SARS-CoV-2 infection causes intestinal cell damage: Role of interferon's imbalance.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the newly emerging lung disease pandemic COVID-19. This viral infection causes a series of respiratory disorders, and although this virus mainly infects respiratory cells, the small intestine can also be an important site of entry or interaction, as enterocytes highly express in angiotensin-2 converting enzyme (ACE) receptors. There are countless reports pointing to the importance of interferons (IFNs) with regard to the mediation of the immune system in viral infection by SARS-CoV-2. Thus, this review will focus on the main cells that make up the large intestine, their specific immunology, as well as the function of IFNs in the intestinal mucosa after the invasion of coronavirus-2.

Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmitted through the respiratory route, but potential extra-respiratory routes of SARS-CoV-2 transmission remain uncertain. Here we inoculated five rhesus macaques with 1 × 106 TCID50 of SARS-CoV-2 conjunctivally (CJ), intratracheally (IT), and intragastrically (IG). Nasal and throat swabs collected from CJ and IT had detectable viral RNA at 1-7 days post-inoculation (dpi). Viral RNA was detected in anal swabs from only the IT group at 1-7 dpi. Viral RNA was undetectable in tested swabs and tissues after intragastric inoculation. The CJ infected animal had a higher viral load in the nasolacrimal system than the IT infected animal but also showed mild interstitial pneumonia, suggesting distinct virus distributions. This study shows that infection via the conjunctival route is possible in non-human primates; further studies are necessary to compare the relative risk and pathogenesis of infection through these different routes in more detail.

Comparative Assessment of Small and Large Intestine Biopsies for Ex Vivo HIV-1 Pathogenesis Studies.

Ex vivo mucosal explants have become a mainstay of HIV-1 studies using human tissue. In this study, we examine the baseline phenotypic and virologic differences between biopsies derived from the small intestine (SI) and large intestine (LI) for use in ex vivo explant studies. To do this, we collected endoscopic mucosal biopsies from both SI and LI from the same healthy, HIV-seronegative participants. Mucosal mononuclear cell phenotypes and quantity were compared using flow cytometry. Comparative HIV-1 infectibility of the explants was assessed using an ex vivo explant HIV-1 infection assay. We found that all biopsies had similar numbers of T cells per biopsy. While the percentage of CD4+ T cells from SI biopsies expressed significantly more activation markers (CD38, HLA-DR) and HIV coreceptors (CXCR4, CCR5), the absolute numbers of activated CD4+ T cells were similar between both sites. LI explants, however, supported more efficient HIV-1 infection, as evidenced by earlier rise in p24 accumulation and greater percent of infected explants at limiting infectious doses. These results suggest that explants from LI biopsies support more efficient HIV-1 infection than SI biopsies, despite similar numbers of available, activated HIV-1 target cells. These findings highlight important differences in LI and SI explants, which must be considered in designing and interpreting ex vivo HIV-1 infection studies, and suggest that factors within the tissue other than target cell number and activation state may play a role in regulating HIV-1 infection.

Reactive oxygen species-induced parthanatos of immunocytes by human cytomegalovirus-associated substance.

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV-infected lesions has yet to be established, it is here shown that substances produced by HCMV-infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV-associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV-infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX-2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP-ribose polymerase-1 (PARP-1)-mediated cell death, or parthanatos. HCMVAIS-induced cell death is accompanied by PARP-1 activation in a caspase-independent manner, nuclear translocation of apoptosis-inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS-induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV-positive foci in serial section samples of a large intestine with HCMV infection was TUNEL-positive, cleaved caspase 3-negative and CD45-positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV-infected cells through ROS-induced parthanatos by HCMVAIS.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

Characterization of cells expressing lymphatic marker LYVE-1 in macaque large intestine during simian immunodeficiency virus infection identifies a large population of nonvascular LYVE-1(+)/DC-SIGN(+) cells.

Abstract LYVE-1 is a marker expressed by lymphatic endothelial cells (LECs) that line the lymphatic endothelium. Through studies designed to examine potential changes in expression of LYVE-1 in cynomolgus macaque colon tissues during the course of simian immunodeficiency virus (SIV) infection, we discovered that LYVE-1 was expressed by heterogenous populations of cells. As revealed by in situ hybridization (ISH), LYVE-1 mRNA levels in colon were decreased in macaques with AIDS compared with acutely infected or uninfected macaques. In the submucosal layer of the colon, approximately half of the LYVE-1-expressing cells co-expressed the dendritic cell (DC) marker, DC-SIGN/CD209, and this percentage did not change appreciably during infection. Subsets of cells expressing LYVE-1 also co-expressed macrophage markers, such as CD68 and the macrophage mannose receptor (MMR)/CD206, in both the colon and lymph nodes. LECs, DCs, and macrophages that co-expressed LYVE-1 were observed in colon and lymph node from uninfected, healthy animals as well as in tissues with SIV-driven inflammation. These findings provide further definition of the phenotypic overlap between LECs and antigen presenting cells, reveal the heterogeneity within the population of cells expressing the lymphatic marker LYVE-1, and show that SIV modulates this population of cells in a mucosal surface across which the virus is acquired.

Large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection.

Both rectal and vaginal mucosal surfaces serve as transmission routes for pathogenic microorganisms. Vaccination through large intestinal mucosa, previously proven protective for both of these mucosal sites in animal studies, can be achieved successfully by direct intracolorectal (i.c.r.) administration, but this route is clinically impractical. Oral vaccine delivery seems preferable but runs the risk of the vaccine's destruction in the upper gastrointestinal tract. Therefore, we designed a large intestine-targeted oral delivery with pH-dependent microparticles containing vaccine nanoparticles, which induced colorectal immunity in mice comparably to colorectal vaccination and protected against rectal and vaginal viral challenge. Conversely, vaccine targeted to the small intestine induced only small intestinal immunity and provided no rectal or vaginal protection, demonstrating functional compartmentalization within the gut mucosal immune system. Therefore, using this oral vaccine delivery system to target the large intestine, but not the small intestine, may represent a feasible new strategy for immune protection of rectal and vaginal mucosa.

The large intestine as a major reservoir for simian immunodeficiency virus in macaques with long-term, nonprogressing infection.

Although patients with human immunodeficiency virus type 1 infection who are receiving antiretroviral therapy and those with long-term, nonprogressive infection (LTNPs) usually have undetectable viremia, virus persists in tissue reservoirs throughout infection. However, the distribution and magnitude of viral persistence and replication in tissues has not been adequately examined. Here, we used the simian immunodeficiency virus (SIV) macaque model to quantify and compare viral RNA and DNA in the small (jejunum) and large (colon) intestine of LTNPs. In LTNPs with chronic infection, the colon had consistently higher viral levels than did the jejunum. The colon also had higher percentages of viral target cells (memory CD4(+) CCR5(+) T cells) and proliferating memory CD4(+) T cells than did the jejunum, whereas markers of cell activation were comparable in both compartments. These data indicate that the large intestine is a major viral reservoir in LTNPs, which may be the result of persistent, latently infected cells and higher turnover of naive and central memory CD4(+) T cells in this major immunologic compartment.

Shedding and serologic responses following primary and secondary inoculation of house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with low-pathogenicity avian influenza virus.

Waterfowl and shorebirds are well-recognized natural reservoirs of low-pathogenicity avian influenza viruses (LPAIV); however, little is known about the role of passerines in avian influenza virus ecology. Passerines are abundant, widespread, and commonly come into contact with free-ranging birds as well as captive game birds and poultry. We inoculated and subsequently challenged house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with wild-bird origin LPAIV H3N8 to evaluate their potential role in transmission. Oropharyngeal shedding was short lived, and was detected in more starlings (97.2%) than sparrows (47.2%; n=36 of each). Cloacal shedding was rare in both species (8.3%; n=36 of each) and no cage-mate transmission occurred. Infectious LPAIV was cultured from oropharyngeal and cloacal swabs and gastrointestinal and respiratory tissues from both species. Seroconversion was detected as early as 3 days post inoculation (d.p.i.) (16.7% of sparrows and 0% of starlings; n=6 each); 50% of these individuals seroconverted by 5 d.p.i., and nearly all birds (97%; n=35) seroconverted by 28 d.p.i. In general, pre-existing homologous immunity led to reduced shedding and increased antibody levels within 7 days of challenge. Limited shedding and lack of cage-mate transmission suggest that passerines are not significant reservoirs of LPAIV, although species differences apparently exist. Passerines readily and consistently seroconverted to LPAIV, and therefore inclusion of passerines in epidemiological studies of influenza outbreaks in wildlife and domestic animals may provide further insight into the potential involvement of passerines in avian influenza virus transmission ecology.

Progressive transformation of the germinal center of extranodal organs: a clinicopathological, immunohistochemical, and genotypic study of 14 cases.

Progressive transformation of germinal center (PTGC) usually affects the peripheral lymph nodes. Little is known about the extranodal PTGC. To clarify the clinicopathological and molecular findings of extranodal PTGC, we studied 14 such cases. Using formalin-fixed, paraffin-embedded sections, we carried out histological and immunohistochemical examinations, as well as in situ hybridization (ISH) and polymerase chain reaction (PCR). Eleven patients were female, and three were male. They were between 44 and 77 years old, with a mean age of 62 years. The large intestine (n=7) was the most frequently involved tissue, followed by skin (n=2) and subcutaneous soft tissue (n=2). Oral cavity, Waldeyer ring, and orbit were affected in one case each. Histologically, 13 cases contained both early stage PTGC and late stage PTGC. The remaining 14th case contained only late stage PTGC. Expansion of the marginal zone was identified in three cases. Immunohistochemical study demonstrated the reactive nature of the B-cells in all 14 lesions. However, PCR study revealed immunoglobulin heavy chain (IgH) gene rearrangement in one of our 14 cases. There was no development of B-cell lymphoma in one lesion with IgH rearrangement. ISH study demonstrated Epstein-Barr virus-encoded small RNA+ cells in three lesions. Compared with PTGC of the peripheral lymph node, PTGC of extranodal sites was characterized by a female predominance, an older age group, and the presence of numerous PTGC at the affected sites. However, the histological findings of extranodal PTG were similar to those of lymph node PTGC. The clinicopathological findings of the extranodal PTGCs appeared to be different from those of lymph node PTGC.

Simian immunodeficiency virus-induced intestinal cell apoptosis is the underlying mechanism of the regenerative enteropathy of early infection.

The enteropathic manifestations of the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV) in late infection are usually due to infection by other microbes, but in early infection the viruses themselves cause an enteropathy by heretofore undetermined mechanisms. Here we report that SIV induces massive apoptosis of intestinal epithelial cells lining the small and large bowel, thus identifying apoptosis as the driving force behind the regenerative pathology of early infection. We found that apoptosis of gut epithelium paralleled the previously documented apoptosis and massive depletion of CD4 T cells in gut lamina propria, triggered by established mechanisms of gut epithelial cell apoptosis and, at peak, possibly by virus interactions with GPR15/Bob, an intestinal epithelial cell-associated alternative coreceptor for SIV and HIV-1. Apoptosis in early SIV infection is thus the common theme of the pathological processes that quickly afflict the innate as well as adaptive arms of the gut immune system.

Emergence of influenza A virus variants after prolonged shedding from pheasants.

We previously demonstrated the susceptibility of pheasants to infection with influenza A viruses of 15 hemagglutinin (HA) subtypes: 13/23 viruses tested were isolated for >or=14 days, all in the presence of serum-neutralizing antibodies; one virus (H10) was shed for 45 days postinfection. Here we confirmed that 20% of pheasants shed low-pathogenic influenza viruses for prolonged periods. We aimed to determine why the antibody response did not clear the virus in the usual 3 to 10 days, because pheasants serve as a long-term source of influenza viruses in poultry markets. We found evidence of virus replication and histological changes in the large intestine, bursa of Fabricius, and cecal tonsil. The virus isolated 41 days postinfection was antigenically distinct from the parental H10 virus, with corresponding changes in the HA and neuraminidase. Ten amino acid differences were found between the parental H10 and the pheasant H10 virus; four were in potential antigenic sites of the HA molecule. Prolonged shedding of virus by pheasants results from a complex interplay between the diversity of virus variants and the host response. It is often argued that vaccination pressure is a mechanism that contributes to the generation of antigenic-drift variants in poultry. This study provided evidence that drift variants can occur naturally in pheasants after prolonged shedding of virus, thus strengthening our argument for the removal of pheasants from live-bird retail markets.

Detection of Epstein-Barr virus in lower gastrointestinal tract lymphomas: a study in Malaysian patients.

BACKGROUND: Many studies in the literature have shown that Epstein-Barr virus (EBV) is associated with several human lymphoid and epithelial malignancies. However, the prevalence of EBV in non-Hodgkin lymphoma (NHL) of the lower gastrointestinal (GI) tract has not been fully elucidated. AIM: The aim of this study was to determine the presence and distribution of EBV in formalin-fixed paraffin-embedded tissue samples obtained from 18 Malaysian patients diagnosed with NHL of the lower GI tract. METHODS: The GI tract lymphoma tissue samples analyzed for the presence of EBV were divided into the following groups: NHL of the small intestine (seven cases); NHL of the ileocecum (ten cases); and NHL of the rectum (one case). The presence of EBV-encoded RNA (EBER) in all of the above tissue samples was tested for using conventional in situ hybridization technology. RESULTS: Two of 18 cases (11.1%) of NHL of the lower GI tract demonstrated positive signals for EBV/EBER. In the first positive case, EBV/EBER signals were located in lymphoma cells in the serosa layer of the small intestine. In the second EBV/EBER-positive case, EBV/EBER signals were detected in diffuse B-cell lymphoma of the ileocecum. CONCLUSION: These findings demonstrate a rare association between EBV and lower GI tract lymphomas in this group of Malaysian patients.

Detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease.

Clinical signs of a winter dysentery-like syndrome in 6- to 9-month-old cattle in 3 feedlots included acute onset of diarrhea with high morbidity and low mortality, respiratory tract problems that included dyspnea, coughing, and nasal discharge, and high rectal temperatures. Bovine coronavirus was detected by use of an ELISA and immune electron microscopy in fecal and nasal swab samples and by immunohistochemical analysis of intestinal sections collected from calves during necropsy. Bovine coronavirus should be considered in the differential diagnoses for diseases that cause acute onset of bloody diarrhea in feedlot cattle.

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.