Expression profiling & functional characterization of candidate miRNAs in serum exosomes among Indians with & without HIV-tuberculosis coinfection.

Background & objectives Despite the evidence of population differences in miRNA expression, limited information is available about the expression profile of miRNAs in Indian tuberculosis (TB) patients. The present study aimed to investigate the expression profile of candidate serum exosomal microRNAs in Indian patients with and without HIV-TB coinfection. Methods The pool samples of serum exosomes of study participants (HIV-TB coinfection, extra-pulmonary TB, HIV mono-infection, pulmonary TB) and healthy humans were processed for the isolation of total RNA followed by miRNA analysis using miRCURY LNA human focus PCR panel by real-time PCR. The significantly altered miRNAs were identified using differential expression analysis. The target genes prediction and potential functional analysis of exclusively differentially expressed miRNAs were performed using bioinformatics tools. Results The expression profile of 57, 58, 49 and 11 miRNAs was significantly altered in exosome samples of HIV-TB coinfected, extra-pulmonary TB, HIV mono-infected and pulmonary TB patients compared to healthy controls, respectively. The set of three (hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-92a-3p), three (hsa-miR-20a-5p, hsa-let-7e-5p, hsa-miR-26a-5p) and four (hsa-miR-21-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-146a-5p) miRNAs were exclusively significantly differentially expressed in study participants with HIV-TB coinfection, extra-pulmonary TB and pulmonary TB, respectively. Most of the target genes of exclusively differentially expressed miRNAs were enriched in pathways in cancer, MAPK signalling pathway and Ras signalling pathway. Interpretation & conclusions The present study demonstrates a distinct expression profile of miRNAs in serum exosomes of the study participants and identified crucial miRNAs which may have a significant impact on the biomarker analysis and pathogenesis of TB in Indian patients.

Polymorphisms in the genes encoding RLR and TLR3 and CMV DNAemia in subjects coinfected with human immunodeficiency virus and cytomegalovirus.

Cytomegalovirus (CMV) is a pathogen that is common worldwide and is often present in individuals infected with human immunodeficiency virus (HIV). Pattern recognition receptors (PRRs) are host sensors that activate the immune response against infectious agents. However, it is unclear whether PRR single-nucleotide polymorphisms (SNPs) are associated with the occurrence of CMV DNAemia in subjects coinfected with HIV and CMV. HIV/CMV-coinfected patients with and without CMV DNAemia were recruited for this study. The DDX58 rs10813831 and IFIH1 (rs3747517 and rs1990760) polymorphisms were genotyped using the TaqMan Allelic Discrimination Assay, whereas the DDX58 rs12006123 and TLR3 (rs3775291 and rs3775296) SNPs were analyzed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. A mutation present in at least one allele of the DDX58 rs12006123 SNP occurred at least two times more frequently in HIV/CMV-coinfected patients with CMV DNAemia than in coinfected subjects without CMV DNAemia (OR, 2.50; 95% CI, 1.33-4.68; p = 0.004, in the dominant model). A higher level of CMV DNAemia was observed in subjects who had the heterozygous (GA) or homozygous recessive (AA) genotype for the DDX58 rs12006123 SNP compared with those who had the wild-type (GG) genotype (p = 0.0003). Moreover, in subjects with a mutation detected in at least one allele of the DDX58 rs12006123 SNP, a lower serum IFN-ß concentration was found compared with those who had a wild-type (GG) genotype for this polymorphism (p = 0.024). The DDX58 rs12006123 SNP is associated with CMV DNAemia in HIV/CMV-coinfected patients.

Impact of Hepatitis Delta Virus Infection on the Selection of Hepatitis B Surface Antigen Mutations.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences (n = 55) were included in our study ([HIV/HBV-positive (n = 50) and HIV/HBV/HDV-positive (n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations (L95W, W156Q, C221Y) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations (W199R, C221A, C221S, W223G) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

Pharmacogenetic determinants of tenofovir diphosphate and lamivudine triphosphate concentrations in people with HIV/HBV coinfection.

The nucleos(t)ide analogs require phosphorylation to the pharmacologically active anabolites in cells. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) in genes that encode transporters and phosphodiesterase (PDE) enzymes involved in tenofovir (TFV), disoproxil fumarate (TDF), and lamivudine (3TC) disposition will be associated with concentrations of their phosphate anabolites and virologic response. Individuals with human immunodeficiency virus (HIV) and hepatitis B virus (HBV) coinfection receiving TDF/3TC-containing antiretroviral therapy were enrolled. Steady-state TFV diphosphate (TFV-DP) and 3TC triphosphate (3TC-TP) concentrations in peripheral blood mononuclear cells (PBMCs) and dried blood spot samples were quantified. The relationship between genetic variants and TFV-DP and 3TC-TP concentrations as well as with virologic response were examined using multivariable linear regression. Of the 136 participants (median age 43 years; 63% females), 6.6% had HBV non-suppression, and 7.4% had HIV non-suppression. The multidrug resistance protein 2 (encoded by ABCC2 rs2273697) SNP was associated with 3TC-TP concentrations in PBMCs. The human organic anion transporter-1 (encoded by SLC28A2) rs11854484 SNP was associated with HIV non-suppression, and when evaluated together with SNPs with marginal associations (ABCC2 rs717620 and PDE1C rs30561), participants with two or three variants compared to those with none of these variants had an adjusted odds ratio of 48.3 (confidence interval, 4.3-547.8) for HIV non-suppression. None of the SNPs were associated with HBV non-suppression. Our study identified ABCC2 SNP to be associated with 3TC-TP concentrations in PBMCs. Also, a combination of genetic variants of drug transporters and PDE was associated with HIV non-suppression.

Immunogenetic Profile Associated with Patients Living with HIV-1 and Epstein-Barr Virus (EBV) in the Brazilian Amazon Region.

Viral coinfection among HIV-positive patients, coupled with the development of AIDS, remains a major public health problem. The synergism between the presence of HIV and other viruses has consequences in relation to changes in the severity of the infection, as well as changes in the natural course of both infections. Several polymorphisms present in genes that encode cytokines have a relevant influence on their transcription and consequently on the production of such immunological molecules. The present study evaluated the influence of SNPs located in the promoter regions of genes encoding the cytokines INF-É£, TNF, IL-6, IL-4, and IL-2, as well as their respective plasma concentrations, in patients infected with HIV and/or EBV in the state of Pará. Additionally, this study described the epidemiological profile and compared CD4+ and CD8+ T lymphocyte counts among the groups studied. The associative analysis between the SNPs and plasma cytokine concentrations in different groups showed statistical relevance for three polymorphisms: rs2069762 (IL2), where the GG genotype demonstrated higher IL-2 levels in HIV mono-infected individuals; rs2243250 (IL4), where the CT genotype showed higher IL-4 levels in the control group; and rs2069705 (IFNG), where the TT genotype showed higher IFN-γ levels in the coinfected group. Regarding SNP associations with CD4+/CD8+ counts, significant findings were observed in HIV mono-infected individuals: the rs2069705 (IFNG) polymorphism was linked to higher CD4+ counts with the CT genotype, and rs1799964 (TNF) was associated with higher CD8+ counts with the CC genotype. Therefore, this study provides evidence that the rs2069705 (IFNG) SNP is associated with elevated IFN-γ levels, which may have pathogenic consequences, as depletion of this cytokine is concerning for people living with HIV due to its antiviral properties.

Prevalence of HBsAg among Moroccan HIV-1 infected patients and APOBEC3G variant frequencies in HIV-1/HBV co-infection.

INTRODUCTION: Human immunodeficiency virus (HIV) / hepatitis B virus (HBV) causes higher rates of liver disease compared to infection with just one virus. Co-infection can accelerate the progression to liver fibrosis or hepatocellular carcinoma and disturb the treatment response. APOBEC3G is a host defense factor which interferes with HIV-1 and HBV. We aimed to determine the prevalence of hepatitis B surface antigen (HBsAg) among HIV-infected patients and seronegative controls, and screen the HIV/HBV population for APOBEC3G variants rs8177832, rs35228531 and rs2294367, previously associated with HIV-1 infection susceptibility in Morocco. METHODOLOGY: A case control study was conducted on 404 individuals (204 HIV-infected and 200 eligible blood donors) from April to November 2021. HBsAg was measured on the Roche Cobas e411 automatic analyzer (Roche Diagnostics, Basel, Switzerland) and APOBEC3G polymorphisms were identified using the TaqMan genotyping allelic discrimination method. Fisher Exact test, odds ratio (OR) with 95% confidence interval (CI), and haplotype frequencies were calculated. RESULTS: Of the 204 HIV-1 seropositive patients and 200 controls, 4.9% (95%CI: 2.38-8.83) and 2.50% (95% CI: 0.82-5.74) were HBsAg-positive respectively. There was a significant association between increasing age (> 40 years) and HBV infection among controls (p = 0.04). The distribution of genotypes and alleles frequencies of APOBEC3G variants was heterogenous and five different haplotypes with frequencies ≥ 5% were obtained, of which ACC (rs8177832, rs35228531, rs2294367) was the most prevalent. CONCLUSIONS: HBV co-infection is common among HIV-1 infected individuals in Morocco. Efforts should be made to prevent, treat and control HBV transmission in this population.

Two complete 1918 influenza A/H1N1 pandemic virus genomes characterized by next-generation sequencing using RNA isolated from formalin-fixed, paraffin-embedded autopsy lung tissue samples along with evidence of secondary bacterial co-infection.

The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus-influenza virus co-infection. IMPORTANCE: This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

Co-infection of Aspergillus ochraceopetaliformis strain RCEF7483 by a novel chrysovirus and a known partitivirus.

An airborne microflora isolate, Aspergillus ochraceopetaliformis RCEF7483, was found to harbor seven dsRNA elements, indicating co-infection with a novel chrysovirus and a known partitivirus. Sequence analysis and RT-PCR confirmed dsRNA5-7 as components of Aspergillus ochraceous virus (AOV), a member of the Partitiviridae family. In light of its distinct host, we have designated it Aspergillus ochraceopetaliformis partitivirus 1 (AoPV1). The dsRNA segments, named dsRNA1-4, with lengths of 3706 bp, 3410 bp, 3190 bp, and 3158 bp, respectively, constitute the genome of a novel chrysovirus designated Aspergillus ochraceopetaliformis chrysovirus 1 (AoCV1). The dsRNA1-4 segments contain five open-reading frames (ORF1-5). Specifically, ORF1 encodes a putative RNA-dependent RNA polymerase (RdRp) with a length of 1112 amino acids, and ORF2 encodes a putative coat protein (CP) spanning 976 amino acids. Additionally, ORF3-5 encode hypothetical proteins (HP1, HP2, and HP3) with lengths of 108, 843, and 914 amino acids, respectively. Comparative analysis revealed the highest similarity of dsRNA1-4 with corresponding proteins in Aspergillus terreus chrysovirus 1 (AtCV1) (RdRp, 66.58%; CP, 51.02%; HP2, 61.80%; and HP3, 41.30%). Due to falling below the threshold for a new species in the Chrysoviridae, we propose that dsRNA1-4 in A. ochraceopetaliformis strain RCEF7483 constitute the novel chrysovirus AoCV1. Moreover, phylogenetic analysis using RdRp amino acid sequences placed AoCV1 within the Alphachrysovirus genus of the Chrysoviridae family, clustering with AtCV1 and other alphachrysoviruses. Our study contributes to the understanding of mycoviruses in A. ochraceopetaliformis and expands our knowledge of the diversity and evolution of chrysoviruses in fungal hosts.

Synchronous Epidermodysplasia Verruciformis and Intraepithelial Lesion of the Vulva Is Caused by Coinfection With Alpha-Human Papillomavirus and Beta-Human Papillomavirus Genotypes and Facilitated by Mutations in Cell-Mediated Immunity Genes.

CONTEXT.­: There have been exceedingly few reports of epidermodysplasia verruciformis (EV) or EV-like lesions in the vulva. We describe the first observation of vulvar lesions displaying synchronous EV-like histology and conventional high-grade squamous intraepithelial lesion (HSIL), a finding hitherto unreported in medical literature. OBJECTIVES.­: To describe this novel vulvar lesion with hybrid features of HSIL and EV, attempt to confirm the hypothesis of coinfection with α and ß human papillomavirus (α-HPV and ß-HPV) genotypes, and describe relevant underlying genetic mutations. DESIGN.­: Cases were retrospectively selected from our institutional archive. Detailed review of clinical information, histologic examination, and whole genome sequencing (WGS) were performed. RESULTS.­: Five samples from 4 different patients were included. Three of 4 patients had a history of either iatrogenic immune suppression or prior immune deficiency, and all 3 featured classic HSIL and EV changes within the same lesion. One patient had no history of immune disorders, presented with EV-like changes and multinucleated atypia of the vulva, and was the sole patient without conventional HSIL. By WGS, several uniquely mappable reads pointed toward infection with multiple HPV genotypes, including both α-HPVs and ß-HPVs. Mutations in genes implicated in cell-mediated immunity, such as DOCK8, CARMIL2, MST1, and others, were also found. CONCLUSIONS.­: We provide the first description of vulvar lesions harboring simultaneous HSIL and EV features in the English-language literature, a phenomenon explained by coinfection with α-HPV and ß-HPV genotypes. The finding of EV-like changes in a vulvar specimen should prompt assessment of the patient's immune status.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Virome Data Explorer: A web resource to longitudinally explore respiratory viral infections, their interactions with other pathogens and host transcriptomic changes in over 100 people.

Viral respiratory infections are an important public health concern due to their prevalence, transmissibility, and potential to cause serious disease. Disease severity is the product of several factors beyond the presence of the infectious agent, including specific host immune responses, host genetic makeup, and bacterial coinfections. To understand these interactions within natural infections, we designed a longitudinal cohort study actively surveilling respiratory viruses over the course of 19 months (2016 to 2018) in a diverse cohort in New York City. We integrated the molecular characterization of 800+ nasopharyngeal samples with clinical data from 104 participants. Transcriptomic data enabled the identification of respiratory pathogens in nasopharyngeal samples, the characterization of markers of immune response, the identification of signatures associated with symptom severity, individual viruses, and bacterial coinfections. Specific results include a rapid restoration of baseline conditions after infection, significant transcriptomic differences between symptomatic and asymptomatic infections, and qualitatively similar responses across different viruses. We created an interactive computational resource (Virome Data Explorer) to facilitate access to the data and visualization of analytical results.

Effects of RNA silencing during antagonism between citrus exocortis viroid and citrus bark cracking viroid in Etrog citron (Citrus medica).

Citrus exocortis viroid (CEVd) and citrus bark cracking viroid (CBCVd) are two important viroids that infect citrus plants and frequently occur as mixed infections in orchards. However, the mechanism of antagonism between the two viroids in mixed infections remains unclear. The CEVd/CBCVd-citron system and small RNA sequencing (sRNA-seq) were used to study the antagonism. When CBCVd was inoculated before CEVd, the CEVd titre was significantly reduced and the symptoms were attenuated. Viroid-derived sRNAs (vd-sRNAs) from CEVd and CBCVd were predominantly 21-nucleotide (nt) and 22-nt in length and had similar 5' base biases. Homologous sequences of the two viroids in the terminal right (TR) region are rich in vd-sRNAs, and the high frequency vd-sRNAs selected from the CBCVd TR region can be used to degrade the transcripts of CEVd in vivo directly. These results suggest that RNA silencing may play an important role in the antagonism of the two viroids, thus deepening our understanding of the molecular interaction of long noncoding RNAs in woody plants.

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Severity of Schistosoma haematobium co-infection with malaria in school-children is potentially modulated by host CD14 gene variants.

OBJECTIVE: Schistosomiasis remains a chronic disease of global importance, especially in many rural areas of the world where co-infection with Plasmodium falciparum is common. It is critical to decipher the role of single or co-infected disease scenarios on immune system regulation in such individuals and how such co-infections can either ameliorate or complicate immune response and the consequent disease outcome. First, 10 ml of urine samples, collected between 10:00 am and 2:00 pm, was filtered for diagnosis of schistosomiasis, while egg count, indicative of disease severity, was determined by microscopy. Furthermore, genomic DNA samples extracted from dried blood spots collected on filter paper from one hundred and forty-four Schistosoma haematobium-infected school-children was tested for P. falciparum parasite positivity by an allele-specific nested-PCR analysis of merozoite surface protein (msp)-1 and -2 genes and a real-time PCR assay. In addition, among P. falciparum parasite-positive individuals, we carried out a Taqman SNP genotyping assay to extrapolate the effect of host CD14 (-159 C/T; rs2569190) genetic variants on schistosome egg count. RESULTS: Of the 144 individuals recruited, P. falciparum parasite positivity with msp-1 gene were 34%, 43% and 55% for MAD20, RO33 and K1 alleles respectively. Of the co-infected individuals, CD14 genetic variants ranged from 18.8% vs 21.5%, 33.3% vs 44.4%, 9.7% vs 11.8% for single versus schistosome co-infection for the wild type (CC), heterozygous (CT) and mutant (TT) variants respectively. Though the mean egg count for co-infected individuals with CD14 wild type (33.7 eggs per 10 ml of urine) and heterozygote variants (37.5 eggs per 10 ml of urine) were lower than that of schistosome infection alone (52.48 and 48.08 eggs/10 ml of urine respectively), it lacked statistical significance (p-value 0.12 and 0.29), possibly reflecting the benefit of the CD14 activation in schistosome plus malaria co-infection and not schistosome infection alone. In addition, the lower mean egg count in co-infected individuals reveal the benefit of downstream Th1 immune response mitigated by CD14 innate activation that is absent in schistosome infection alone.

MicroRNA signature from extracellular vesicles of HCV/HIV co-infected individuals differs from HCV mono-infected.

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus-host interaction. KEY MESSAGES: HCV and HCV/HIV displayed similar plasma-EV size and concentration. EVs- derived miRNA profile was characterized by NGS. 37 SDE miRNAs between HCV and HCV/HIV were observed. SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.

Evaluation of an inducible knockout system in insect cells based on co-infection and CRISPR/Cas9.

Due to comparably high product titers and low production costs, the baculovirus/insect cell expression system is considered a versatile production platform in the biopharmaceutical industry. Its excellence in producing complex multimeric protein assemblies, including virus-like particles (VLPs), which are considered promising vaccine candidates to counter emerging viral threats, made the system even more attractive. However, the co-formation of budded baculovirus during VLP production poses a severe challenge to downstream processing. In order to reduce the amount of budded baculovirus in the expression supernatant we developed an inducible knockout system based on CRISPR/Cas9 and co-infection with two baculoviral vectors: one bringing along the Cas9 nuclease and the other one having incorporated the sequence for sgRNA expression. With our set-up high titer viruses can be generated separately, as only when both viruses infect cells simultaneously a knockout can occur. When budding essential genes gp64 and vp80 were targeted for knockout, we measured a reduction in baculovirus titer by over 90%. However, as a consequence, we also determined lower overall eYFP fluorescence intensity showing reduced recombinant protein production, indicating that further improvements in engineering as well as purification are required in order to ultimately minimize costs and timeframes for vaccine production utilizing the baculovirus/insect cell expression system.

Mixed infections with new emerging viruses associated with jujube mosaic disease.

BACKGROUND: Jujube is an economically important fruit tree and native to China. Viral disease is a new threat to jujube production, and several new viruses have been identified infecting jujube plants. During our field survey, jujube mosaic disease was widely distributed in Beijing, but the associated causal agents are still unknown. METHODS: Small RNA deep sequencing was conducted to identify the candidate viruses associated with jujube mosaic. Further complete genome sequences of the viruses were cloned, and the genomic characterization of each virus was analyzed. The field distribution of these viruses was further explored with PCR/RT-PCR detection of field samples. RESULTS: Mixed infection of four viruses was identified in a plant sample with the symptom of mosaic and leaf twisting, including the previously reported jujube yellow mottle-associated virus (JYMaV), persimmon ampelovirus (PAmpV), a new badnavirus tentatively named jujube-associated badnavirus (JaBV), and a new secovirus tentatively named jujube-associated secovirus (JaSV). PAmpV-jujube was 14,093 nt in length with seven putative open reading frames (ORFs) and shared highest (79.4%) nucleotide (nt) sequence identity with PAmpV PBs3. Recombination analysis showed that PAmpV-jujube was a recombinant originating from plum bark necrosis stem pitting-associated virus isolates nanjing (KC590347) and bark (EF546442). JaBV was 6449 bp in length with conserved genomic organization typical of badnaviruses. The conserved RT and RNAse H region shared highest 67.6% nt sequence identity with jujube mosaic-associated virus, which was below the 80% nt sequence identity value used as the species demarcation threshold in Badnavirus. The genome of JaSV composed of two RNA molecules of 5878 and 3337 nts in length, excluding the polyA tails. Each genome segment contained one large ORF that shared homology and phylogenetic identity with members of the family Secoviridae. Field survey showed JYMaV and JaBV were widely distributed in jujube trees in Beijing. CONCLUSION: Two new viruses were identified from jujube plants, and mixed infections of JYMaV and JaBV were common in jujube in Beijing.

Genetic complementation fosters evolvability in complex fitness landscapes.

The ability of natural selection to optimize traits depends on the topology of the genotype-fitness map (fitness landscape). Epistatic interactions produce rugged fitness landscapes, where adaptation is constrained by the presence of low-fitness intermediates. Here, we used simulations to explore how evolvability in rugged fitness landscapes is influenced by genetic complementation, a process whereby different sequence variants mutually compensate for their deleterious mutations. We designed our model inspired by viral populations, in which genetic variants are known to interact frequently through coinfection. Our simulations indicate that genetic complementation enables a more efficient exploration of rugged fitness landscapes. Although this benefit may be undermined by genetic parasites, its overall effect on evolvability remains positive in populations that exhibit strong relatedness between interacting sequences. Similar processes could operate in contexts other than viral coinfection, such as in the evolution of ploidy.

PBMCs gene expression signature of advanced cirrhosis with high risk for clinically significant portal hypertension in HIV/HCV coinfected patients: A cross-control study.

BACKGROUND: Patients with advanced cirrhosis are at high risk of developing clinically significant portal hypertension (CSPH). We analyzed the gene expression profile of peripheral blood mononuclear cells (PBMCs) from HIV/HCV coinfected patients to identify a gene expression signature of advanced cirrhosis with high risk for CSPH. METHODS: We conducted a cross-sectional study on 68 patients. Liver stiffness measurement (LSM) was used to stratify patients into < 12.5 kPa (no cirrhosis, n = 19), 12.5 - 24.9 kPa (cirrhosis, n = 20), and ≥ 25 kPa (advanced cirrhosis with high risk for CSPH, n = 29). Besides, we further evaluated LSM < 25 kPa (n = 39) vs. ≥ 25 kPa (n = 29). Total RNA was extracted from PBMCs, and poly(A) RNA sequencing was performed. Two significant differentially expressed (SDE) transcripts were validated by quantitative PCR in a different cohort (n = 46). RESULTS: We found 60 SDE transcripts between patients with LSM < 12.5 kPa and ≥ 25 kPa. Partial least squares discriminant analysis showed that those 60 SDE transcripts collectively discriminated LSM ≥ 25 kPa, with an area under the receiver operating characteristic curve (AUROC) of 0.84. Eight genes had an AUROC ≥ 0.75 for LSM ≥ 25 kPa: five were positively associated with LSM values (SCAMP1, ABHD17B, GPR146, GTF2A1, and TMEM64), while three were inversely associated (ZFHX2-AS1, MDK, and STAG3L2). We validated the two SDE transcripts with the highest discrimination capacity in a different cohort, finding significant differences between < 25 kPa and ≥ 25 kPa (MDK (p = 0.006) and STAG3L2 (p = 0.021)). CONCLUSIONS: A gene expression signature of 60 transcripts was associated with advanced cirrhosis with high risk for CSPH in HIV/HCV coinfected patients.

Mixed Infection of Blackcurrant with a Novel Cytorhabdovirus and Black Currant-Associated Nucleorhabdovirus.

A virome screen was performed on a new breeding line, KB1, of blackcurrant. Rhabdovirus-like particles were observed by electron microscopy in ultrathin sections of flower stalks, and the complete genome sequence of a novel virus, provisionally named blackcurrant rhabdovirus 2 (BCRV2), was determined and verified using high-throughput sequencing. The genomic organization of BCRV2 was characteristic of cytorhabdoviruses (family Rhabdoviridae) and included seven genes: 3 ́- N-P´-P-P3-M-G-L -5 ́. BLASTP analysis revealed that the putative L protein had the highest amino acid sequence identity (75 %) with strawberry virus 2. BCRV2 was detected in Cryptomyzusgaleopsidis, but efficient transmission by this aphid was not confirmed. Of note, we observed coinfection of the KB1 line with blackcurrant-associated rhabdovirus (BCaRV) by RT-PCR. This is likely the first evidence of the presence of a cyto- and a nucleorhabdovirus in a single host.