Porcine reproductive and respiratory syndrome virus genetic variability a management and diagnostic dilemma

As genetic analysis becomes less expensive, more comprehensive diagnostics such as whole genome sequencing (WGS) will become available to the veterinary practitioner. The WGS elucidates more about porcine reproductive and respiratory syndrome virus (PRRSV) beyond the traditional analysis of open reading frame (ORF) 5 Sanger sequencing. The veterinary practitioner will require a more complete understanding of the mechanics and consequences of PRRSV genetic variability to interpret the WGS results. More recently, PRRSV recombination events have been described in the literature. The objective of this review is to provide a comprehensive outlook for swine practitioners that PRRSV mutates and recombines naturally causing genetic variability, review the diagnostic cadence when suspecting recombination has occurred, and present theory on how, why, and where industry accepted management practices may influence recombination. As practitioners, it is imperative to remember that PRRS viral recombination is occurring continuously in swine populations. Finding a recombinant by diagnostic analysis does not ultimately declare its significance. The error prone replication, mutation, and recombination of PRRSV means exact clones may exist; but a quasispecies swarm of variable strains also exist adding to the genetic diversity. PRRSV nonstructural proteins (nsps) are translated from ORF1a and ORF1b. The arterivirus nsps modulate the hosts’ immune response and are involved in viral pathogenesis. The strains that contribute the PRRSV replicase and transcription complex is driving replication and possibly recombination in the quasispecies swarm. Furthermore, mutations favoring the virus to evade the immune system may result in the emergence of a more fit virus. More fit viruses tend to become the dominant strains in the quasispecies swarm. In theory, the swine management practices that may exacerbate or mitigate recombination include immunization strategies, swine movements, regional swine density, and topography. Controlling PRRSV equates to managing the quasispecies swarm and its interaction with the host. Further research is warranted on the frequency of recombination and the genome characteristics impacting the recombination rate. With a well-defined understanding of these characteristics, the clinical implications from recombination can be detected and potentially reduced; thus, minimizing recombination and perhaps the emergence of epidemic strains.

Unusual SARS-CoV-2 intra-host diversity reveals lineages superinfection

The SARS-CoV-2 has infected almost 200 million people worldwide by July 2021 and the pandemic has been characterized by infection waves of viral lineages showing distinct fitness profiles. The simultaneous infection of a single individual by two distinct SARS-CoV-2 lineages provides a window of opportunity for viral recombination and the emergence of new lineages with differential phenotype. Several hundred SARS-CoV-2 lineages are currently well characterized but two main factors have precluded major coinfection/codetection analysis thus far: i) the low diversity of SARS-CoV-2 lineages during the first year of the pandemic which limited the identification of lineage defining mutations necessary to distinguish coinfecting viral lineages; and the ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we have put together a large sequencing dataset from Brazilian samples covering a period of 18 May 2020 to 30 April 2021 and probed it for unexpected patterns of high intrasample/intrahost variability. It enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming their plausibility with the co-circulating lineages at the timeframe investigated. These coinfections represent around 0.61% of all samples investigated. Although our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, it is likely an underestimation and coinfection rates warrants further investigation.

Co-variation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa

Adaptation of viruses to their environments occurs through the acquisition of both novel Single-Nucleotide Variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

Pandemic-Scale Phylogenomics Reveals Elevated Recombination Rates in the SARS-CoV-2 Spike Region

Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses. During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral recombination. Low SARS-CoV-2 mutation rates make detecting recombination difficult. Here, we develop and apply a novel phylogenomic method to exhaustively search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. We investigate a 1.6M sample tree, and identify 606 recombination events. Approximately 2.7% of sequenced SARS-CoV-2 genomes have recombinant ancestry. Recombination breakpoints occur disproportionately in the Spike protein region. Our method empowers comprehensive real time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.

Biofilms and coronaviruses reservoirs: a perspective review

Bats are a key reservoir of coronaviruses (CoVs), including the agent of the severe acute respiratory syndrome, SARS-CoV-2, responsible for the recent deadly viral pneumonia pandemic. However, understanding how bats can harbor several microorganisms without developing illnesses is still a matter under discussion. Viruses and other pathogens are often studied as stand-alone even though it is known that, in nature, they mostly live in multi-species associations called biofilms - both externally and within the host. Microorganisms in biofilms are enclosed by an extracellular matrix that confers protection and improves survival. Previous studies have shown that viruses can secondarily colonize preexisting biofilms, and viral biofilms have also been already described. In this review, we raise the perspective that CoVs can persistently infect bats due to occurrence in biofilm structures. This phenomenon potentially provides an optimal environment for non-pathogenic and well-adapted viruses to interact with the host, as well as for viral recombination. Biofilms can also enhance virion viability in extracellular environments, such as in fomites and aquatic sediments, allowing viral persistence and dissemination. Moreover, understanding CoVs biofilm lifestyle in reservoirs might contribute to explain several burning questions that remain unanswered including persistence and transmissibility by highly pathogenic emerging CoVs.

Analysis of Synchronous and Asynchronous In Vitro Infections with Homologous Murine Norovirus Strains Reveals Time-Dependent Viral Interference Effects

Viral recombination is a key mechanism in the evolution and diversity of noroviruses. In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Here, we determine the effect of a temporal separation of in vitro infections with the two homologous murine norovirus strains MNV-1 WU20 and CW1 on the composition of nascent viral populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-h delays). Then, 24 h after initial co-or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows for the establishment of a barrier that reduces or prevents superinfection.

Early-Transmitted Variants and Their Evolution in a HIV-1 Positive Couple: NGS and Phylogenetic Analyses

We had access to both components of a couple who became infected with human immunodeficiency virus (HIV)-1 through sexual behavior during the early initial phase of infection and before initiation of therapy. We analyzed blood samples obtained at the time of diagnosis and after six months of combined antiretroviral therapy. Next-generation sequencing (NGS) and phylogenetic analyses were used to investigate the transmission and evolution of HIV-1 quasispecies. Phylogenetic analyses were conducted using Bayesian inference methods. Both partners were infected with an HIV-1 B subtype. No evidence of viral recombination was observed. The lowest intrapersonal genetic distances were observed at baseline, before initiation of therapy, and in particular in the V1V2 fragment (distances ranging from 0.102 to 0.148). One HIV-1 single variant was concluded to be dominant in all of the HIV-1 regions analyzed, although some minor variants could be observed. The same tree structure was observed both at baseline and after six months of therapy. These are the first extended phylogenetic analyses performed on both members of a therapy-naïve couple within a few weeks of infection, and in which the effect of antiretroviral therapy on viral evolution was analyzed. Understanding which HIV-1 variants are most likely to be transmitted would allow a better understanding of viral evolution, possibly playing a role in vaccine design and prevention strategies.

Global patterns of recombination across human viruses

Viral recombination is a major evolutionary mechanism driving adaptation processes, such as the ability of host-switching. Understanding global patterns of recombination could help to identify underlying mechanisms and to evaluate the potential risks of rapid adaptation. Conventional approaches (e.g. those based in Linkage Disequilibrium) are computationally demanding or even intractable when sequence alignments include hundreds of sequences, common in viral datasets. We present a comprehensive analysis of recombination across 30 genomic alignments from viruses infecting humans. In order to scale the analysis and avoid the computational limitations of conventional approaches, we apply newly-developed topological data analysis methods able to infer recombination rates for large datasets. We show that viruses such as ZEBOV and MARV consistently displayed low levels of recombination, while high levels of recombination were observed in Sarbecoviruses, HBV, HEV, Rhinovirus A and HIV. We observe that recombination is more common in positive single stranded RNA viruses than in negatively single stranded RNA ones. Interestingly, the comparison across multiple viruses suggests an inverse correlation between genome length and recombination rate. Positional analyses of recombination breakpoints along viral genomes, combined with our approach, detected at least 39 non-uniform patterns of recombination (i.e. cold or hotspots) in 18 viral groups. Among these, noteworthy hotspots are found in MERS-CoV and Sarbecoviruses (at spike, Nucleocapsid and ORF8). In summary, we have developed a fast pipeline to measure recombination that, combined with other approaches, has allowed us to find both common and lineage-specific patterns of recombination among viruses with potential relevance in viral adaptation.

The coronavirus proofreading exoribonuclease mediates extensive viral recombination

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.