Diversity Of Virus Populations Research Articles

Infectious bronchitis virus: an overview of the "chicken coronavirus".

Infectious bronchitis virus (IBV) is a highly contagious avian Gammacoronavirus that affects mainly chickens (Gallus gallus) but can circulate in other avian species. IBV constitutes a significant threat to the poultry industry, causing reduced egg yield, growth and mortality levels that can vary in impact. The virus can be transmitted horizontally by inhalation or direct/indirect contact with infected birds or contaminated fomites, vehicles, farm personnel and litter (Figure 1). The error-prone viral polymerase and recombination mechanisms mean diverse viral population results, with multiple genotypes, serotypes, pathotypes and protectotypes. This significantly complicates control and mitigation strategies based on vigilance in biosecurity and the deployment of vaccination.

Exploring the gut DNA virome in fecal immunochemical test stool samples reveals associations with lifestyle in a large population-based study

Stool samples for fecal immunochemical tests (FIT) are collected in large numbers worldwide as part of colorectal cancer screening programs. Employing FIT samples from 1034 CRCbiome participants, recruited from a Norwegian colorectal cancer screening study, we identify, annotate and characterize more than 18000 DNA viruses, using shotgun metagenome sequencing. Only six percent of them are assigned to a known taxonomic family, with Microviridae being the most prevalent viral family. Linking individual profiles to comprehensive lifestyle and demographic data shows 17/25 of the variables to be associated with the gut virome. Physical activity, smoking, and dietary fiber consumption exhibit strong and consistent associations with both diversity and relative abundance of individual viruses, as well as with enrichment for auxiliary metabolic genes. We demonstrate the suitability of FIT samples for virome analysis, opening an opportunity for large-scale studies of this enigmatic part of the gut microbiome. The diverse viral populations and their connections to the individual lifestyle uncovered herein paves the way for further exploration of the role of the gut virome in health and disease.

A broad wastewater screening and clinical data surveillance for virus-related diseases in the metropolitan Detroit area in Michigan

BackgroundPeriodic bioinformatics-based screening of wastewater for assessing the diversity of potential human viral pathogens circulating in a given community may help to identify novel or potentially emerging infectious diseases. Any identified contigs related to novel or emerging viruses should be confirmed with targeted wastewater and clinical testing.ResultsDuring the COVID-19 pandemic, untreated wastewater samples were collected for a 1-year period from the Great Lakes Water Authority Wastewater Treatment Facility in Detroit, MI, USA, and viral population diversity from both centralized interceptor sites and localized neighborhood sewersheds was investigated. Clinical cases of the diseases caused by human viruses were tabulated and compared with data from viral wastewater monitoring. In addition to Betacoronavirus, comparison using assembled contigs against a custom Swiss-Prot human virus database indicated the potential prevalence of other pathogenic virus genera, including: Orthopoxvirus, Rhadinovirus, Parapoxvirus, Varicellovirus, Hepatovirus, Simplexvirus, Bocaparvovirus, Molluscipoxvirus, Parechovirus, Roseolovirus, Lymphocryptovirus, Alphavirus, Spumavirus, Lentivirus, Deltaretrovirus, Enterovirus, Kobuvirus, Gammaretrovirus, Cardiovirus, Erythroparvovirus, Salivirus, Rubivirus, Orthohepevirus, Cytomegalovirus, Norovirus, and Mamastrovirus. Four nearly complete genomes were recovered from the Astrovirus, Enterovirus, Norovirus and Betapolyomavirus genera and viral species were identified.ConclusionsThe presented findings in wastewater samples are primarily at the genus level and can serve as a preliminary “screening” tool that may serve as indication to initiate further testing for the confirmation of the presence of species that may be associated with human disease. Integrating innovative environmental microbiology technologies like metagenomic sequencing with viral epidemiology offers a significant opportunity to improve the monitoring of, and predictive intelligence for, pathogenic viruses, using wastewater.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Climate change and vector-borne diseases: a multi-omics approach of temperature-induced changes in the mosquito.

Climate change and globalization contribute to the expansion of mosquito vectors and their associated pathogens. Long spared, temperate regions have had to deal with the emergence of arboviruses traditionally confined to tropical regions. Chikungunya virus (CHIKV) was reported for the first time in Europe in 2007, causing a localized outbreak in Italy, which then recurred repeatedly over the years in other European localities. This raises the question of climate effects, particularly temperature, on the dynamics of vector-borne viruses. The objective of this study is to improve the understanding of the molecular mechanisms set up in the vector in response to temperature. We combine three complementary approaches by examining Aedes albopictus mosquito gene expression (transcriptomics), bacterial flora (metagenomics) and CHIKV evolutionary dynamics (genomics) induced by viral infection and temperature changes. We show that temperature alters profoundly mosquito gene expression, bacterial microbiome and viral population diversity. We observe that (i) CHIKV infection upregulated most genes (mainly in immune and stress-related pathways) at 20°C but not at 28°C, (ii) CHIKV infection significantly increased the abundance of Enterobacteriaceae Serratia marcescens at 28°C and (iii) CHIKV evolutionary dynamics were different according to temperature. The substantial changes detected in the vectorial system (the vector and its bacterial microbiota, and the arbovirus) lead to temperature-specific adjustments to reach the ultimate goal of arbovirus transmission; at 20°C and 28°C, the Asian tiger mosquito Ae. albopictus was able to transmit CHIKV at the same efficiency. Therefore, CHIKV is likely to continue its expansion in the northern regions and could become a public health problem in more countries than those already affected in Europe.

Coliphages of the human urinary microbiota.

Due to its frequent association with urinary tract infections (UTIs), Escherichia coli is the best characterized constituent of the urinary microbiota (urobiome). However, uropathogenic E. coli is just one member of the urobiome. In addition to bacterial constituents, the urobiome of both healthy and symptomatic individuals is home to a diverse population of bacterial viruses (bacteriophages). A prior investigation found that most bacterial species in the urobiome are lysogens, harboring one or more phages integrated into their genome (prophages). Many of these prophages are temperate phages, capable of entering the lytic cycle and thus lysing their bacterial host. This transition from the lysogenic to lytic life cycle can impact the bacterial diversity of the urobiome. While many phages that infect E. coli (coliphages) have been studied for decades in the laboratory setting, the coliphages within the urobiome have yet to be cataloged. Here, we investigated the diversity of urinary coliphages by first identifying prophages in all publicly available urinary E. coli genomes. We detected 3,038 intact prophage sequences, representative of 1,542 unique phages. These phages include both novel species as well as species also found within the gut microbiota. Ten temperate phages were isolated from urinary E. coli strains included in our analysis, and we assessed their ability to infect and lyse urinary E. coli strains. We also included in these host range assays other urinary coliphages and laboratory coliphages. The temperate phages and other urinary coliphages were successful in lysing urinary E. coli strains. We also observed that coliphages from non-urinary sources were most efficient in killing urinary E. coli strains. The two phages, T2 and N4, were capable of lysing 83.5% (n = 86) of strains isolated from females with UTI symptoms. In conclusion, our study finds a diverse community of coliphages in the urobiome, many of which are predicted to be temperate phages, ten of which were confirmed here. Their ability to infect and lyse urinary E. coli strains suggests that urinary coliphages may play a role in modulating the E. coli strain diversity of the urobiome.

Abstract 3013: The immunogenetic basis of hepatitis B virus-related hepatocellular carcinoma

Abstract Background: The reported associations of genetic variants within the human leukocyte antigen (HLA) region with hepatis B virus (HBV)-related hepatocellular carcinoma (HCC) are not consistent, and the structure-function patterns involved in this disease remain unclear. Methods: Utilizing genotype data from 706 HBV-related HCC cases and 6197 chronic HBV carriers without HCC collected from three resources in Taiwan, we imputed the classical HLA alleles at 2-field resolution and analyzed the molecular landscape of HLA complexes by deeply dissecting their genotypic configurations, functional divergence, and HBV-antigen binding capabilities in associations with the HCC risk. Logistic regression models were used, and all models were adjusted for sex, age, and population structure using the top 10 principal components. We also tested the underlying biological implications of our findings by investigating the HLA binding affinities and HLA effect on control of viral replication (i.e, HBV DNA load), viral population diversity (i.e, quasispecies), and inflammation (i.e, chemokines, cytokines, and soluble programmed cell death 1 [sPD-1]-associated T cell exhaustion). Results: We found high accuracy (≥95% concordance) of HLA imputation at all HLA class I and class II alleles. The strongest evidence for an association with HCC was observed for HLA-DQB1*03:01 (adjusted OR= 1.35 in an additive genetic model, P=1.3×10-6). Three amino acids at specific positions within or near the peptide-binding groove of DQB1 molecules might explain the observed allele association, which leads to loss of binding HBV nucleocapsid protein (i.e., low binding affinity predicted by NetMHCIIpan 4.0 for HLA-DQB1*03:01). HLA-DQB1*03:01 was significantly associated with a higher HBV DNA load (P= 4.3×10-4) and an increased level of serum sPD-1 (P=6.8×10-4), but not with increased levels of chemokines, cytokines, and viral complexity (i.e., Shannon entropy for HBV quasispecies, all P values>0.05). Analysis of HLA zygosity confirmed that HLA-DQB1 heterozygosity was associated with a decreased risk of HCC (adjusted OR=0.77, P= 0.015). Analysis of HLA evolutionary divergence (HED) showed that high HED at HLA-DRB1 loci was marginally associated with a decreased risk of HCC among HLA-DQB1*03:01 non-carriers (P=0.05). Conclusion: Our data shed light on the immunogenetic risk associated with HBV-related HCC. The effect of HLA-DQB1*03:01 could be partly explained by its low binding affinity with HBV nucleocapsid antigen, and/or its effect on viral load control and sPD-1, but not by its control on other inflammation markers (chemokines and cytokines) and viral population diversity. The association between HLA-DQB1 heterozygosity and HCC risk could be independent of the HLA-DQB1*03:01 effect, which indicates underlying mechanisms related to increased immunosurveillance in clearing potential viral antigens or neoantigens. Citation Format: Zhiwei Liu, Chih-Jen Huang, Yu-Han Huang, Mei-Hung Pan, Mei-Hsuan Lee, Mathias Viard, Allan Hildesheim, Ruth M. Pfeiffer, Mary Carrington, Chien-Jen Chen, Tobias L. Lenz, Hwai-I Yang. The immunogenetic basis of hepatitis B virus-related hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3013.

Cencurut virus: A novel Orthonairovirus from Asian house shrews (Suncus murinus) in Singapore

Orthonairovirus is a genus of viruses in the family Nairoviridae, order Bunyavirales, with a segmented circular RNA genome. They typically infect birds and mammals and are primarily transmitted by ectoparasites such as ticks. Four of nine Orthonairovirus genogroups can infect humans, with Crimean-Congo hemorrhagic fever virus infections displaying case fatality rates up to 40%. Here, we discover and describe a novel Orthonairovirus as Cencurut virus (CENV). CENV was detected in 34 of 37 Asian house shrews (Suncus murinus) sampled in Singapore and in a nymphal Amblyomma helvolum tick collected from an infected shrew. Pairwise comparison of CENV S, M, and L segments had 95.0 to 100% nucleotide and 97.5 to 100% amino acid homology within CENV genomes, suggesting a diverse viral population. Phylogenetic analysis of the individual gene segments showed that CENV is related to Erve, Lamgora, Lamusara, and Thiafora viruses, with only 49.0 to 58.2% nucleotide and 41.7 to 61.1% amino acid homology, which has previously been detected in other shrew species from France, Gabon, and Senegal respectively. The high detection frequency suggests that CENV is endemic among S. murinus populations in Singapore. The discovery of CENV, from a virus family with known zoonotic potential, underlines the importance of surveillance of synanthropic small mammals that are widely distributed across Southeast Asia.

Evolutional transition of HBV genome during the persistent infection determined by single-molecule real-time sequencing.

Although HBV infection is a serious health issue worldwide, the landscape of HBV genome dynamics in the host has not yet been clarified. This study aimed to determine the continuous genome sequence of each HBV clone using a single-molecule real-time sequencing platform, and clarify the dynamics of structural abnormalities during persistent HBV infection without antiviral therapy. Twenty-five serum specimens were collected from 10 untreated HBV-infected patients. Continuous whole-genome sequencing of each clone was performed using a PacBio Sequel sequencer; the relationship between genomic variations and clinical information was analyzed. The diversity and phylogeny of the viral clones with structural variations were also analyzed. The whole-genome sequences of 797,352 HBV clones were determined. The deletion was the most common structural abnormality and concentrated in the preS/S and C regions. Hepatitis B e antibody (anti-HBe)-negative samples or samples with high alanine aminotransferase levels have significantly diverse deletions than anti-HBe-positive samples or samples with low alanine aminotransferase levels. Phylogenetic analysis demonstrated that various defective and full-length clones evolve independently and form diverse viral populations. Single-molecule real-time long-read sequencing revealed the dynamics of genomic quasispecies during the natural course of chronic HBV infections. Defective viral clones are prone to emerge under the condition of active hepatitis, and several types of defective variants can evolve independently of the viral clones with the full-length genome.

Superinfection exclusion creates spatially distinct influenza virus populations.

Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.

P915 Uncovering the role of Archaea in the pathogenesis of Inflammatory bowel disease

Abstract Background Microbial dysbiosis is a major factor that contributes to the course of Inflammatory Bowel Disease (IBD), a complex class of relapsing-remitting chronic inflammatory diseases. The body's overall homeostasis is significantly influenced by the human microbiota, which is a diverse population of bacteria, viruses, fungi, protists, and archaea. Recent research shows that archaea can affect both health and disease, by directly interacting with the host as well as other members of the microbial community. Although the role of bacteria, viruses, and fungi has already been studied, the archaeome remains the under-discovered part of the gut microbiota, especially in the context of IBD. Methods In this study, we profiled the gut archaea using archaeal 16S rRNA gene region in a cohort of pediatric patients with different forms of IBD including Ulcerative colitis (UC), Crohn’s disease (CD), unclassified IBD (IBDU) and healthy controls. We determined the compositional, diversity, and taxonomic alterations of archaea in pediatric patients with IBD and compared to healthy controls. Results Euryarchaeota, and Crenarchaeota, were the major archaeal phyla identified in our samples, other minor phyla identified were Halobacterota and Thermoplasmatota. At the genus level, Methanobrevibacter and Methanosphaera were the most abundant archaea, respectively. Overall, we did not observe any significant difference in the archeal alpha diversity within the various groups. There was however a significant difference between the overall community structure of the archaea between controls and IBD patients (p<0.05). Conclusion A thorough understanding of the role played by the archaea during the pathogenesis of IBD may open the door to the identification of new processes and, ultimately, the development of creative therapeutic targets that can complement the currently used treatments for IBD patients.

Genetic diversity of the dengue virus population in dengue fever and dengue hemorrhagic fever patients.

The error-prone replication of dengue virus (DENV) in host results in the highly diverse viral population. Together with the host factor, intra-host diversity may influence the disease severity. Therefore, it is worth investigating whether there is a correlation between intra-host genetic diversity and disease severity. To investigate the genetic diversity in DENV for four serotypes of the dengue population from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) using next-generation sequencing (NGS) technology. Forty RNA samples categorized into eight groups by severity and serotypes were sequenced and analyzed for genetic variation. Analysis on the hot-cold genomic regions, selection pressure and correlation between genotype and disease severity were performed in this study. Comparison between the NGS data of the DF and DHF specimens showed conservation between their major populations with the consensus sequences for DF and DHF sharing 99% similarity. However, the minor populations in DF and DHF were more diverse. Many genes in DF had an #NS/#S ratio higher than in DHF. Only NS4B of DENV1 DF has #NS/#S ratio higher than one. Hot regions of the DF were detected in NS3 of DENV1, DENV2 and Envelope of DENV3, whereas the hot regions of the DHF samples were detected in the small region in 3'UTR of DENV2 and DENV3. Various explorations of the variations of DF and DHF were performed in this study. However, we have not yet found any specific characteristics of intra-host diversity associated with disease severity.

Genetic Diversity of Viral Populations Associated with Ananas Germplasm and Improvement of Virus Diagnostic Protocols.

Pineapple (Ananas comosus L. [Merr.]) accessions from the U.S. Tropical Plant Genetic Resources and Disease Research (TPGRDR) in Hilo, Hawaii were subjected to RNA-sequencing to study the occurrence of viral populations associated with this vegetatively propagated crop. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named "pineapple secovirus C" (PSV-C) and "pineapple secovirus D" (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7485 bp) corresponding to a previously reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing ten members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events across the quintuple gene block, while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an important advance for surveillance of viral infections of pineapple.

Imaging and biopsy of HIV-infected individuals undergoing analytic treatment interruption.

BackgroundHIV persistence during antiretroviral therapy (ART) is the principal obstacle to cure. Lymphoid tissue is a compartment for HIV, but mechanisms of persistence during ART and viral rebound when ART is interrupted are inadequately understood. Metabolic activity in lymphoid tissue of patients on long-term ART is relatively low, and increases when ART is stopped. Increases in metabolic activity can be detected by 18F-fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and may represent sites of HIV replication or immune activation in response to HIV replication.MethodsFDG-PET imaging will be used to identify areas of high and low metabolic uptake in lymphoid tissue of individuals undergoing long-term ART. Baseline tissue samples will be collected. Participants will then be randomized 1:1 to continue or interrupt ART via analytic treatment interruption (ATI). Image-guided biopsy will be repeated 10 days after ATI initiation. After ART restart criteria are met, image-guided biopsy will be repeated once viral suppression is re-achieved. Participants who continued ART will have a second FDG-PET and biopsies 12–16 weeks after the first. Genetic characteristics of HIV populations in areas of high and low FDG uptake will be assesed. Optional assessments of non-lymphoid anatomic compartments may be performed to evaluate HIV populations in distinct anatomic compartments.Anticipated resultsWe anticipate that PET standardized uptake values (SUV) will correlate with HIV viral RNA in biopsies of those regions and that lymph nodes with high SUV will have more viral RNA than those with low SUV within a patient. Individuals who undergo ATI are expected to have diverse viral populations upon viral rebound in lymphoid tissue. HIV populations in tissues may initially be phylogenetically diverse after ATI, with emergence of dominant viral species (clone) over time in plasma. Dominant viral species may represent the same HIV population seen before ATI.DiscussionThis study will allow us to explore utility of PET for identification of HIV infected cells and determine whether high FDG uptake respresents areas of HIV replication, immune activation or both. We will also characterize HIV infected cell populations in different anatomic locations. The protocol will represent a platform to investigate persistence and agents that may target HIV populations.Study protocol registrationIdentifier: NCT05419024.

Quantitative and Qualitative Changes in the Deformed Wing Virus Population in Honey Bees Associated with the Introduction or Removal of Varroa destructor.

Varroa destructor is an ectoparasitic mite associated with significant losses of honeybee colonies globally. The mite vectors a range of pathogenic viruses, the most important of which is the Deformed wing virus (DWV). In the absence of Varroa, DWV exists as a low-level, highly diverse virus population. However, when transmitted by Varroa, certain variants become highly elevated, and may become near-clonal and cause symptomatic infections. Mite transmission between colonies can occur when parasitised workers drift from or rob adjacent hives. These activities can result in elevated mite levels, but the resulting change in the DWV population, the primary determinant of winter colony losses, has not been determined. In reciprocal studies, we investigated the influence of the removal of mites, or their acquisition, on the DWV population. When mites were removed from heavily infested colonies, there was a striking and rapid reduction in virus load. Conversely, siting Varroa-naïve colonies in a mite-infested apiary resulted in the acquisition of mites and concomitant changes in the virus population. We observed both near-clonal and highly divergent virus populations regardless of titre, suggesting changes were stochastic and colony-specific. Our findings have implications for the outcome of strategies in areas with total or patchy implementation of Varroa control plans.

Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest

Alfalfa (Medicago sativa L.) is one of the most extensively cultivated forage legumes in the world. It is currently the third most valuable field crop in the United States with an estimated value of over $9.3 billion. Alfalfa productivity is limited by various infectious diseases that can reduce forage yield and quality and shorten stand life. The crop can frequently be infected with a diverse array of pathogens and other organisms that have distinct life cycles, biology, and mode of action. Among them are many coinfecting viruses, that greatly contribute to the heterogeneity of within-host pathogenic communities, representing a ubiquitous and abundant background for all other host–pathogen interactions. Regrettably, the impact of viral diseases, their role in alfalfa health and involvement in the severity of multi-pathogen infections are often underestimated and not well understood. As high-throughput sequencing approaches have been developed, opportunities to delve into these complex interactions can be realized. In this work, we have characterized a diversity of viral populations in several commercial alfalfa production fields located in the U.S. Pacific Northwest. At least 45 distinct viruses have been identified in all alfalfa samples. Among them some were known to infect the crop prior to this study, and others were designated as emerging, novel and viruses integrated into the alfalfa genome. Known viruses included alfalfa mosaic virus, pea streak virus and bean leafroll virus, while among emerging and novel agents were alfalfa virus S, cherry virus Trakiya, several rhabdoviruses and others. Additional biological and impact studies will be needed to determine if newly identified viruses, especially those that have not been reported from alfalfa before, should be considered pathogens of this crop.

Viral Population Diversity during Co-Infection of Foot-And-Mouth Disease Virus Serotypes SAT1 and SAT2 in African Buffalo in Kenya.

African buffalo are the natural reservoirs of the SAT serotypes of foot-and-mouth disease virus (FMDV) in sub-Saharan Africa. Most buffalo are exposed to multiple FMDV serotypes early in life, and a proportion of them become persistently infected carriers. Understanding the genetic diversity and evolution of FMDV in carrier animals is critical to elucidate how FMDV persists in buffalo populations. In this study, we obtained oropharyngeal (OPF) fluid from naturally infected African buffalo, and characterized the genetic diversity of FMDV. Out of 54 FMDV-positive OPF, 5 were co-infected with SAT1 and SAT2 serotypes. From the five co-infected buffalo, we obtained eighty-nine plaque-purified isolates. Isolates obtained directly from OPF and plaque purification were sequenced using next-generation sequencing (NGS). Phylogenetic analyses of the sequences obtained from recombination-free protein-coding regions revealed a discrepancy in the topology of capsid proteins and non-structural proteins. Despite the high divergence in the capsid phylogeny between SAT1 and SAT2 serotypes, viruses from different serotypes that were collected from the same host had a high genetic similarity in non-structural protein-coding regions P2 and P3, suggesting interserotypic recombination. In two of the SAT1 and SAT2 co-infected buffalo identified at the first passage of viral isolation, the plaque-derived SAT2 genomes were distinctly grouped in two different genotypes. These genotypes were not initially detected with the NGS from the first passage (non-purified) virus isolation sample. In one animal with two SAT2 haplotypes, one plaque-derived chimeric sequence was found. These findings demonstrate within-host evolution through recombination and point mutation contributing to broad viral diversity in the wildlife reservoir. These mechanisms may be critical to FMDV persistence at the individual animal and population levels, and may contribute to the emergence of new viruses that have the ability to spill-over to livestock and other wildlife species.

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: A first attempt to phylogeographic inference for a marine mollusc disease.

The genetic diversity of viral populations is a key driver of the spatial and temporal diffusion of viruses; yet, studying the diversity of whole genomes from natural populations still remains a challenge. Phylodynamic approaches are commonly used for RNA viruses harboring small genomes but have only rarely been applied to DNA viruses with larger genomes. Here, we used the Pacific oyster mortality syndrome (a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and an innovative combination of bioinformatics tools, we de novo assembled twenty-one OsHV-1 new genomes. Combining quantification of major and minor genetic variations, phylogenetic analysis, and ancestral state reconstruction of discrete traits approaches, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oléron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. We demonstrate that phylodynamic approaches can be applied to aquatic DNA viruses to determine how epidemiological, immunological, and evolutionary processes act and potentially interact to shape their diversity patterns.

Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster.

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. Here, we sought to study this evolutionary arms-race and assessed the impact of the innate immune system on viral population diversity and evolution, using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). We isogenized eight fly genotypes generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV, and the virus was serially passaged ten times via reinfection in naïve flies. Viral population diversity was studied after each viral passage by high-throughput sequencing, and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. We found that the absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, our results indicate that the innate immune system as a whole, and not specific antiviral defense pathways in isolation, generally constrains viral diversity and evolution.

Human cytomegalovirus multiple-strain infections and viral population diversity in haematopoietic stem cell transplant recipients analysed by high-throughput sequencing

Human cytomegalovirus (HCMV) is an important opportunistic pathogen in allogeneic haematopoietic stem cell transplant (HSCT) recipients. High-throughput sequencing of target-enriched libraries was performed to characterise the diversity of HCMV strains present in this high-risk group. Forty-four HCMV-DNA-positive plasma specimens (median viral input load 321 IU per library) collected at defined time points from 23 HSCT recipients within 80 days of transplantation were sequenced. The genotype distribution for 12 hypervariable HCMV genes and the number of HCMV strains present (i.e. single- vs. multiple-strain infection) were determined for 29 samples from 16 recipients. Multiple-strain infection was observed in seven of these 16 recipients, and five of these seven recipients had the donor (D)/recipient (R) HCMV-serostatus combination D + R + . A very broad range of genotypes was detected, with an intrahost composition that was generally stable over time. Multiple-strain infection was not associated with particular virological or clinical features, such as altered levels or duration of antigenaemia, development of acute graft-versus-host disease or increased mortality. In conclusion, despite relatively low viral plasma loads, a high frequency of multiple-strain HCMV infection and a high strain complexity were demonstrated in systematically collected clinical samples from this cohort early after HSCT. However, robust evaluation of the pathogenic role of intrahost viral diversity and multiple-strain infection will require studies enrolling larger numbers of recipients.