises colony losses in the future.