Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.

A cell atlas of the adult female Aedes aegypti midgut revealed by single-cell RNA sequencing.

Aedes aegypti is a primary vector for transmitting various arboviruses, including Yellow fever, dengue and Zika virus. The mosquito midgut is the principal organ for blood meal digestion, nutrient absorption and the initial site of arbovirus infection. Although a previous study delineated midgut's transcriptome of Ae. aegypti at the single-nucleus resolution, there still lacks an established protocol for isolating and RNA sequencing of single cells of Ae. aegypti midgut, which is required for investigating arbovirus-midgut interaction at the single-cell level. Here, we established an atlas of the midgut cells for Ae. aegypti by single-cell RNA sequencing. We annotated the cell clusters including intestinal stem cells/enteroblasts (ISC/EB), cardia cells (Cardia), enterocytes (EC, EC-like), enteroendocrine cells (EE), visceral muscle (VM), fat body cells (FBC) and hemocyte cells (HC). This study will provide a foundation for further studies of arbovirus infection in mosquito midgut at the single-cell level.

Alert for pharynx-centered gastrointestinal and respiratory cross-infection and cryptic cross-transmission routes of 2019-nCoV.

We proposed that the pharynx, as a common organ of the respiratory and digestive tracts, may be a respiratory and digestive tract cross cryptic transmission pathway for 2019-nCoV infection from the nasal cavities to the pharynx and lung, then to nasal cavities by aerosol (respiratory route) to the pharynx and the gastrointestinal tract, then to the oral cavity by feces (fecal-oral route) and to pharynx, lungs, or gastrointestinal tract.

Regulation of host/pathogen interactions in the gastrointestinal tract by type I and III interferons.

Interferons (IFNs) are an integral component of the host innate immune response during viral infection. Recent advances in the study of type I and III IFNs suggest that though both types counteract viral infection, type III IFNs act predominantly at epithelial barrier sites, while type I IFNs drive systemic responses. The dynamics and specific roles of type I versus III IFNs have been studied in the context of infection by a variety of enteric pathogens, including reovirus, rotavirus, norovirus, astrovirus, and intestinal severe acute respiratory syndrome coronavirus 2, revealing shared patterns of regulatory influence. An important role for the gut microbiota, including the virome, in regulating homeostasis and priming of intestinal IFN responses has also recently emerged.

Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions.

Salmonella is a primary enteric pathogen related to the contamination of poultry and other food products in numerous foodborne outbreaks. The continuous emergence of multidrug-resistant bacteria has become a serious issue due to the overuse of antibiotics. Hence, lytic phages are considered alternative biocontrol agents against these bacterial superbugs. Here, two Salmonella phages-S4lw and D5lw-were subjected to genomic and biological characterization and further encapsulated to improve the stability under acidic conditions mimicking gastrointestinal conditions. The two lytic phages, S4lw and D5lw, taxonomically belong to new species under the Guernseyvirinae and Ackermannviridae families, respectively. Each phage showed antimicrobial activities against diverse Salmonella spp., such as S. Enteritidis and S. Typhimurium, achieving 1.7-3.4 log reduction after 2-6 h of treatment. The phage cocktail at a multiplicity of infection (MOI) of 100 or 1000 completely inhibited these Salmonella strains for at least 14 h at 25 °C. Additionally, the bead-encapsulated phage cocktail could withstand low pH and different simulated gut environments for at least 1 h. Overall, the newly isolated phages can potentially mitigate Salmonella spp. under the gastrointestinal environments through encapsulation and may be further applied via oral administration to resolve common antimicrobial resistance issues in the poultry production chain.

Longitudinal analysis of the enteric virome in paediatric subjects from the Free State Province, South Africa, reveals early gut colonisation and temporal dynamics.

The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut.

Altered Immunoglobulin Repertoire and Decreased IgA Somatic Hypermutation in the Gut during Chronic HIV-1 Infection.

Humoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection. IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

The SARS-CoV-2 Alpha variant exhibits comparable fitness to the D614G strain in a Syrian hamster model.

Late 2020, SARS-CoV-2 Alpha variant emerged in United Kingdom and gradually replaced G614 strains initially involved in the global spread of the pandemic. In this study, we use a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeed to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicate to almost the same level and induced a comparable disease and immune response. A slight fitness advantage is noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium. This study highlights the need to combine data from different laboratories using various animal models to decipher the biological properties of newly emerging SARS-CoV-2 variants.

Protein expression of transmembrane protease serine 4 in the gastrointestinal tract and in healthy, cancer, and SARS­CoV­2 infected lungs.

In addition to the angiotensin­converting enzyme 2 (ACE2), a number of host cell entry mediators have been identified for severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), including transmembrane protease serine 4 (TMPRSS4). The authors have recently demonstrated the upregulation of TMPRSS4 in 11 different cancers, as well as its specific expression within the central nervous system using in silico tools. The present study aimed to expand the initial observations and, using immunohistochemistry, TMPRSS4 protein expression in the gastrointestinal (GI) tract and lungs was further mapped. Immunohistochemistry was performed on tissue arrays and lung tissues of patients with non­small cell lung cancer with concurrent coronavirus disease 2019 (COVID­19) infection using TMPRSS4 antibody. The results revealed that TMPRSS4 was abundantly expressed in the oesophagus, stomach, small intestine, jejunum, ileum, colon, liver and pancreas. Moreover, the extensive TMPRSS4 protein expression in the lungs of a deceased patient with COVID­19 with chronic obstructive pulmonary disease and bronchial carcinoma, as well in the adjacent normal tissue, was demonstrated for the first time, at least to the best of our knowledge. On the whole, the immunohistochemistry data of the present study suggest that TMPRSS4 may be implicated in the broader (pulmonary and extra­pulmonary) COVID­19 symptomatology; thus, it may be responsible for the tropism of this coronavirus both in the GI tract and lungs.

Genome binning of viral entities from bulk metagenomics data.

Despite the accelerating number of uncultivated virus sequences discovered in metagenomics and their apparent importance for health and disease, the human gut virome and its interactions with bacteria in the gastrointestinal tract are not well understood. This is partly due to a paucity of whole-virome datasets and limitations in current approaches for identifying viral sequences in metagenomics data. Here, combining a deep-learning based metagenomics binning algorithm with paired metagenome and metavirome datasets, we develop Phages from Metagenomics Binning (PHAMB), an approach that allows the binning of thousands of viral genomes directly from bulk metagenomics data, while simultaneously enabling clustering of viral genomes into accurate taxonomic viral populations. When applied on the Human Microbiome Project 2 (HMP2) dataset, PHAMB recovered 6,077 high-quality genomes from 1,024 viral populations, and identified viral-microbial host interactions. PHAMB can be advantageously applied to existing and future metagenomes to illuminate viral ecological dynamics with other microbiome constituents.

Cytomegalovirus cell tropism and clinicopathological characteristics in gastrointestinal tract of patients with HIV/AIDS.

BACKGROUND: Cytomegalovirus (CMV) has been recognized as one of the frequently occurring opportunistic infections (OIs) reported in the patients having human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). In addition, it has been identified as the factor leading to gastrointestinal (GI) tract disorder among HIV/AIDS population. CMV exhibits broad cell tropism in different organs. This study evaluated the CMV cell tropism and clinicopathological characteristics of CMV infection in the different GI regions in HIV/AIDS cases. METHODS: Using nucleic acid in situ hybridization (ISH), CMV was detected in the gastrointestinal mucosal biopsy samples. The paraffin-embedded samples were stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC), respectively. RESULTS: A total of 32 HIV/AIDS patients were enrolled in this study. Fourteen of these patients underwent gastroscopy, while the remaining eighteen received colonoscopy. CMV-infected cells were observed at 46 GI sites. Among them, the colon was the region with the highest susceptibility to GI CMV infection (n = 12, 26.1%). The CMV giant cell inclusion bodies were detected in epithelial cells and mesenchymal cells, including histiocytes, smooth muscle cells, fibroblasts, and endothelial cells. In the duodenum, there were markedly more positive epithelial cells than mesenchymal cells (p = 0.033). In contrast, in the esophagus (p = 0.030), cardia (p = 0.003), rectum (p = 0.019), colon (p < 0.001), and cecum (p < 0.001), there were notably less positive epithelial cells than mesenchymal cells. The expression levels of PDGFRα and Nrp2 in the mesenchymal cells were higher than the epithelial cells in cardia, cecum, colon, sigmoid, and rectum, especially in the areas with ulcers. However, Nrp2 in the epithelial cells was higher than that in the duodenum. Moreover, the positive CMV DNA in peripheral blood was related to the CMV-positive cell count, as well as the ulceration in GI tract (p = 0.035 and 0.036, respectively). CONCLUSIONS: The colon has been identified as the GI site with the highest susceptibility to CMV infection. There are different CMV-infected cells in the different sites of the GI that relate to the expression level of PDGFRα and Nrp2. CMV DNA positive in the blood is related to the positive CMV cell count, as well as ulceration in the GI tract.

Intestinal viral infections of nSARS-CoV2 in the Indian community: Risk of virus spread in India.

In December 2019, novel severe acute respiratory syndrome coronavirus 2 (nSARS-CoV-2) virus outbreaks emerged from Wuhan, China, and spread all over the world, including India. Molecular diagnosis of Coronavirus Disease 2019 (COVID) 19 for densely and highly populated countries like India is time-consuming. A few reports have described the successful diagnosis of nSARS-CoV-2 virus from sewage and wastewater samples contaminated with fecal matter, suggesting the diagnosis of COVID 19 from the same to raise an alarm about the community transmission of virus for implementation of evacuation and lockdown strategies. So far, the association between the detection of virus and its concentration in stool samples with severity of the disease and the presence or absence of gastrointestinal symptoms have been rarely reported. We led the search utilizing multiple databases, specifically PubMed (Medline), EMBASE, and Google Scholar. We conducted a literature survey on gastrointestinal infection and the spread of this virus through fecal-oral transmission. Reports suggested that the existence and persistence of nSARS-CoV-2 in anal/rectal swabs and stool specimens for a longer period of time than in nasopharyngeal swabs provides a strong tenable outcome of gastrointestinal contamination and dissemination of this infection via potential fecal-oral transmission. This review may be helpful to conduct further studies to address the enteric involvement and excretion of nSARS-CoV-2 RNA in feces and control the community spread in both COVID-19 patients ahead of the onset of symptoms and in asymptomatic individuals through wastewater and sewage surveillance as an early indication of infection. The existence of the viral genome and active viral particle actively participate in genomic variations. Hence, we comprehended the enteric spread of different viruses amongst communities with special reference to nSARS-CoV-2.

Alternative Splicing Landscape of Small Brown Planthopper and Different Response of JNK2 Isoforms to Rice Stripe Virus Infection.

Alternative splicing (AS) is a frequent posttranscriptional regulatory event occurring in response to various endogenous and exogenous stimuli in most eukaryotic organisms. However, little is known about the effects of insect-transmitted viruses on AS events in insect vectors. The present study used third-generation sequencing technology and RNA sequencing (RNA-Seq) to evaluate the AS response in the small brown planthopper Laodelphax striatellus to rice stripe virus (RSV). The full-length transcriptome of L. striatellus was obtained using single-molecule real-time sequencing technology (SMRT). Posttranscriptional regulatory events, including AS, alternative polyadenylation, and fusion transcripts, were analyzed. A total of 28,175 nonredundant transcript isoforms included 24,950 transcripts assigned to 8,500 annotated genes of L. striatellus, and 5,000 of these genes (58.8%) had AS events. RNA-Seq of the gut samples of insects infected by RSV for 8 d identified 3,458 differentially expressed transcripts (DETs); 2,185 of these DETs were transcribed from 1,568 genes that had AS events, indicating that 31.4% of alternatively spliced genes responded to RSV infection of the gut. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, experienced exon skipping, resulting in three transcript isoforms. These three isoforms differentially responded to RSV infection during development and in various organs. Injection of double-stranded RNAs targeting all or two isoforms indicated that three or at least two JNK2 isoforms facilitated RSV accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors. IMPORTANCE Alternative splicing (AS) is a regulatory mechanism that occurs after gene transcription. AS events can enrich protein diversity to promote the reactions of the organisms to various endogenous and exogenous stimulations. It is not known how insect vectors exploit AS events to cope with transmitted viruses. The present study used third-generation sequencing technology to obtain the profile of AS events in the small brown planthopper Laodelphax striatellus, which is an efficient vector for rice stripe virus (RSV). The results indicated that 31.4% of alternatively spliced genes responded to RSV infection in the gut of planthoppers. One of the c-Jun N-terminal kinase (JNK) genes, JNK2, produced three transcript isoforms by AS. These three isoforms showed different responses to RSV infection, and at least two isoforms facilitated viral accumulation in planthoppers. These results implied that AS events could participate in the regulation of complex relationships between viruses and insect vectors.