Inter-host Diversity Research Articles

Longitudinal follow-up of HPV16 sequence after cervical infection: Low intrahost variation and no correlation with clinical evolution.

Human papillomavirus (HPV) 16 exhibits different variants that may differ in their carcinogenic risk. To identify some high-risk variants, we sequenced and compared HPV16 whole genomes obtained from a longitudinal cohort of 34 HPV16-infected women who had either spontaneously cleared their infection (clearance group or "C"), or developed cervical high-grade lesions following a viral persistence (group persistence or "P"). Phylogenetic analysis of paired samples obtained at the beginning (C0 or P0) and at the end (C2 or P2) of the follow-up (median intervals between C0-C2 and between P0-P2 were 16 and 36.5 months, respectively) revealed a low genetic variability within the host compared to the genetic interhost diversity. By comparing our HPV16 sequences to a reference sequence, we observed 301 different substitutions, more often transitions (60.9%) than transversions (39.1%), that occurred throughout the viral genome, but with a low frequency in E6 and E7 oncogenes (10 and 9 substitutions), suggesting a high conservation of these genes. Deletions and insertions were mostly observed in intergenic regions of the virus. The only significant substitution found between the subgroups C2 and P2 was observed in the L2 gene (L330F), with an unclear biological relevance. Our results suggest a low longitudinal intrahost evolution of HPV16 sequences and no correlation between genetic variations and clinical evolution.

Low Intrahost and Interhost Genetic Diversity of Carnivore Protoparvovirus 1 in Domestic Cats during a Feline Panleukopenia Outbreak.

Feline panleukopenia (FPL), a highly contagious and frequently fatal disease of cats, is caused by Feline parvovirus (FPV) and Canine parvovirus (CPV). We characterised the diversity of these Carnivore protoparvovirus 1 variants in 18 faecal samples collected from domestic cats with FPL during an outbreak, using targeted parvoviral DNA metagenomics to a mean depth of >10,000 × coverage per site. All samples comprised FPV alone. Compared with the reference FPV genome, isolated in 1967, 44 mutations were detected. Ten of these were nonsynonymous, including 9 in nonstructural genes and one in VP1/VP2 (Val232Ile), which was the only one to exhibit interhost diversity, being present in five sequences. There were five other polymorphic nucleotide positions, all with synonymous mutations. Intrahost diversity at all polymorphic positions was low, with subconsensus variant frequencies (SVF) of <1% except for two positions (2108 and 3208) in two samples with SVF of 1.1–1.3%. Intrahost nucleotide diversity was measured across the whole genome (0.7–1.5%) and for each gene and was highest in the NS2 gene of four samples (1.2–1.9%). Overall, intrahost viral genetic diversity was limited and most mutations observed were synonymous, indicative of a low background mutation rate and strong selective constraints.

Single-gene long-read sequencing illuminates Escherichia coli strain dynamics in the human intestinal microbiome.

Gut microbiome is of major interest due to its close relationship to health and disease. Bacteria usually vary in gene content, leading to functional variations within species, so resolution higher than species-level methods is needed for ecological and clinical relevance. We design a protocol to identify strains in selected species with high discrimination and in high numbers by amplicon sequencing of the flagellin gene. We apply the protocol to fecal samples from a human diet trial, targeting Escherichia coli. Across the 119 samples from 16 individuals, there are 1,532 amplicon sequence variants (ASVs), but only 32 ASVs are dominant in one or more fecal samples, despite frequent dominant strain turnover. Major strains in an intestine are found to be commonly accompanied by a large number of satellite cells, and many are identified as potential extraintestinal pathogens. The protocol could be used to track epidemics or investigate the intra- or inter-host diversity of pathogens.

SARS-CoV-2 genomic diversity and the implications for qRT-PCR diagnostics and transmission.

The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 lineages that have spread throughout the world. In this study, we investigated 129 RNA-seq data sets and 6928 consensus genomes to contrast the intra-host and inter-host diversity of SARS-CoV-2. Our analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights intra-host single nucleotide variant (iSNV) and SNP similarity, albeit with differences in C > U changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of insertions and deletions contribute to the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Novel Picobirnaviruses in Respiratory and Alimentary Tracts of Cattle and Monkeys with Large Intra- and Inter-Host Diversity.

Picobirnaviruses (PBVs) are mostly found in animal alimentary samples. In this study, among 576 respiratory specimens from 476 mammals and 100 chickens, genogroup I PBVs were detected in three cattle and three monkeys, and a genogroup II PBV-positive sample was collected from one cattle specimen. More than one PBV sequence type was observed in two and one genogroup I PBV-positive samples from cattle and monkeys, respectively. Twenty-four complete/near-complete segments 2 (nine from respiratory and 15 from alimentary samples) from the cattle and monkey genogroup I PBVs and one complete segment 2 from the cattle genogroup II PBV were sequenced. Similar to other studies, the cattle PBVs also showed a high diversity. In contrast, the monkey PBVs observed in this study were clustered into three distinct clades. Within each clade, all the sequences showed >99% amino acid identities. This unique phenomenon is probably due to the fact that monkeys in our locality reside in separated troops with minimal inter-troop contact.

Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing.

Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Correction: Characterization of intra- and inter-host norovirus P2 genetic variability in linked individuals by amplicon sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0201850.].

Is the trophosome of Ridgeia piscesae monoclonal?

The hydrothermal vent tubeworm Ridgeia piscesae relies on intracellular chemolithoautotrophic symbionts for its nutrition. Yet, little is known about symbiont diversity within and between individual worms. We report several lines of molecular evidence for multiple genotypes of very closely related symbionts within the trophosome of the R. piscesae. We examined the distribution of genotypic variants (insertions, deletions, and substitutions) in whole genome shotgun sequences of symbionts from the trophosome of a unique individual R. piscesae and the pooled sequences of five other tubeworms of the same species. Observed heterogeneity is unlikely to be the result of recent point or structural mutations of a monoclonal symbiont lineage. To assess inter-host diversity we examined single nucleotide polymorphisms (SNPs) in pyrosequences of the highly variable regions V1 to V3 of the symbiont 16S rRNA gene across 53 individual hosts from two vent sites. Most of the identified SNPs were found in more than one individual, and one seemed to be region specific. Two of the identified SNPs were also present in metagenomic data generated from high-throughput sequencing of trophosome material from an individual R. piscesae. Finally, we observed compositional and structural variations of CRISPR spacers within a CRISPR array.

High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination.

Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

Sobre a evolução do vírus da bronquite infecciosa das galinhas em células VERO

Avian infectious bronchitis virus (IBV) (Nidovirales: Coronaviridae) is a chicken Gammacoronavirus with the highestevolution rate in the genus and, despite the recently reported proofreading activity of its polymerase, intra and interhost diversity is a well-documented phenomenon. This study aimed to assess the genetic variation of serial passages of a variant genotype IBV strain in vitro. Strain CRG-BETA, propagated in chicken embryos, was inoculated in VERO cells monolayers up to the 4th passage and each passage was monitored with an RT-PCR targeted to the S1 gene (nt 705to 1094) and an RT-PCR to the protein 5a mRNA. All passages were positive to RT-PCRs to S1 and passages 1 to 3 to5a mRNA; S1 sequences showed no polymorphism. The finding of IBV mRNA in the cell cultures demonstrates that the CRG-BETA IBV strain is replicating in the VERO cells and regarding S1 sequence analysis, the lack of nucleotide mutations shows that CRG-BETA might have reached a fixed status. As a conclusion, different genotypes of IBV present different evolutionary patterns not only in vivo as previously known, but also in vitro, as described herein.

Previously Transmitted HIV-1 Strains Are Preferentially Selected During Subsequent Sexual Transmissions

A genetic bottleneck is known to exist for human immunodeficiency virus (HIV) at the point of sexual transmission. However, the nature of this bottleneck and its effect on viral diversity over time is unclear. Interhost and intrahost HIV diversity was analyzed in a stable population in Rakai, Uganda, from 1994 to 2002. HIV-1 envelope sequences from both individuals in initially HIV-discordant relationships in which transmission occurred later were examined using Sanger sequencing of bulk polymerase chain reaction (PCR) products (for 22 couples), clonal analysis (for 3), and next-generation deep sequencing (for 9). Intrahost viral diversity was significantly higher than changes in interhost diversity (P < .01). The majority of HIV-1-discordant couples examined via bulk PCR (16 of 22 couples), clonal analysis (3 of 3), and next-generation deep sequencing (6 of 9) demonstrated that the viral populations present in the newly infected recipient were more closely related to the donor partner's HIV-1 variants found earlier during infection as compared to those circulating near the estimated time of transmission (P = .03). These findings suggest that sexual transmission constrains viral diversity at the population level, partially because of the preferential transmission of ancestral as opposed to contemporary strains circulating in the transmitting partner. Future successful vaccine strategies may need to target these transmitted ancestral strains.

Homogeneity of Powassan virus populations in naturally infected Ixodes scapularis

Powassan virus (POWV, Flaviviridae: Flavivirus) is the sole North American member of the tick-borne encephalitis complex and consists of two distinct lineages that are maintained in ecologically discrete enzootic transmission cycles. The underlying genetic mechanisms that lead to niche partitioning in arboviruses are poorly understood. Therefore, intra- and interhost genetic diversity was analyzed to determine if POWV exists as a quasispecies in nature and quantify selective pressures within and between hosts. In contrast to previous reports for West Nile virus (WNV), significant intrahost genetic diversity was not observed. However, pN (0.238) and d(N)/d(S) ratios (0.092) for interhost diversity were similar to those of WNV. Combined, these data suggest that purifying selection and/or population bottlenecks constrain quasispecies diversity within ticks. These same selective and stochastic mechanisms appear to drive minor sequence changes between ticks. Moreover, Powassan virus populations seem not to be structured as quasispecies in naturally infected adult deer ticks.

Diversity of naturally occurring Epstein-Barr virus revealed by nucleotide sequence polymorphism in hypervariable domains in the BamHI K and N subgenomic regions.

The extent of nucleotide sequence microheterogeneity varies among subgenomic regions of Epstein-Barr virus (EBV). We examined, in EBV-carrying lymphoid cell lines, the extent of polymorphism in EBV DNA fragments amplified from the BamHI E, K, N and Z regions, and then investigated the diversity of the more hypervariable regions in tissues and body fluids. In cell lines, sequence dissimilarities in a genotype-specifying fragment of the EBNA-3C gene varied from < 1-4% within each genotype; dissimilarities in the first intron of the BZLF- 1 gene were < 2% within each genotype. By contrast, dissimilarities in a C-terminal unique domain of the EBNA-1 gene, and in a fragment that encompasses and is upstream of the LMP-1 start codon, varied between 2 and 7% and were not genotype-specific. The sequence diversity in BamHI K and N regions was then examined in tissues and body fluids by single-strand conformation polymorphism (SSCP) analysis and cycle sequencing. Extensive inter-host diversity was observed, whether the host was co-infected by human immunodeficiency virus (HIV) or not. In the oral cavity of HIV-infected patients, inter-compartmental EBV diversity could be demonstrated, even between sites that were anatomically proximate. Studies of BamHI K clones derived from EBV in oral lesions revealed infection by multiple variants. Identification of hypermutable loci within the EBV genome such as those located in the BamHI K and N regions should permit fine discrimination of individual EBV variants.

VacA genotypes and genetic diversity in clinical isolates of Helicobacter pylori.

Genetic diversity in Helicobacter pylori strains may affect the function and antigenicity of virulence factors associated with bacterial infection and, ultimately, disease outcome. In this study, DNA diversity of H. pylori isolates was examined by analysis of vacA genotypes and by restriction fragment length polymorphism (RFLP) analysis of H. pylori-associated genes (vacA, cagA,flaA, ureAB, and ureCD). Thirty-seven H. pylori isolates from 26 patients were successfully classified into distinct vacA allelic genotypes. The signal sequence allele sl (31 of 37) predominated over the s2 allele (6 of 37) and was significantly associated with the occurrence (past or present) of gastric ulcers. A novel midregion allele, designated as m3, has been identified in two H. pylori isolates which could not be typed with midregion allele m1- or m2-specific primers. Additionally, significant nucleotide diversity yielding different amino acid sequences was demonstrated by DNA sequencing of vacA fragments from clinical isolates of H. pylori. Furthermore, RFLP analysis of 45 H. pylori isolates (including 15 paired isolates) obtained from antrum and corpus biopsy specimens from 30 individual patients showed remarkably high interhost diversity (one patient, one H. pylori strain) and intrahost identity in gene sequences coding for VacA, CagA, flagellin, and urease. Only in a single patient was a minor genotypic variation at different anatomic sites within the stomach identified. These data warrant the detailed analysis of the effect of genetic diversity on the function and antigenicity of H. pylori-associated virulence factors.