Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids.

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.

Porcine kobuvirus enhances porcine epidemic diarrhea virus pathogenicity and alters the number of intestinal lymphocytes in piglets.

Recent epidemiological studies have discovered that a lot of cases of porcine epidemic diarrhea virus (PEDV) infection are frequently accompanied by porcine kobuvirus (PKV) infection, suggesting a potential relationship between the two viruses in the development of diarrhea. To investigate the impact of PKV on PEDV pathogenicity and the number of intestinal lymphocytes, piglets were infected with PKV or PEDV or co-infected with both viruses. Our findings demonstrate that co-infected piglets exhibit more severe symptoms, acute gastroenteritis, and higher PEDV replication compared to those infected with PEDV alone. Notably, PKV alone does not cause significant intestinal damage but enhances PEDV's pathogenicity and alters the number of intestinal lymphocytes. These results underscore the complexity of viral interactions in swine diseases and highlight the need for comprehensive diagnostic and treatment strategies addressing co-infections.

An isotonic protein solution favorably modulated the porcine intestinal immune response and cellular adhesion markers and reduced PEDV shedding in vivo.

Porcine epidemic diarrhea virus (PEDV) causes immensely large economic losses worldwide in the swine industry. PEDV attacks the intestine, disrupts intestinal epithelium morphology and barrier integrity, and results in profound diarrhea and high mortality. A commercially available isotonic protein solution (IPS) (Tonisity Px) has anecdotally been reported to be effective in supportive treatment of piglets with active PEDV infections. This study evaluated the effects of supplementing (or not) the drinking water of 14 day old PEDV-infected piglets with the IPS on the content of E-cadherin, fibronectin, interferon-alpha (IFN-α), and matrix metalloproteinase 9 (MMP-9) in duodenal tissue. The content of PEDV DNA in feces was also measured. Though both groups had similar PEDV shedding at day 1, IPS piglets had significantly lower PEDV shedding at day 5, 14 and 21. The IPS group also had a shorter duration of PEDV virus shedding. Levels of E-cadherin and fibronectin, both of which are structural proteins in the intestine, remained unchanged from baseline in the IPS group, whereas the same molecules decreased significantly in the control group. IFN-α, an antiviral cytokine, and MMP-9, an enzyme that aids in tissue remodeling, were increased at days 5 and 14 post infection, and then decreased at day 21 post-infection in the IPS group compared to control. Overall, the IPS used in this study enhanced epithelial intercellular adhesion (E-cadherin) and extracellular matrix structure (fibronectin), resulted in significantand favorable changes in MMP-9 activity, and favorably modulated IFN-α production. This is the first report of this panel of biomarkers, especially MMP-9 and IFN-α, in the face of in vivo PEDV infection. This is also the first report to investigate a commercially available swine product that does not need to be administered in solid feed, and that is already registered for use throughout Asia, Europe, South America, and North America. Overall, the results of this study serve to clarify the behavior of 4 key biomarkers in the presence of in vivo PEDV infection. The results also indicate that IPS (Tonisity Px) supplementation is a viable intervention to modulate the porcine intestinal immune response with favorable effects on the intestine.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G.

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3-86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26-506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G.

Profiling the human intestinal environment under physiological conditions.

The spatiotemporal structure of the human microbiome1,2, proteome3 and metabolome4,5 reflects and determines regional intestinal physiology and may have implications for disease6. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals7. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Structural atlas of a human gut crassvirus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a severe global health challenge. We isolate and characterize two previously unidentified lytic phages, P24 and P39, with large burst sizes active against ST11 KL64, a major CRKP lineage. P24 and P39 represent species of the genera Przondovirus (Studiervirinae subfamily) and Webervirus (Drexlerviridae family), respectively. P24 and P39 together restrain CRKP growth to nearly 8 h. Phage-resistant mutants exhibit reduced capsule production and decreased virulence. Modifications in mshA and wcaJ encoding capsule polysaccharide synthesis mediate P24 resistance whilst mutations in epsJ encoding exopolysaccharide synthesis cause P39 resistance. We test P24 alone and together with P39 for decolonizing CRKP using mouse intestinal colonization models. Bacterial load shed decrease significantly in mice treated with P24 and P39. In conclusion, we report the characterization of two previously unidentified lytic phages against CRKP, revealing phage resistance mechanisms and demonstrating the potential of lytic phages for intestinal decolonization.

Advances in understanding of the innate immune response to human norovirus infection using organoid models.

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.

The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans.

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

Diaphorina citri flavi-like virus localization, transmission, and association with Candidatus Liberibacter asiaticus in its psyllid host.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

Intestinal Abnormalities in Patients With SARS-CoV-2 Infection: Histopathologic Changes Reflect Mechanisms of Disease.

Approximately 20% of patients with symptomatic syndrome-associated coronavirus-2 (SARS-CoV-2) infection have gastrointestinal bleeding and/or diarrhea. Most are managed without endoscopic evaluation because the risk of practitioner infection outweighs the value of biopsy analysis unless symptoms are life-threatening. As a result, much of what is known about the gastrointestinal manifestations of coronavirus disease-2019 (COVID-19) has been gleaned from surgical and autopsy cases that suffer from extensive ischemic injury and/or poor preservation. There are no detailed reports describing any other gastrointestinal effects of SARS-CoV-2 even though >3,000,000 people have died from COVID-19 worldwide. The purpose of this study is to report the intestinal findings related to SARS-CoV-2 infection by way of a small case series including one with evidence of direct viral cytopathic effect and 2 with secondary injury attributed to viral infection. Infection can be confirmed by immunohistochemical stains directed against SARS-CoV-2 spike protein, in situ hybridization for spike protein-encoding RNA, and ultrastructural visualization of viruses within the epithelium. It induces cytoplasmic blebs and tufted epithelial cells without inflammation and may not cause symptoms. In contrast, SARS-CoV-2 infection can cause gastrointestinal symptoms after the virus is no longer detected, reflecting systemic activation of cytokine and complement cascades rather than direct viral injury. Reversible mucosal ischemia features microvascular injury with hemorrhage, small vessel thrombosis, and platelet-rich thrombi. Systemic cytokine elaboration and dysbiosis likely explain epithelial cell injury that accompanies diarrheal symptoms. These observations are consistent with clinical and in vitro data and contribute to our understanding of the protean manifestations of COVID-19.

SARS-CoV-2 Spike Protein in Intestinal Cells of a Patient with Coronavirus Disease 2019 Multisystem Inflammatory Syndrome.

A previously healthy 12-year-old boy had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related multisystem inflammatory syndrome (MIS-C) that was rapidly fatal. Autopsy revealed the presence of a large intracardiac thrombus. SARS-CoV-2 spike protein was detected in intestinal cells, supporting the hypothesis that viral presence in the gut may be related to the immunologic response of MIS-C.

SARS-CoV-2 infection of intestinal epithelia cells sensed by RIG-I and DHX-15 evokes innate immune response and immune cross-talk.

SARS-CoV-2 causes a spectrum of clinical symptoms from respiratory damage to gastrointestinal disorders. Intestinal infection of SARS-CoV-2 triggers immune response. However, the cellular mechanism that how SARS-CoV-2 initiates and induces intestinal immunity is not understood. Here, we exploited SARS-CoV-2-GFP/ΔN trVLP pseudo-virus system and demonstrated that RIG-I and DHX15 are required for sensing SARS-CoV-2 and inducing cellular immune response through MAVS signaling in intestinal epithelial cells (IECs) upon SARS-CoV-2 infection. NLRP6 also engages in the regulation of SARS-CoV-2 immunity by producing IL-18. Furthermore, primary cellular immune response provoked by SARS-CoV-2 in IECs further cascades activation of MAIT cells and produces cytotoxic cytokines including IFN-γ, granzyme B via an IL-18 dependent mechanism. These findings taken together unveil molecular basis of immune recognition in IECs in response to SARS-CoV-2, and provide insights that intestinal immune cross-talk with other immune cells triggers amplified immunity and probably contributes to immunopathogenesis of COVID-19.

Rotavirus NSP1 Contributes to Intestinal Viral Replication, Pathogenesis, and Transmission.

Rotavirus (RV)-encoded nonstructural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of the simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups in both the 129sv and C57BL/6 backgrounds. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. The defective replication of the NSP1-null RV in small intestinal tissues occurred as early as 1 day postinfection. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development. IMPORTANCE Rotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate, for the first time in a pathologically valid homologous small animal model, that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon or inflammasome signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo, making it an attractive target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.

Systemic and Intestinal Viral Reservoirs in CD4+ T Cell Subsets in Primary SIV Infection.

The HIV reservoir size in target CD4+ T cells during primary infection remains unknown. Here, we sorted peripheral and intestinal CD4+ T cells and quantified the levels of cell-associated SIV RNA and DNA in rhesus macaques within days of SIVmac251 inoculation. As a major target cell of HIV/SIV, CD4+ T cells in both tissues contained a large amount of SIV RNA and DNA at day 8-13 post-SIV infection, in which productive SIV RNA highly correlated with the levels of cell-associated SIV DNA. Memory CD4+ T cells had much higher viral RNA and DNA than naïve subsets, yet memory CD4+ T cells co-expressing CCR5 had no significant reservoir size compared with those that were CCR5-negative in blood and intestine. Collectively, memory CD4+ T cells appear to be the major targets for primary infection, and viral reservoirs are equally distributed in systemic and lymphoid compartments in acutely SIV-infected macaques.

Porcine Deltacoronavirus Utilizes Sialic Acid as an Attachment Receptor and Trypsin Can Influence the Binding Activity.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Studies showed that PDCoV uses porcine aminopeptidase N (pAPN) as an entry receptor, but the infection of pAPN-knockout cells or pigs with PDCoV revealed that pAPN might be not a critical functional receptor, implying there exists an unidentified receptor involved in PDCoV infection. Herein, we report that sialic acid (SA) can act as an attachment receptor for PDCoV invasion and facilitate its infection. We first demonstrated that the carbohydrates destroyed on the cell membrane using NaIO4 can alleviate the susceptibility of cells to PDCoV. Further study showed that the removal of SA, a typical cell-surface carbohydrate, could influence the PDCoV infectivity to the cells significantly, suggesting that SA was involved in the infection. The results of plaque assay and Western blotting revealed that SA promoted PDCoV infection by increasing the number of viruses binding to SA on the cell surface during the adsorption phase, which was also confirmed by atomic force microscopy at the microscopic level. In in vivo experiments, we found that the distribution levels of PDCoV and SA were closely relevant in the swine intestine, which contains huge amount of trypsin. We further confirmed that SA-binding capacity to PDCoV is related to the pre-treatment of PDCoV with trypsin. In conclusion, SA is a novel attachment receptor for PDCoV infection to enhance its attachment to cells, which is dependent on the pre-treatment of trypsin on PDCoV. This study paves the way for dissecting the mechanisms of PDCoV-host interactions and provides new strategies to control PDCoV infection.