ABSTRACT
The hepatitis E virus (HEV) is infecting over 20 million people annually with a high morbidity especially in pregnant women and immune-suppressed individuals. While HEV genotype 1 (HEV-1) infects only humans, genotype 3 (HEV-3) is zoonotic and commonly transmitted from infected animals to humans. Whereas a few reverse genetics systems enabling targeted genome manipulations exist for HEV-3, those for HEV-1 are still very limited, mainly because of inefficient cell culture replication. Here, the generation of HEV-1 strain Sar55 and HEV-3 strain 47832mc by transfecting in vitro-transcribed and capped virus genomes into different cell lines was attempted. Culture supernatants of colon-derived colorectal adenocarcinoma cell line Caco-2 contained HEV-1 and HEV-3 capable of infecting Caco-2 cells. Density gradient centrifugation analyses of culture supernatants confirmed that HEV-1 particles were quasi-enveloped in analogy to HEV-3 and that non-virion-associated capsid protein was secreted from cells. Following transfection or infection of Caco-2 cells, HEV-1 consistently reached higher titers than HEV-3 in culture supernatants, but HEV-1 generated by transfection of Caco-2 cells was unable to efficiently infect hepatoma cell lines PLC/PRF/5 or HuH7-Lunet BLR. Taken together, our results indicate that HEV-1 is able to exert a complete replication cycle in Caco-2 cells. An efficient cell culture system for this genotype will be useful for studying species tropism, but further research is required to determine the significance of HEV-1 replication in colon-derived cells.
Subject(s)
Genotype , Hepatitis E virus , Reverse Genetics , Virus Replication , Humans , Hepatitis E virus/genetics , Hepatitis E virus/physiology , Caco-2 Cells , Reverse Genetics/methods , Colon/virology , Genome, Viral , Hepatitis E/virologyABSTRACT
There are no other bovine coronavirus (BCoV) infection models except calves, which makes efficacy evaluation of vaccines and pathogenic mechanism research of BCoV inconvenient owing to their high value and inconvenient operation. This study aimed to establish a mouse model of BCoV infection. BCoV was used to infect 4-week-old male BALB/c mice and the optimal infection conditions were screened, including the following infection routes: gavage, intraperitoneal injection, and tail vein injection at doses of 1 × 108 TCID50, 2 × 108 TCID50 and 4 × 108 TCID50. Using the optimal infection conditions, BALB/c mice were infected with BCoV, and their body weight, blood routine, inflammatory factors, autopsy, virus distribution, and viral load were measured at 1, 3, 5, and 7 days after infection. The results showed that the optimal conditions for infecting BALB/c mice with BCoV HLJ-325 strain were continuous oral gavage for 3 days with a dose of 4 × 108 TCID50. On the 7th day after infection, there was significant extensive consolidation of the lungs and thinning of the colon wall. Significant inflammation was observed in various organs, especially in the colon and alveoli, where a large number of inflammatory cells infiltrate. Both BCoV Ag and nucleic acid are positive in visceral organs. The viral load in the colon and lungs was significantly higher than that in the other organs (p < 0.001). BCoV-infected mice showed a decreasing trend in body weight starting from day 5, and there was a significant difference compared to the control group on days 6 and 7 (p < 0.001). The total number of white blood cells and lymphocytes began to decrease and was significantly lower than that in the control group 24 h after infection (p < 0.001), and gradually returned to the control level. The cytokine TNF-α, IL-1ß, and IL-6 showed an increasing trend, significantly higher than the control group on day 5 and 7 (p < 0.001). These results indicate that the BCoV HLJ-325 strain can infect BALB/c mice and cause inflammatory reactions and tissue lesions. The most significant effect was observed on the seventh day after infection with a dose of 4 × 108 TCID50 and three consecutive gavages. This study established, for the first time, a BALB/c mouse model of BCoV infection, providing a technical means for evaluating the immune efficacy of BCoV vaccines and studying their pathogenic mechanisms.
Subject(s)
Coronavirus Infections , Coronavirus, Bovine , Disease Models, Animal , Lung , Mice, Inbred BALB C , Viral Load , Animals , Mice , Male , Lung/pathology , Lung/virology , Coronavirus Infections/pathology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cattle , Disease Susceptibility , Colon/pathology , Colon/virology , Interleukin-6/blood , Interleukin-1beta , Tumor Necrosis Factor-alpha , Cytokines/metabolism , Cytokines/blood , Body WeightABSTRACT
Microbiomes feature complex interactions between diverse bacteria and bacteriophages. Synthetic microbiomes offer a powerful way to study these interactions; however, a major challenge is obtaining a representative bacteriophage population during the bacterial isolation process. We demonstrate that colony isolation reliably excludes virulent viruses from sample sources with low virion-to-bacteria ratios such as feces, creating "virulent virus-free" controls. When the virulent dsDNA virome is reintroduced to a 73-strain synthetic gut microbiome in a bioreactor model of the human colon, virulent viruses target susceptible strains without significantly altering community structure or metabolism. In addition, we detected signals of prophage induction that associate with virulent predation. Overall, our findings indicate that dilution-based isolation methods generate synthetic gut microbiomes that are heavily depleted, if not devoid, of virulent viruses and that such viruses, if reintroduced, have a targeted effect on community assembly, metabolism, and prophage replication.
Subject(s)
Bacteria , Bacteriophages , Feces , Gastrointestinal Microbiome , Bacteriophages/genetics , Bacteriophages/physiology , Humans , Feces/microbiology , Feces/virology , Bacteria/virology , Bacteria/genetics , Prophages/genetics , Prophages/physiology , Virome , Bioreactors/microbiology , Bioreactors/virology , Colon/microbiology , Colon/virology , Microbiota , VirulenceABSTRACT
BACKGROUND: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). METHODS: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. FINDINGS: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. INTERPRETATION: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. FUNDING: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).
Subject(s)
Endogenous Retroviruses , HIV Infections , HIV-1 , Humans , HIV Infections/virology , HIV Infections/immunology , HIV Infections/genetics , HIV-1/genetics , Endogenous Retroviruses/genetics , Male , Female , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , Adult , Middle Aged , Colon/metabolism , Colon/virology , Colon/pathology , Long Interspersed Nucleotide Elements/genetics , Retroelements/genetics , Gene Expression Profiling , Gene Expression Regulation , Gastrointestinal MicrobiomeABSTRACT
The Omicron variant of SARS-CoV-2, characterized by multiple subvariants including BA.1, XBB.1.5, EG.5, and JN.1, became the predominant strain in early 2022. Studies indicate that Omicron replicates less efficiently in lung tissue compared to the ancestral strain. However, the infectivity of Omicron in the gastrointestinal tract is not fully defined, despite the fact that 70% of COVID-19 patients experience digestive disease symptoms. Here, using primary human colonoids, we found that, regardless of individual variability, Omicron infects colon cells similarly or less effectively than the ancestral strain or the Delta variant. The variant induced limited type III interferon expression and showed no significant impact on epithelial integrity. Further experiments revealed inefficient cell-to-cell spread and spike protein cleavage in the Omicron spike protein, possibly contributing to its lower infectious particle levels. The findings highlight the variant-specific replication differences in human colonoids, providing insights into the enteric tropism of Omicron and its relevance to long COVID symptoms.
Subject(s)
COVID-19 , Colon , Epithelial Cells , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , SARS-CoV-2/pathogenicity , Colon/virology , COVID-19/virology , Epithelial Cells/virology , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/genetics , Virus Replication , Interferon LambdaABSTRACT
IMPORTANCE: Alterations of the gut microbiome can have significant effects on gastrointestinal homeostasis leading to various diseases and symptoms. Increased understanding of rotavirus infection in relation to the microbiota can provide better understanding on how microbiota can be used for clinical prevention as well as treatment strategies. Our volumetric 3D imaging data show that antibiotic treatment and its consequent reduction of the microbial load does not alter the extent of rotavirus infection of enterocytes in the small intestine and that restriction factors other than bacteria limit the infection of colonocytes.
Subject(s)
Colon , Gastrointestinal Microbiome , Rotavirus Infections , Animals , Humans , Colon/virology , Gastrointestinal Tract , Intestine, Small/virology , Rotavirus , MiceABSTRACT
Introduction: Human immunodeficiency virus type 1 (HIV) transmission mostly occurs through the genital and intestinal mucosae. Although HIV-1 transmission has been extensively investigated, gaps remain in understanding the initial steps of HIV entry through the colonic mucosa. We previously showed that HIV can selectively trigger mononuclear phagocytes (MNP) to migrate within colonic epithelial cells to sample virions. Mucosal exposure to human seminal plasma (HSP), rich in pro- and anti-inflammatory cytokines, chemokines and growth factors, may as well induce alterations of the colonic mucosa and recruit immune cells, hence, affecting pathogen sampling and transmission. Methods: Here, we studied the role of HSP on the paracellular intestinal permeability by analyzing the distribution of two proteins known to play a key role in controlling the intestinal barrier integrity, namely the tight junctions-associated junctional adhesion molecule (JAM-A) and the adherents junction associated protein E-cadherin (E-CAD), by immunofluorescence and confocal microscopy. Also, we evaluated if HSP promotes the recruitment of MNP cells, specifically, the CD11c and CD64 positive MNPs, to the apical side of the human colonic mucosa. At this scope, HSP of HIV-infected and uninfected individuals with known fertility status was tested for cytokines, chemokines and growth factors concentration and used in an ex vivo polarized colonic tissue culture system to mimic as closely as possible the physiological process. Results: HSP showed statistically significant differences in cytokines and chemokines concentrations between the three groups of donors, i.e. HIV infected, or uninfected fertile or randomly identified. Nevertheless, we showed that in the ex vivo tissue culture HSP in general, neither affected the morphological structure of the colonic mucosa nor modulated the paracellular intestinal permeability. Interestingly, CD11c+ MNP cells migrated to the apical surface of the colonic epithelium regardless, if incubated with HIV-infected or -uninfected HSPs, while CD64+ MNP cells, did not change their distribution within the colonic mucosa. Discussion: In conclusion, even if HSP did not perturb the integrity of the human colonic mucosa, it affected the migration of a specific subset of MNPs that express CD11c towards the apical side of the colonic mucosa, which in turn may be involved in pathogen sampling.
Subject(s)
Cell Movement , Colon , HIV Infections , Intestinal Mucosa , Monocytes , Semen , Humans , Cadherins/immunology , Cytokines/immunology , Epithelium/immunology , HIV Infections/immunology , HIV Infections/transmission , HIV Infections/virology , Junctional Adhesion Molecules , Phagocytes/immunology , Semen/immunology , Monocytes/immunology , CD11c Antigen/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , Colon/immunology , Colon/virology , HIV-1/immunology , Cell Movement/immunology , Virus Internalization , Host-Pathogen Interactions/immunologyABSTRACT
HIV/SIV (simian immunodeficiency virus) infection leads to a loss of CD4+ T helper (Th) cells in number and function that begins during the acute phase and persists through the chronic phase of infection. In particular, there is a drastic decrease of Th17 and Th22 cells in the HIV/SIV-infected gastrointestinal (GI) tract as a source of interleukin (IL)-17 and IL-22. These cytokines are vital in the immune response to extracellular pathogens and maintenance of the GI tract. However, innate lymphoid cells (ILCs) are a source of IL-17 and IL-22 during the early stages of an immune response in mucosal tissue and remain vital cytokine producers when the immune response is persistent. Here, we wanted to determine whether ILCs are a source of IL-17 and IL-22 in the SIV-infected colon and could compensate for the loss of Th17 and Th22 cells. As a control, we evaluated the frequency and number of ILCs expressing interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα). We determined the frequency and number of cytokine expressing ILC subsets and T cell subsets within leukocytes from the colons of uninfected as well as acute and chronic SIV-infected colons without in vitro mitogenic stimulation. In the present study, we find that: (1) the frequency of IL-22, IFNγ, and TNFα but not IL-17 producing ILCs is increased in the acutely infected colon and remains high during the chronically infected colon relative to cytokine expressing ILCs in the uninfected colon, (2) ILCs are a significant source of IL-22, IFNγ, and TNFα but not IL-17 when CD4+ T lymphocytes in the gut lose their capacity to secrete these cytokines during SIV infection, and (3) the changes in the cytokines expressed by ILCs relative to CD4+ T cells in the infected colon were not due to increases in the frequency or number of ILCs in relation to T lymphocytes found in the tissue.
Subject(s)
Cytokines , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Colon/pathology , Colon/virology , Cytokines/immunology , HIV Infections , Immunity, Innate , Interferon-gamma , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/immunology , Th17 Cells , Tumor Necrosis Factor-alphaABSTRACT
Here, we report the appearance of natural killer B (NKB) cells within the colon during simian immunodeficiency virus (SIV) infection of susceptible monkeys. Using RNA sequencing (RNAseq) and flow cytometry, we show that NKB cells are unique cells with features and functions of both NK and B cells. NKB cells express receptors and ligands found on B cells that are important for (i) antigen presentation; (ii) activities associated with class switching, affinity maturation, and B-cell memory formation in secondary lymphoid follicles; and (iii) antigen recognition. The predominant immunoglobulins (Igs) expressed on NKB cells are IgA, although NKB cells can express surface IgM and IgG. There is dominant lambda expression over the kappa light chain characteristic of mucosal B cells. In addition to B-cell aspects, NKB cells express NK cell activation receptors and Fas ligand. We show in this study that NKB cells express perforin and granzymes and lyse cells in a lytic assay. In addition to NK cell cytolytic function, NKB cells also produce the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin-18 (IL-18). Finally, we noted the increased capacity of NKB cells to proliferate compared to NK cells and CD8+ T cells from the SIV-infected colon. The increased proliferation and inflammatory cytokine production may be related to the relatively high expression levels of IL-15 receptor beta, IL-7 receptor, IL-18 receptor, and 41BB relative to the same receptors on CD8 and NK cells. The properties of NKB cells may point to their role in the enhanced inflammation observed in the SIV-infected gut. IMPORTANCE There is low-level but significant mucosal inflammation in the gastrointestinal tract secondary to human immunodeficiency virus (HIV) infection that has long-term consequences for the infected host. This inflammation most likely originates from the immune response that appears as a consequence of HIV. Here, we show in an animal model of HIV that the chronically SIV-infected gut contains cytotoxic natural killer B cells that produce inflammatory cytokines and proliferate during infection.
Subject(s)
Killer Cells, Natural , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , CD8-Positive T-Lymphocytes/immunology , Colon/cytology , Colon/immunology , Colon/virology , Cytokines/metabolism , Inflammation/pathology , Killer Cells, Natural/immunology , Macaca mulatta , Receptors, Natural Killer Cell/metabolism , Simian Immunodeficiency Virus/immunologyABSTRACT
In the United States, most new cases of human immunodeficiency virus (HIV) belong to the at-risk group of gay and bisexual men. Developing therapies to reverse viral latency and prevent spread is paramount for the HIV cure agenda. In gay and bisexual men, a major, yet poorly characterized, route of HIV entry is via transport across the colonic epithelial barrier. While colonic tears and paracellular transport contribute to infection, we hypothesize that HIV entry through the colonic mucosa proceeds via a process known as transcytosis, involving (i) virion binding to the apical surface of the colonic epithelium, (ii) viral endocytosis, (iii) transport of virions across the cell, and (iv) HIV release from the basolateral membrane. Using Caco-2 colonic epithelial cells plated as a polarized monolayer in transwells, we characterized the mechanism of HIV transport. After exposing the monolayer to HIV apically, reverse transcription quantitative PCR (RT-qPCR) of the viral genome present in the basolateral chamber revealed that transport is dose dependent, cooperative, and inefficient, with released virus first detectable at 12 h. Inefficiency may be associated with >50% decline in detectable intracellular virus that correlates temporally with increased association of the virion with lysosomal-associated membrane protein 1 (LAMP-1+) endosomes. Microscopy revealed green fluorescent protein (GFP)-labeled HIV within the confines of the epithelial monolayer, with no virus detectable between cells, suggesting that viral transport is transcellular. Treatment of the monolayer with endocytosis inhibitors, cholesterol reducing agents, and small interfering RNA (siRNA) to caveolin showed that viral endocytosis is mediated by caveolin-coated endosomes contained in lipid rafts. These results indicate that HIV transport across the intestinal epithelial barrier via transcytosis is a viable mechanism for viral spread and a potential therapeutic target. IMPORTANCE Despite the success of combination antiretroviral therapy in suppressing HIV replication and the emergence and effectiveness of PrEP-based prevention strategies, in 2018, 37,968 people in the United States received a new HIV diagnosis, accompanied by 15,820 deaths. While the annual number of new diagnoses decreased 7% from 2014 to 2018, 14% of people with HIV did not know they were infected. Gay and bisexual men accounted for 69% of all HIV diagnoses and 83% of diagnoses among males. Due to the scope of the HIV epidemic, determining and understanding precise routes of infection and the mechanisms of viral spread are paramount to ending the epidemic. Since transcellular transport of HIV across an intact colonic epithelial barrier is poorly understood, our overall goal is to characterize the molecular events involved in HIV transcytosis across the intestinal epithelial cell.
Subject(s)
Colon , Endocytosis , HIV Infections , HIV , Intestinal Mucosa , Caco-2 Cells , Caveolins/metabolism , Colon/immunology , Colon/virology , Endosomes/metabolism , HIV/metabolism , HIV Infections/metabolism , HIV Infections/prevention & control , HIV Infections/transmission , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , MaleABSTRACT
Acute HIV-1 infection (AHI) results in the widespread depletion of CD4+ T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4+ immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death-associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4+ iNKT cells were depleted faster and more profoundly than conventional CD4+ T cells, and the preferential infection of CD4+ iNKT cells over conventional CD4+ T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4+ iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Colon/immunology , Colon/virology , HIV Infections/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , Natural Killer T-Cells/immunology , Adolescent , Adult , Disease Progression , Female , HIV Infections/virology , HIV-1/immunology , Humans , Male , Middle Aged , Persistent Infection/immunology , Persistent Infection/virology , Young AdultABSTRACT
BACKGROUND: Data about cytomegalovirus (CMV) colitis in children are scarce. We aimed to describe the characteristics of childhood CMV colitis in terms of risk factors, clinical symptoms, diagnosis, therapeutic approaches, and outcomes. METHODS: Inflammatory bowel disease (IBD) and non-IBD patients with CMV colitis diagnosed by histology and tissue CMV PCR at 2 tertiary centers between January 2017 and November 2019 were studied. Clinical and laboratory data were retrieved from medical records. Underlying conditions, immune status, response to therapy and outcomes were described and followed up to 6 months after diagnosis. RESULTS: A total of 16 children (8 non-IBD, 7 ulcerative colitis and 1 Crohn's disease) with CMV colitis were included. All patients had persistent diarrhea (bloody in 13 cases). There was a significant age difference between IBD and non-IBD children (P < 0.05). The final diagnosis in 1 patient was immunodeficiency with a mutation in JAK1 gene. Three children were categorized as apparently immunocompromised and 4 children as apparently immunocompetent. Ulcer was not visible in 2 children from the non-IBD group. The mean fecal calprotectin level of IBD children was significantly higher than that of non-IBD children (376.12 ± 231.21 µg/g vs. 160.96 ± 69.94 µg/g, P < 0.05). After follow-up, 1 patient died because of another reason. Ganciclovir was used in 14 of 16 children for 3 weeks and the treatment was continued with valganciclovir in selected 6 children. CONCLUSIONS: CMV colitis is a rare but overlooked cause of prolonged diarrhea in immunocompetent and immunocompromised children. CMV colitis might present without any ulcer formation at colonoscopy in infants.
Subject(s)
Colitis/diagnosis , Colitis/virology , Cytomegalovirus Infections/complications , Immunocompetence , Immunocompromised Host , Adolescent , Child , Colon/pathology , Colon/virology , Diarrhea/pathology , Diarrhea/virology , Female , Humans , Infant , Male , Opportunistic Infections/diagnosis , Opportunistic Infections/virology , Retrospective Studies , Risk Factors , Tertiary Care Centers/statistics & numerical dataABSTRACT
Mucosal exposure to infected semen accounts for the majority of HIV-1 transmission events, with rectal intercourse being the route with the highest estimated risk of transmission. Yet, the impact of semen inflammation on colorectal HIV-1 transmission has never been addressed. Here we use cynomolgus macaques colorectal tissue explants to explore the effect of leukocytospermia, indicative of male genital tract inflammation, on SIVmac251 infection. We show that leukocytospermic seminal plasma (LSP) has significantly higher concentration of a number of pro-inflammatory molecules compared to normal seminal plasma (NSP). In virus-exposed explants, LSP enhance SIV infection more efficiently than NSP, being the increased viral replication linked to the level of inflammatory and immunomodulatory cytokines. Moreover, LSP induce leukocyte accumulation on the apical side of the colorectal lamina propria and the recruitment of a higher number of intraepithelial dendritic cells than with NSP. These results suggest that the outcome of mucosal HIV-1 infection is influenced by the inflammatory state of the semen donor, and provide further insights into mucosal SIV/HIV-1 pathogenesis.
Subject(s)
Colon/virology , Dendritic Cells/virology , Rectum/virology , Semen/virology , Simian Immunodeficiency Virus/physiology , Virus Replication/physiology , Animals , Colon/metabolism , Cytokines/metabolism , HIV Infections/metabolism , HIV Infections/transmission , HIV Infections/virology , HIV-1/physiology , Leukocytes/metabolism , Leukocytes/pathology , Leukocytes/virology , Macaca mulatta , Male , Rectum/metabolism , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Acquired Immunodeficiency Syndrome/transmission , Simian Acquired Immunodeficiency Syndrome/virology , Tissue Culture TechniquesABSTRACT
Infection with EBV has been associated with various inflammatory disorders including inflammatory bowel diseases (IBD). Contribution of this virus to intestinal disease processes has not been assessed. We previously detected that EBV DNA triggers proinflammatory responses via the activation of endosomal Toll-like receptor (TLR) signaling. Hence, to examine the colitogenic potential of EBV DNA, we used the dextran sodium sulfate (DSS) mouse colitis model. C57BL/6J mice received either DSS-containing or regular drinking water. Mice were then administered EBV DNA by rectal gavage. Administration of EBV DNA to the DSS-fed mice aggravated colonic disease activity as well as increased the damage to the colon histologic architecture. Moreover, we observed enhanced expression of IL-17A, IFNγ and TNFα in colon tissues from the colitis mice (DSS-treated) given the EBV DNA compared to the other groups. This group also had a marked decrease in expression of the CTLA4 immunoregulatory marker. On the other hand, we observed enhanced expression of endosomal TLRs in colon tissues from the EBV DNA-treated colitis mice. These findings indicate that EBV DNA exacerbates proinflammatory responses in colitis. The ubiquity of EBV in the population indicates that possible similar responses may be of pertinence in a relevant proportion of IBD patients.
Subject(s)
DNA, Viral/genetics , Epstein-Barr Virus Infections/complications , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/pathogenicity , Inflammatory Bowel Diseases/virology , Animals , Colon/immunology , Colon/pathology , Colon/virology , Disease Models, Animal , Disease Progression , Female , Herpesvirus 4, Human/immunology , Inflammatory Bowel Diseases/immunology , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Mice , Mice, Inbred C57BL , Signal Transduction , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolismABSTRACT
Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.
Subject(s)
Colitis, Ulcerative/virology , Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Colitis, Ulcerative/etiology , Colon/virology , Cytomegalovirus Infections/complications , Female , Humans , Male , Middle Aged , Mutation , Polymorphism, Genetic , Prevalence , Retrospective Studies , Young AdultABSTRACT
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of endogenous lipids with promising physiological functions in mammals. We previously introduced a new type of lipids to this family called short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs), branching specific to the C2 carbon of a long-chain fatty acid (≥C20). In this study, we discovered a homologous series of SFAHFAs comprising C16-C26 hydroxy fatty acids esterified with short-chain fatty acids (C2-C5) in mouse colon contents. The detected SFAHFAs were characterized by high-resolution mass spectrometry with MSn analysis. The double-bond position of monounsaturated SFAHFAs was determined by the epoxidation reaction of samples with m-chloroperoxybenzoic acid and their MSn analysis. Further, the measurement of SFAHFA concentration in the colon contents of mice infected with influenza A/Puerto Rico/8/34 (H1N1; PR8) virus revealed a significant increase in their levels compared to native control. A strong correlation was observed between hydroxy fatty acid and SFAHFAs. Detection, characterization, and profiling of these new SFAHFA levels in relation with pandemic H1N1; PR8 influenza virus will contribute to the in-depth study of their function and metabolism.
Subject(s)
Colon/chemistry , Fatty Acids, Volatile/analysis , Fatty Acids, Volatile/chemistry , Mass Spectrometry/methods , Orthomyxoviridae Infections/metabolism , Animals , Chlorobenzoates/chemistry , Colon/metabolism , Colon/virology , Epoxy Compounds/chemistry , Esters/analysis , Esters/chemistry , Fatty Acids, Volatile/metabolism , Influenza A Virus, H1N1 Subtype/pathogenicity , Male , Mice, Inbred C57BL , Multivariate AnalysisABSTRACT
Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.
Subject(s)
Colon/virology , HIV Antibodies , HIV Infections , Immunoglobulin A, Secretory , Mucous Membrane/virology , Animals , Macaca mulatta , Mucous Membrane/immunology , Positron Emission Tomography Computed Tomography , RectumABSTRACT
Clinical characteristics of intestinal ulcers complicated with Epstein-Barr virus (EBV) infection remain poorly studied. This study is aimed at providing further insight into clinical features of this patient cohort. The presence of serum EBV DNA was assessed in 399 patients with colonic ulcers, of which 30 cases were positive. In EBV-positive patients, the EBV-encoded RNA (EBER) was detected in intestinal tissues of 13 patients (EBER-positive group). The test was negative in 17 patients (EBER-negative group). Acute EBV infection rate in patients with colonic ulcer was 7.52%. Age and sex differences between two groups were not statistically significant. Fever, abdominal lymph node enlargement, and crater-like gouged ulcer morphology were more common in the EBER-positive group (P < 0.05). The albumin level in the EBER-positive group was significantly lower compared to that in the EBER-negative group (P < 0.05). The copy count of EBV DNA in the blood of patients from the EBER-positive group was higher, and the prognosis was worse (P < 0.05). Clinical manifestations were more severe in the EBER-positive group. Endoscopic, histopathological, and biochemical findings were also more serious in this group of patients. The findings point to the importance of assessing the EBER expression in patients with intestinal ulcers of various etiology. EBER positivity should be viewed as a diagnostic marker of more severe condition requiring more aggressive treatment.
Subject(s)
Colitis, Ulcerative/pathology , Epstein-Barr Virus Infections/pathology , Adolescent , Adult , Aged , Colitis, Ulcerative/complications , Colitis, Ulcerative/virology , Colon/pathology , Colon/virology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/epidemiology , Epstein-Barr Virus Infections/virology , Female , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/isolation & purification , Humans , Male , Middle Aged , PrognosisABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.
Subject(s)
COVID-19/pathology , Colon/pathology , Intestinal Mucosa/pathology , Organoids/pathology , Cell Line , Colon/virology , Epithelial Cells/virology , Humans , Induced Pluripotent Stem Cells/cytology , Inflammation/virology , Intestinal Mucosa/virology , Models, Biological , Organoids/cytology , Organoids/virology , SARS-CoV-2 , Virus Replication/physiologyABSTRACT
INTRODUCTION: Cytomegalovirus (CMV) infection in patients with inflammatory bowel disease (IBD) is reactivated by the use of immunosuppressive drugs. CMV infection may produce IBD flares refractory to standard therapy. OBJECTIVE: The aim of our study was to assess the efficacy and safety of faecal microbiota transplantation (FMT) for the treatment of CMV colitis in patients with ulcerative colitis (UC) flare. MATERIAL AND METHODS: A total of 8 children, with mild to severe UC, positive for CMV PCR in colonic biopsies, received 50-100 ml FMT by nasogastric tube on 5 consecutive days in each of 2 weeks. During the study, the subjects were treated with 5ASA and FMT. Immunosuppressant therapy was withdrawn, when CMV colitis was diagnosed by positive DNA PCR in colonic tissues. The clinical response was defined as a decrease of Paediatric UC Activity Index by ≥20 points. RESULTS: At the 6th week of the study, negative colonic CMV DNA PCR was measured after 10 infusions in 7/8 patients. For one boy, 20 infusions were administered to assess CMV elimination from colonic biopsies. A clinical response was observed in 3/8 patients, with clinical remission in 3/8 patients. Faecal calprotectin decreased significantly in 3 patients. CRP normalized in 2 patients after 6 weeks. No serious adverse effects were observed during and after infusions. CONCLUSIONS: FMT seems to be an effective and safe treatment option for CMV colitis in children with UC. This is the first study to demonstrate the application of FMT as a new therapeutic option for CMV colitis.