Near-full Length Research Articles

Detection, characterization, and phylogenetic analysis of novel astroviruses from endemic Malagasy fruit bats

Bats (order: Chiroptera) are known to host a diverse range of viruses, some of which present a human public health risk. Thorough viral surveillance is therefore essential to predict and potentially mitigate zoonotic spillover. Astroviruses (family: Astroviridae) are an understudied group of viruses with a growing amount of indirect evidence for zoonotic transfer. Astroviruses have been detected in bats with significant prevalence and diversity, suggesting that bats may act as important astrovirus hosts. Most astrovirus surveillance in wild bat hosts has, to date, been restricted to single-gene PCR detection and concomitant Sanger sequencing; additionally, many bat species and many geographic regions have not yet been surveyed for astroviruses at all. Here, we use metagenomic Next Generation Sequencing (mNGS) to detect astroviruses in three species of Madagascar fruit bats, Eidolon dupreanum, Pteropus rufus, and Rousettus madagascariensis. We detect numerous partial sequences from all three species and one near-full length astrovirus sequence from Rousettus madagascariensis, which we use to characterize the evolutionary history of astroviruses both within bats and the broader mammalian clade, Mamastrovirus. Taken together, applications of mNGS implicate bats as important astrovirus hosts and demonstrate novel patterns of bat astrovirus evolutionary history, particularly in the Southwest Indian Ocean region.

Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C

IntroductionHIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C.MethodsPrimer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability.ResultsThe in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2–6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region.ConclusionsWe developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.

188 Characterization of the Vagino-Uterine Microbiota in Pregnant and Non-Pregnant Beef Cattle Using 16S Rrna Gene Sequencing and Culturing

Abstract Emerging evidence suggests that urogenital microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. The objectives of this study were to: 1) characterize the vaginal and uterine microbiota between beef cattle that became pregnant via artificial insemination (AI) and those that did not; 2) identify differentially abundant taxa between pregnant and non-pregnant cattle; and 3) characterize the culturable fraction of the vaginal and uterine microbiota using extensive culturing. Vaginal and uterine swab samples were collected from two cohorts of Angus-crossbred cattle: mature cows (n =100) and heifers (n = 72, vaginal swab only) at the time of AI. At 35 days post-AI, pregnancy diagnoses were made via ultrasound. A subset of vaginal and uterine swabs from cows (27 non-pregnant and 31 pregnant) and heifers (26 non-pregnant and 33 pregnant) were selected and processed for microbiota assessment using 16S rRNA gene (V3-V4) sequencing. For culturing, a subset of cryopreserved vaginal and uterine swabs (128) were plated onto three different agar types and cultured, and isolates were identified by near-full length 16S rRNA gene sequencing. Sequencing results revealed 11 taxa that were more abundant in the vaginas of heifers that failed to become pregnant via AI than heifers that did become pregnant. While there was no significant difference in vaginal microbiota community structure (P = 0.21) between pregnant and non-pregnant cows, non-pregnant cows tended to have greater microbial richness and Shannon diversity (P ≤ 0.08) compared with pregnant cows. No differentially abundant taxa were detected in the vaginas of pregnant versus non-pregnant cows. Pregnant cows had a distinct uterine microbiota community structure (R2 = 0.032, P = 0.008) but similar alpha diversity (P > 0.1) compared with non-pregnant cows. Twenty-eight differentially abundant taxa were observed in the uterine microbiota between pregnant and non-pregnant groups, with 11 of them more abundant in pregnant cows. Methanobrevibacter ruminantium (archaea) and Fusobacterium necrophorum were among these 11 positive pregnancy-associated taxa. Although community structure was significantly different between the vagina and uterus, 277 “core taxa” (present in ≥ 80% of all samples) were shared between the two communities. A total of 733 bacterial isolates were recovered from vaginal (512) and uterine (221) swabs under aerobic (n = 363, 83 different species) and anaerobic (n = 370, 69 species) culturing. Potential pathogenic isolates within the Trueperella, Mannheimia and Histophilus genera were most exclusively identified from vaginal swabs. Only two Lactobacillus isolates were recovered. Overall, our results indicate that pregnancy-associated taxonomic signatures are present in the bovine uterine and vaginal microbiota. Among 39 pregnancy status-associated taxa, 41% were unclassified at the genus level, suggesting that shotgun metagenomics and culturomics are needed to identify and characterize the pregnancy-associated taxa in cattle.

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization.

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediatedLong-readSequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.

Comparative genotyping of SARS-CoV-2 among Egyptian patients: near-full length genomic sequences versus selected spike and nucleocapsid regions.

Several tools have been developed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genotyping based on either whole genome or spike sequencing. We aimed to highlight the molecular epidemiological landscape of SARS-CoV-2 in Egypt since the start of the pandemic, to describe discrepancies between the 3 typing tools: Global Initiative on Sharing Avian Influenza Data (GISAID), Nextclade, and Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) and to assess the fitness of spike and nucleocapsid regions for lineage assignment compared to the whole genome. A total of 3935 sequences isolated from Egypt (March 2020-2023) were retrieved from the GISAID database. A subset of data (n = 1212) with high coverage whole genome was used for tool discrimination and agreement analyses. Among 1212 sequences, the highest discriminatory power was 0.895 for PANGOLIN, followed by GISAID (0.872) and Nextclade (0.866). There was a statistically significant difference (p = 0.0418) between lineages assigned via spike (30%) and nucleocapsid (46%) compared to their whole genome-assigned lineages. The first 3 pandemic waves were dominated by B.1, followed by C.36 and then C.36.3, while the fourth to sixth waves were dominated by the B.1.617.2, BA, and BA.5.2 lineages, respectively. Current shift in lineage typing to recombinant forms. The 3 typing tools showed comparable discrimination among SARS-CoV-2 lineages. The nucleocapsid region could be used for lineage assignment.

The First Genomic Characterization of the Chikungunya Virus in Saudi Arabia

BackgroundChikungunya is an arboviral infection caused by the Chikungunya virus (CHIKV) transmitted to humans by mosquitoes of Aedes spp. CHIKV has been confined to African countries and South-East Asia up to 2004, but since then, the pathogen has become more global, and its high morbidity rate has become more visible. Saudi Arabia is not an endemic region of CHIKV, and the virus’s origin is not yet fully understood. This study aimed to characterize the genome of CHIKV from samples detected in Jeddah in 2018.MethodTwenty-two sets of primers were designed to amplify near-full length genome of CHIKV. RT-PCR was conducted from clinical samples. Two samples were used for studying near complete genome sequence while the remaining samples were used to study the E1 gene. Different bioinformatics tools were utilized.ResultsPhylogenetic analysis showed that the CHIKV strains clustered with strains isolated from Kenya during 2017–2018 and belonged to ECSA genotype. E1: L136F, K211E and I317V mutations were identified in our strains. Also, E2: M74I, A76T, and V264A mutations were documented. Additionally, the capsid N79S substitution was also detected.ConclusionThe genome of CHIKV was analyzed for the first time in Saudi Arabia to better understand the origin of the CHIKV and its genetic diversity, which showed high similarity with IE—a subclade of CHIKV strains detected in Mombasa (Kenya) indicating its possible origin.

The Metagenomic Analysis of Viral Diversity in Colorado Potato Beetle Public NGS Data.

The Colorado potato beetle (CPB) is one of the most serious insect pests due to its high ecological plasticity and ability to rapidly develop resistance to insecticides. The use of biological insecticides based on viruses is a promising approach to control insect pests, but the information on viruses which infect leaf feeding beetles is scarce. We performed a metagenomic analysis of 297 CPB genomic and transcriptomic samples from the public National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database. The reads that were not aligned to the reference genome were assembled with metaSPAdes, and 13314 selected contigs were analyzed with BLAST tools. The contigs and non-aligned reads were also analyzed with Kraken2 software. A total of 3137 virus-positive contigs were attributed to different viruses belonging to 6 types, 17 orders, and 32 families, matching over 97 viral species. The annotated sequences can be divided into several groups: those that are homologous to genetic sequences of insect viruses (Adintoviridae, Ascoviridae, Baculoviridae, Dicistroviridae, Chuviridae, Hytrosaviridae, Iflaviridae, Iridoviridae, Nimaviridae, Nudiviridae, Phasmaviridae, Picornaviridae, Polydnaviriformidae, Xinmoviridae etc.), plant viruses (Betaflexiviridae, Bromoviridae, Kitaviridae, Potyviridae), and endogenous retroviral elements (Retroviridae, Metaviridae). Additionally, the full-length genomes and near-full length genome sequences of several viruses were assembled. We also found sequences belonging to Bracoviriform viruses and, for the first time, experimentally validated the presence of bracoviral genetic fragments in the CPB genome. Our work represents the first attempt to discover the viral genetic material in CPB samples, and we hope that further studies will help to identify new viruses to extend the arsenal of biopesticides against CPB.

Unexpected finding of Fusobacterium varium as the dominant Fusobacterium species in cattle rumen: potential implications for liver abscess etiology and interventions.

Fusobacterium varium has been generally overlooked in cattle rumen microbiome studies relative to the presumably more abundant liver abscess-causing Fusobacterium necrophorum. However, F. varium was found to be more abundant in the rumen fluid of cattle and under culture conditions tailored to enrich F. necrophorum. Using near-full length 16S ribosomal ribonucleic acid sequencing, we demonstrate that F. varium grows under restrictive conditions commonly used to enumerate F. necrophorum, suggesting that previous F. necrophorum abundance assessment may have been inaccurate and that F. varium may be an underestimated member of the ruminal bacterial community. Fusobacterium varium were not as susceptible as F. necrophorum to in-feed antibiotics conventionally used in feedlots. Exposure to tylosin, the current gold standard for liver abscess reduction strategies in cattle, consistently hindered growth of the F. necrophorum strains tested by over 67% (P < 0.05) relative to the unexposed control. In contrast, F. varium strains were totally or highly resistant (0%-13% reduction in maximum yield, P < 0.05). Monensin, an ionophore antibiotic, had greater inhibitory activity against F. necrophorum than F. varium. Finally, preliminary genomic analysis of two F. varium isolates from the rumen revealed the presence of virulence genes related to those of pathogenic human F. varium isolates associated with active invasion of mammalian cells. The data presented here encourage further investigation into the ecological role of F. varium within the bovine rumen and potential role in liver abscess development, and proactive interventions.

Theileria terrestris nov. sp.: A Novel Theileria in Lowland Tapirs (Tapirus terrestris) from Two Different Biomes in Brazil.

The low-land tapir (Tapirus terrestris) is the largest wild terrestrial mammal found in Brazil. Although T. terrestris has been already reported as a host of hemoparasites, the occurrence and genetic identity of Piroplasmida agents in this species is still cloudy. Although it is reported that Theileria equi, an endemic equid-infective agent in Brazil, is occurring in lowland tapirs, these reports are probably misconceived diagnoses since they are solely based on small fragments of 18S rRNA that may not achieve accurate topologies on phylogenetic analyses. The present study aimed to detect and investigate the identity of Theileria spp. in tapirs from Pantanal and Cerrado biomes. Blood-DNA samples from tapirs were screened for a partial (~800 bp) 18S rRNA gene fragment from Piroplasmida and 64 (64/122; 52.46% CI: 43.66-61.11%) presented bands of expected size. Samples were submitted to different protocols for molecular characterization, including near-full length 18S rRNA gene (~1500 bp), and the ema-1 gene from T. equi. Eight sequences were obtained for extended fragments (1182-1473 bp) from the 18S rRNA gene. Moreover, three sequences from partial cox-1 and five from partial hsp70 gene were obtained. None of the samples presented amplifications for the ema-1 gene. Phylogenetic and distance analyses from the 18S rRNA sequences obtained demonstrated a clear separation from tapirs' Theileria spp. and T. equi. Phylogenetic analyses of cox-1 and hsp70 sequences obtained herein also showed a unique clade formed by tapir's Theileria spp. Theileria terrestris sp. nov. is positioned apart from all other Theileria species in 18S rRNA, cox-1, and hps70 phylogenetic analyses. This novel proposed species represents a new Piroplasmida clade, yet to be characterized regarding biological features, vectors involved in the transmission cycles, additional vertebrate hosts, and pathogenicity.

Mutation profiling of the c.1521_1523delCTT (p.Phe508del, F508del) cystic fibrosis transmembrane conductance regulator allele using haplotype-resolved long-read next generation sequencing.

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

Cryo-EM structures of the near full-length HER2/HER3 heterodimer reveal a novel allosteric mechanism of activation

The human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) form a potent pro-oncogenic signaling unit upon binding to the extracellular ligand neuregulin-1ß (NRG1ß). Through single particle cryo-electron microscopy (cryo-EM) on graphene oxide (GO) supported grids, we have solved three novel structures of the HER2/HER3 heterodimer in the context of near-full length receptors. These structures represent the first depictions of a singly-liganded HER receptor dimer, of HER2 actively engaged with a co-receptor, and of HER3 in a ligand-bound configuration.

First detection of feline bocaparvovirus 2 and feline chaphamaparvovirus in healthy cats in Turkey

The pet cat’s population and the number of viruses that infect them are increasing worldwide. Recently, feline chaphamaparvovirus (FeChPV, also called fechavirus) and feline bocaparvovirus (FBoV) infections, which are novel parvovirus species, have been reported in cats from different geographic regions. Here, we investigated FBoV 1–3 and FeChPVs in healthy cats in Turkey using PCR, where nuclear phosphoprotein 1 (NP1) is targeted for FBoV and NP for FeChPV. For this purpose, oropharygeal swabs were obtained from 70 healthy cats with different housing status from June 15 to December 1, 2020. After PCR screening tests, six out of 70 cats (5/47 shelter cats; 1/23 domestic cats) were found to be positive for FBOV, while two were positive for FeChPV (1/47 shelter cats; 1/23 domestic cats). No cat was found in which both viruses were detected. The nucleotide (nt) sequence comparison in the 310 base pair (bp) NP gene of the two FeChPVs identified in this study shared a high identity with each other (95.0% nt and 99% aa identities) and with previously reported FeChPVs (92.4–97.1% nt and 98.1–99.0% aa identities), including 313R/2019/ITA, 49E/2019/ITA, VRI_849, 284R/2019/ITA, and IDEXX-1. Here, the near-full length (1489 nt, 495 amino acids-aa) of the VP2 gene of the FechaV/Tur-2020/68 isolate obtained from the study was also sequenced. The nt and aa identity ratio of this isolate with other FeChPVs was 98.0–98.5%-96–96.5%, respectively. Sequences of the 465 bp NP1 gene of the six Turkish FBoV strains shared high identities with each other (99.6–100% nt and 99.3–100% aa identities) and with those of FBoV-2 strains (97.8–99.1% nt and 98.0–100% aa identities), including 16SY0701, 17CC0505-BoV2, HFXA-6, and POR1. All FBoVs detected in this study were classified as genotype 2, similar to the study conducted in Japan and Portugal. Here, the NS1 (partial), NP1, VP1 and VP2 gene of the FBoV-2/TUR/2020–14 strain obtained from the study were also sequenced and the nt and aa sequences showed high identities to the above-mentioned FBoV-2 strain/isolates (> 96%, except for the aa ratio of strain 16SY0701). In conclusion, this study shows that FBoV and FeChPV are present in healthy cats in Turkey, and these viruses can be detected from oropharyngeal swabs. Our findings contribute to further investigation of the prevalence, genotype distribution, and genetic diversity of Turkish FBoVs and FeChPVs, adding to the molecular epidemiology of FBoV and FeChPVs worldwide.

Antigenic Diversity in Theileria parva Populations From Sympatric Cattle and African Buffalo Analyzed Using Long Read Sequencing.

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva, transmitted by the three-host tick Rhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+ and CD4+ T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle (n = 126) and buffalo samples (n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

HIV-1 proviral landscape characterization varies by pipeline analysis.

IntroductionHIV rebounds after cessation of antiretroviral therapy, representing a barrier to cure. To better understand the virus reservoir, analysis pipelines have been developed that categorize proviral sequences as intact or defective, and further determine the precise nature of the sequence defects that may be present. We investigated the effects that different analysis pipelines had on the characterization of HIV‐1 proviral sequences.MethodsWe used single genome amplification to generate near full‐length (NFL) HIV‐1 proviral DNA sequences, defined as amplicons greater than 8000 base pairs in length, isolated from peripheral blood mononuclear cells (PBMC) of treated suppressed participants with HIV‐1. Amplicons underwent direct next‐generation single genome sequencing and were analysed using four HIV‐1 proviral characterization pipelines. Sequences were characterized as intact or defective; defective sequences were assessed for the number and types of defects present. To confirm and extend our findings, 691 proviruses from the Proviral Sequence Database (PSD) were analysed and the ProSeq‐IT tool of the PSD was used to characterize both the participant and PSD proviruses.Results and discussionVirus sequences derived from thirteen ART‐treated virologically suppressed participants with HIV were studied. A total of 693 HIV‐1 proviral sequences were generated, 282 of which were NFL. An average of 53 sequences per participant was analysed. We found that proviruses often harbour multiple sequence defect types (mean 2.7, 95% confidence interval [CI] 2.5, 3.0); the elimination order used by each pipeline affected the percentage of proviruses allotted into each defect category. These differences varied between participants, depending on the number of defect categories present in a given provirus sequence. Pipeline‐specific differences in characterizing the HIV‐1 5′ untranslated region (5′ UTR) led to an overestimation of the number of intact NFL proviral sequences, a finding corroborated in the independent PSD analysis. A comparison of the four published pipelines to ProSeq‐IT found that ProSeq IT was more likely to characterize proviruses as intact.ConclusionsThe choice of pipeline used for HIV‐1 provirus landscape analysis may bias the classification of defective sequences. To improve the comparison of provirus characterizations across research groups, the development of a consensus elimination pipeline should be prioritized.

HIVIntact: a python-based tool for HIV-1 genome intactness inference

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

Next generation sequencing of near-full length genome of norovirus GII.4 from Botswana

Noroviruses are highly diverse, with genotype GII.4 causing most epidemics. This study aimed to investigate the evolutionary dynamics of norovirus genogroup GII strains among acutely infected children under 5 years in Botswana, between 2016 and 2018. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the whole norovirus genome, followed by next-generation sequencing using Oxford Nanopore technology. Twelve samples were successfully analyzed, with 11 identified as norovirus GII.4 Sydney [P31] and one as GII.4 Sydney [P13]. This study generated the first near-full length norovirus sequences in Botswana (93–95% coverage). Our results show that the norovirus GII.4 strains circulating in Botswana are under evolution through recombination and antigenic drift. Recombination in the GII.4 Sydney [P31] and GII.4 Sydney [P13] strains occurred in the ORF1/ORF2 junction and within ORF1, respectively. This study provides the first description of the GII.4 Sydney [P13] recombinant. Amino acid variation in the immunogenic sites was analyzed. Mutations in epitope A correlate with the emergence of novel norovirus GII.4 strains with altered antigenicity. In this study, we identified 43 unique amino acid substitutions in the VP1 region, with six occurring in epitopes, A (G295N, and E368Q) and E (S40T, N412D, N412K and T413H). The shell subdomain of the GII.4 Sydney [P13] variant was closely related to norovirus GII.17. Lastly, we also observed several mutations in the T cell restricted epitopes of both strains. Our study has made a novel contribution to understanding the evolution of norovirus GII.4 in Botswana.

Detection of Virus-Related Sequences Associated With Potential Etiologies of Hepatitis in Liver Tissue Samples From Rats, Mice, Shrews, and Bats.

Hepatitis is a major global health concern. However, the etiology of 10–20% hepatitis cases remains unclear. Some hepatitis-associated viruses, like the hepatitis E virus, are zoonotic pathogens. Rats, shrews, and bats are reservoirs for many zoonotic pathogens. Therefore, understanding the virome in the liver of these animals is important for the investigation of the etiologies of hepatitis and monitoring the emerging zoonotic viruses. In this study, viral metagenomics and PCR methods were used to investigate viral communities in rats, mice, house shrews, and bats livers. Viral metagenomic analysis showed a diverse set of sequences in liver samples, comprising: sequences related to herpesviruses, orthomyxoviruses, anelloviruses, hepeviruses, hepadnaviruses, flaviviruses, parvoviruses, and picornaviruses. Using PCR methods, we first detected hepatovirus sequences in Hipposideros larvatus (3.85%). We also reported the first detection of Zika virus-related sequences in rats and house shrews. Sequences related to influenza A virus and herpesviruses were detected in liver. Higher detection rates of pegivirus sequences were found in liver tissue and serum samples from rats (7.85% and 15.79%, respectively) than from house shrews. Torque teno virus sequences had higher detection rates in the serum samples of rats and house shrews (52.72% and 5.26%, respectively) than in the liver. Near-full length genomes of pegivirus and torque teno virus were amplified. This study is the first to compare the viral communities in the liver of bats, rats, mice, and house shrews. Its findings expand our understanding of the virome in the liver of these animals and provide an insight into hepatitis-related viruses.

A cell-free antigen processing system reveals antigenic factors critical for HIV-1 epitope dominance and informs vaccine design

Abstract Certain CD4+T cell epitopes have been associated with immune control of HIV-1 replication and the presence of anti-Env neutralizing antibodies. However, it remains unclear what antigen-dependent factors influence the dominance of certain epitopes in HIV-1 proteins. To study HIV-1 epitope dominance, we used a cell-free antigen processing system previously developed by our group that utilizes soluble HLA-DR1*01:01 (DR1), HLA-DM (DM), protein antigen and three cathepsins in a reducing and low pH environment to allow for antigen binding and cleavage. We subjected HIV-1 antigens from Gag, Pol, Env and accessory proteins Vif, Nef, Tat, and Rev to this system. Immunoprecipitation of pMHC-II complexes followed by peptide elution and identification via LC-MS/MS revealed a map of DR1-bound epitopes across the near-full length HIV-1 proteome. Most identified epitopes were DM-resistant, and in some cases, DM narrowed the epitope diversity to one species. Importantly, we identified 28 novel epitopes, including several glycopeptides eluted from DR1. We noted epitope “hot spots” in HIV-1 antigens and striking similarities in epitopes from HIV polyproteins (Gag precursor) versus individual subunits (Gag capsid). Analysis of the location of several epitopes from smaller HIV-1 proteins based on their known crystal structures mapped to regions of low protein stability and higher solvent accessibility. Finally, we found that several epitopes induced memory CD4+T cell responses in persons living with HIV-1. Thus, by using a minimalist in vitro processing system, we can interrogate novel HIV-1 epitopes and understand how DM-resistance and antigen structure influence epitope dominance, which has important implications for vaccine design.

Seasonal and Geographical Transitions in Eukaryotic Phytoplankton Community Structure in the Atlantic and Pacific Oceans

Much is known about how broad eukaryotic phytoplankton groups vary according to nutrient availability in marine ecosystems. However, genus- and species-level dynamics are generally unknown, although important given that adaptation and acclimation processes differentiate at these levels. We examined phytoplankton communities across seasonal cycles in the North Atlantic (BATS) and under different trophic conditions in the eastern North Pacific (ENP), using phylogenetic classification of plastid-encoded 16S rRNA amplicon sequence variants (ASVs) and other methodologies, including flow cytometric cell sorting. Prasinophytes dominated eukaryotic phytoplankton amplicons during the nutrient-rich deep-mixing winter period at BATS. During stratification (‘summer’) uncultured dictyochophytes formed ∼35 ± 10% of all surface plastid amplicons and dominated those from stramenopile algae, whereas diatoms showed only minor, ephemeral contributions over the entire year. Uncultured dictyochophytes also comprised a major fraction of plastid amplicons in the oligotrophic ENP. Phylogenetic reconstructions of near-full length 16S rRNA sequences established 11 uncultured Dictyochophyte Environmental Clades (DEC). DEC-I and DEC-VI dominated surface dictyochophytes under stratification at BATS and in the ENP, and DEC-IV was also important in the latter. Additionally, although less common at BATS, Florenciella-related clades (FC) were prominent at depth in the ENP. In both ecosystems, pelagophytes contributed notably at depth, with PEC-VIII (Pelagophyte Environmental Clade) and (cultured) Pelagomonas calceolata being most important. Q-PCR confirmed the near absence of P. calceolata at the surface of the same oligotrophic sites where it reached ∼1,500 18S rRNA gene copies ml–1 at the DCM. To further characterize phytoplankton present in our samples, we performed staining and at-sea single-cell sorting experiments. Sequencing results from these indicated several uncultured dictyochophyte clades are comprised of predatory mixotrophs. From an evolutionary perspective, these cells showed both conserved and unique features in the chloroplast genome. In ENP metatranscriptomes we observed high expression of multiple chloroplast genes as well as expression of a selfish element (group II intron) in the psaA gene. Comparative analyses across the Pacific and Atlantic sites support the conclusion that predatory dictyochophytes thrive under low nutrient conditions. The observations that several uncultured dictyochophyte lineages are seemingly capable of photosynthesis and predation, raises questions about potential shifts in phytoplankton trophic roles associated with seasonality and long-term ocean change.

Classification of 16S rRNA reads is improved using a niche-specific database constructed by near-full length sequencing.

Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150–500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.