Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection.

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.

Establishment, Persistence, and Reactivation of Latent HIV-1 Infection in Renal Epithelial Cells.

HIV-1 persistence in different cell types presents the main obstacle to an HIV-1 cure. We have previously shown that the renal epithelium is a site of HIV-1 infection and that the kidney represents a separate viral compartment from blood. Whether renal cells can harbor latent virus that can be reactivated upon treatment with latency reversing agents (LRAs) is unknown. To address this question, we developed an in vitro HIV-1 latency model in renal tubule epithelial (RTE) cells using a dual color HIV-1 reporter virus, R7/E-/GFP/EF1a-mCherry (R7GEmC), and evaluated the effect of LRAs, both as single agents and in combination, on viral reactivation. Our data show that HIV-1 can establish latency in RTE cells early postinfection. While the pool of latently infected cells expanded overtime, the percentage of productively infected cells declined. Following LRA treatment only a small fraction of latently infected cells, both T cells and RTE cells, could be reactivated, and the drug combinations more effective in reactivating HIV transcription in RTE cells differed from those more active in T cells. Our study demonstrates that HIV can establish latency in RTE cells and that current LRAs are only marginally effective in inducing HIV-1 reactivation. This suggests that further study of LRA dynamics in non-T cells may be warranted to assess the suitability of LRAs as a sterilizing cure strategy. IMPORTANCE Anti-retroviral therapy (ART) has dramatically reduced HIV-related morbidity and mortality. Despite this success, a number of challenges remain, including the long-term persistence of multiple, clinically latent viral reservoirs capable of reactivation in the absence of ART. As efforts proceed toward HIV eradication or functional cure, further understanding of the dynamics of HIV-1 replication, establishment of latency and mechanisms of reactivation in reservoirs harboring the virus throughout the body is necessary. HIV-1 can infect renal epithelial cells and the expression of viral genes in those cells contributes to the development of HIV associated nephropathy (HIVAN) in untreated individuals. The significance of our work is in developing the first model of HIV-1 latency in renal epithelial cells. This model enhances our understanding of HIV-1 latency and persistence in the kidney and can be used to screen candidate latency reversing agents.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Endogenous Retroviruses Augment Amphibian (Xenopus laevis) Tadpole Antiviral Protection.

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

Utilization of HCV viremic donors in kidney transplantation: a chance or a threat?

Kidney transplantation is the treatment of choice in end-stage renal disease. The main issue which does not allow to utilize it fully is the number of organs available for transplant. Introduction of highly effective oral direct-acting antivirals (DAAs) to the treatment of chronic hepatitis C virus infection (HCV) enabled transplantation of HCV viremic organs to naive recipients. Despite an increasing number of reports on the satisfying effects of using HCV viremic organs, including kidneys, they are more often rejected than those from HCV negative donors. The main reason is the presence of HCV viremia and not the quality of the organ. The current state of knowledge points to the fact that a kidney transplant from an HCV nucleic acid testing positive (NAT+) donor to naive recipients is an effective and safe solution to the problem of the insufficient number of organs available for transplantation. It does not, however, allow to draw conclusions as to the long-term consequence of such an approach. This review analyzes the possibilities and limitations of the usage of HCV NAT + donor organs. Abbreviations: DAA: direct-acting antivirals; HCV: hepatitis C virus; NAT: nucleic acid testing; OPTN: Organ Procurement and Transplantation Network; KDIGO: Kidney Disease: Improving Global Outcomes; Ab: antigen; eGFR: estimated glomerular filtration rate; D: donor; R: recipient; CMV: cytomegalovirus; HBV: hepatitis B virus; UNOS: United Network for Organ Sharing; PHS: Public Health Service; EBR/GZR: elbasvir/grazoprevir; SVR: sustained virologic response; RAS: resistance-associated substitutions; SOF: soforbuvir; GLE/PIB: glecaprevir/pibrentasvir; ACR: acute cellular rejection; AR: acute rejection; DSA: donor-specific antibodies; KTR: kidney transplant recipients; AASLD: American Association for the Study of Liver Disease; IDSA: Infectious Diseases Society of America; PPI: proton pump inhibitors; CKD: chronic kidney disease; GN: glomerulonephritis; KAS: The Kidney Allocation system.

A Novel Soluble ACE2 Protein Provides Lung and Kidney Protection in Mice Susceptible to Lethal SARS-CoV-2 Infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2) as a main receptor to enter target cells. The goal of this study was to demonstrate the preclinical efficacy of a novel soluble ACE2 protein with increased duration of action and binding capacity in a lethal mouse model of COVID-19. METHODS: A human soluble ACE2 variant fused with an albumin binding domain (ABD) was linked via a dimerization motif hinge-like 4-cysteine dodecapeptide (DDC) to improve binding capacity to SARS-CoV-2. This novel soluble ACE2 protein (ACE2-1-618-DDC-ABD) was then administered intranasally and intraperitoneally to mice before intranasal inoculation of SARS-CoV-2 and then for two additional days post viral inoculation. RESULTS: Untreated animals became severely ill, and all had to be humanely euthanized by day 6 or 7 and had pulmonary alveolar hemorrhage with mononuclear infiltrates. In contrast, all but one mouse infected with a lethal dose of SARS-CoV-2 that received ACE2-1-618-DDC-ABD survived. In the animals inoculated with SARS-CoV-2 that were untreated, viral titers were high in the lungs and brain, but viral titers were absent in the kidneys. Some untreated animals, however, had variable degrees of kidney proximal tubular injury as shown by attenuation of the proximal tubular brush border and increased NGAL and TUNEL staining. Viral titers in the lung and brain were reduced or nondetectable in mice that received ACE2-1-618-DDC-ABD, and the animals developed only moderate disease as assessed by a near-normal clinical score, minimal weight loss, and improved lung and kidney injury. CONCLUSIONS: This study demonstrates the preclinical efficacy of a novel soluble ACE2 protein, termed ACE2-1-618-DDC-ABD, in a lethal mouse model of SARS-CoV-2 infection that develops severe lung injury and variable degrees of moderate kidney proximal tubular injury.

Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.

Spectrum of Kidney Injury Following COVID-19 Disease: Renal Biopsy Findings in a Single Italian Pathology Service.

The onset of coronavirus disease (COVID-19) as a pandemic infection, has led to increasing insights on its pathophysiology and clinical features being revealed, such as a noticeable kidney involvement. In this study, we describe the histopathological, immunofluorescence, and ultrastructural features of biopsy-proven kidney injury observed in a series of SARS-CoV-2 positive cases in our institution from April 2020 to November 2021. We retrieved and retrospectively reviewed nine cases (two pediatric and seven adults) that experienced nephrotic syndrome (six cases), acute kidney injury (two cases), and a clinically silent microhematuria and leukocyturia. Kidney biopsies were investigated by means of light microscopy, direct immunofluorescence, and electron microscopy. The primary diagnoses were minimal change disease (four cases), acute tubular necrosis (two cases), collapsing glomerulopathy (two cases), and C3 glomerulopathy (one case). None of the cases showed viral or viral-like particles on ultrastructural analysis. Novel and specific histologic features on kidney biopsy related to SARS-CoV-2 infection have been gradually disclosed and reported, harboring relevant clinical and therapeutic implications. Recognizing and properly diagnosing renal involvement in patients experiencing COVID-19 could be challenging (due to the lack of direct proof of viral infection, e.g., viral particles) and requires a proper integration of clinical and pathological data.

Discovery and Evolutionary Analysis of a Novel Bat-Borne Paramyxovirus.

Paramyxoviruses are a group of RNA viruses, such as mumps virus, measles virus, Nipah virus, Hendra virus, Newcastle disease virus, and parainfluenza virus, usually transmitted by airborne droplets that are predominantly responsible for acute respiratory diseases. In this paper, we identified a novel paramyxovirus belonging to genus Jeilongvirus infecting 4/112 (3.6%) bats from two trapping sites of Hainan Province of China. In these animals, the viral RNA was detected exclusively in kidney tissues. This is the first full-length Jeilongvirus genome (18,095 nucleotides) from bats of genus Hipposideros, which exhibits a canonical genome organization and encodes SH and TM proteins. Results, based on phylogenic analysis and genetic distances, indicate that the novel paramyxovirus formed an independent lineage belonging to genus Jeilongvirus, representing, thus, a novel species. In addition, the virus-host macro-evolutionary analysis revealed that host-switching was not only a common co-phylogenetic event, but also a potential mechanism by which rats are infected by bat-origin Jeilongvirus through cross-species virus transmission, indicating a bat origin of the genus Jeilongvirus. Overall, our study broadens the viral diversity, geographical distribution, host range, and evolution of genus Jeilongvirus.

Kidney Injury in COVID-19: Epidemiology, Molecular Mechanisms and Potential Therapeutic Targets.

As of December 2021, SARS-CoV-2 had caused over 250 million infections and 5 million deaths worldwide. Furthermore, despite the development of highly effective vaccines, novel variants of SARS-CoV-2 continue to sustain the pandemic, and the search for effective therapies for COVID-19 remains as urgent as ever. Though the primary manifestation of COVID-19 is pneumonia, the disease can affect multiple organs, including the kidneys, with acute kidney injury (AKI) being among the most common extrapulmonary manifestations of severe COVID-19. In this article, we start by reflecting on the epidemiology of kidney disease in COVID-19, which overwhelmingly demonstrates that AKI is common in COVID-19 and is strongly associated with poor outcomes. We also present emerging data showing that COVID-19 may result in long-term renal impairment and delve into the ongoing debate about whether AKI in COVID-19 is mediated by direct viral injury. Next, we focus on the molecular pathogenesis of SARS-CoV-2 infection by both reviewing previously published data and presenting some novel data on the mechanisms of cellular viral entry. Finally, we relate these molecular mechanisms to a series of therapies currently under investigation and propose additional novel therapeutic targets for COVID-19.

Transcriptome analysis of infected Crandell Rees Feline Kidney (CRFK) cells by canine parvovirus type 2c Laotian isolates.

The advent of RNA sequencing technology provides insight into the dynamic nature of tremendous transcripts within Crandell-Reese feline kidney (CRFK) cells in response to canine parvovirus (CPV-2c) infection. A total of 1,603 genes displayed differentially expressed genes (DEGs), including 789 up-regulated genes and 814 downregulated genes in the infected cells. Gene expression profiles have shown a subtle pattern of defense mechanism and immune response to CPV through significant DEGs when extensively examined via Gene Ontology (GO) and pathway analysis. Prospective GO analysis was performed and identified several enriched GO biological process terms with significant participating roles in the immune system process and defense response to virus pathway. A Gene network was constructed using the 22 most significantly enriched genes of particular interests in defense response to virus pathways to illustrate the key pathways. Eleven genes (C1QBP, CD40, HYAL2, IFNB1, IFNG, IL12B, IL6, IRF3, LSM14A, MAVS, NLRC5) were identified, which are directly related to the defense response to the virus. Results of transcriptome profiling permit us to understand the heterogeneity of DEGs during in vitro experimental study of CPV infection, reflecting a unique transcriptome signature for the CPV virus. Our findings also demonstrate a distinct scenario of enhanced CPV responses in CRFK cells for viral clearance that involved multistep and perplexity of biological processes. Collectively, our data have given a fundamental role in anti-viral immunity as our highlights of this study, thus providing outlooks on future research priorities to be important in studying CPV.

Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility.

Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity.

Investigation of the Crosstalk between GRP78/PERK/ATF-4 Signaling Pathway and Renal Apoptosis Induced by Nephropathogenic Infectious Bronchitis Virus Infection.

This study aims to explore the crosstalk between GRP78/PERK/ATF-4 signaling pathway and renal apoptosis induced by nephropathogenic infectious bronchitis virus (NIBV). Hy-Line brown chickens were divided into two groups (Con, n = 100 and Dis, n = 200). At 28 days of age, each chicken in the Dis group was intranasally injected with SX9 strain (10-5/0.2 ml). Venous blood and kidney tissues were collected at 1, 5, 11, 18 and 28 days postinfection. Our results showed that NIBV infection upregulated the levels of creatinine, uric acid, and calcium (Ca2+) levels. Histopathological examination revealed severe hemorrhage and inflammatory cell infiltration near the renal tubules. Meanwhile, NIBV virus particles and apoptotic bodies were observed by ultramicro electron microscope. In addition, RT-qPCR and Western blot showed that NIBV upregulated the expression of GRP78, PERK, eIF2α, ATF-4, CHOP, Caspase-3, Caspase-9, P53, Bax, and on the contrary, downregulated the expression of Bcl-2. Furthermore, immunofluorescence localization analysis showed that the positive expression of Bcl-2 protein was significantly decreased. Correlation analysis indicated that endoplasmic reticulum (ER) stress gene expression, apoptosis gene expression, and renal injury were potentially related. Taken together, NIBV infection can induce renal ER stress and apoptosis by activating of GRP78/PERK/ATF-4 signaling pathway, leading to kidney damage. IMPORTANCE Nephropathogenic infectious bronchitis virus (NIBV) induced renal endoplasmic reticulum stress in chickens. NIBV infection induced kidney apoptosis in chickens. GRP78/PERK/ATF-4 signaling pathway is potentially related to renal apoptosis induced by NIBV.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Goose nephritic astrovirus infection increases autophagy, destroys intercellular junctions in renal tubular epithelial cells, and damages podocytes in the kidneys of infected goslings.

Goose nephritic astrovirus (GNAstV) has recently been identified, which causes kidney swelling and visceral gout in goslings. However, the pathological changes in kidney tissue due to GNAstV infection have not yet been described. In the study, fifty goslings were orally infected with GNAstV, and fifty goslings received PBS as a control. Kidney tissue was collected at different days following infection (dpi) to assess the injury. GNAstV infection reduced body weight, increased the relative weight of the kidney, and increased serum uric acid and creatinine levels. GNAstV was found within renal epithelial cells, and the viral load in the kidney peaked at 7 dpi. Pale and swollen kidney tissue was observed in infected goslings, especially at 5 and 7 dpi. GNAstV infection caused degeneration and necrosis of renal epithelial cells, structural destruction of the brush border, glycogen deposition in the glomerular mesangium, increased fibrosis, and infiltration of inflammatory cells into the renal interstitium. Moreover, swollen mitochondria, broken mitochondrial ridges, autophagosomes, and autophagolysosomes were observed under ultrahistopathological examination. GNAstV infection increased levels of LC3B, ATG5, and Beclin 1, and decreased p62, and downregulated WT1 mRNA and upregulated desmin mRNA. At early stages, GNAstV infection decreased expression of intercellular junction-related genes, including ZO-1, occludin, claudin-10, and catenin-α2. In conclusion, GNAstV infection causes renal epithelial cell autophagy, destruction of brush border and intercellular junctions, podocyte damage, and increased fibrosis, ultimately resulting in damage to the kidney.

Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor-like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Circulation of three genotypes and identification of unique mutations in neutralizing epitopes of infectious bronchitis virus in chickens in Bangladesh.

Infectious bronchitis (IB) is a highly contagious respiratory disease caused by a gammacoronavirus that has been circulating for many years in chickens in Bangladesh, resulting in significant economic losses. The aim of this study was to detect and characterize infectious bronchitis virus (IBV) from clinical outbreaks and surveillance samples. Real-time RT-PCR was used to detect IBV in pooled lung and tracheal tissue samples (n = 78), oropharyngeal swabs (n = 19), and pooled fecal samples (n = 13) from live-bird markets. Both respiratory and nephropathogenic forms of IB were suspected at necropsy (n = 7) from clinical outbreaks. Sequencing of hypervariable regions (HVR1-2 and HVR3) of the region of the spike gene (S) encoding the S1 subunit of five isolates revealed circulation of the Mass-like, QX-like, and 4/91-like genotypes of IBV in Bangladesh. Each genotype was extremely variable, as shown by separate clustering of the viruses in a phylogenetic tree and high nucleotide (nt) sequence divergence (38.8-41.2% and 25.7-37.4% in the HVR1-2 and HVR3 sequence, respectively). The unique mutation G65E was observed in each Mass-like isolate, and Y328S was observed in each 4/91-like Bangladeshi isolate. Three neutralizing epitope sites were predicted within the HVRs that differed significantly among the three genotypes. In addition, one Bangladeshi isolate carried fixed mutations at 294F and 306Y, like other pathogenic QX-like IBVs, which could affect epitopes involved in neutralization, facilitating virus circulation among vaccinated flocks. Therefore, continuous screening and genotype characterization will be necessary to track the epidemiology of IBV and control IB infection in Bangladesh.

COVID-19 impact on the renal system: Pathophysiology and clinical outcomes.

Coronavirus disease (COVID-19) causes many deleterious effects throughout the body. Prior studies show that the incidence of acute kidney injury in COVID-19 patients could be as high as 25%. There are also autopsy reports showing evidence of viral tropism to the renal system. In this regard, COVID-19 can damage the kidneys and increase a patient's risk of requiring dialysis. Available evidence suggests that renal involvement in COVID-19 infection is not uncommon, and there has been an increased incidence of chronic kidney disease related to the pandemic. In this literature analysis, we address COVID-19 and its effects on the renal system, including the pathophysiologic mechanisms. We also address current studies on the causes of injury to the renal system, the cause of kidney failure, its effect on mortality, the impact on dialysis patients, and the impact on renal transplant patients. COVID-19 disease may have unique features in individuals on chronic dialysis and kidney transplant recipients, requiring increased vigilance in limiting viral transmission in perioperative, in-patient, and dialysis center settings.

Porcine Deltacoronavirus Enters Porcine IPI-2I Intestinal Epithelial Cells via Macropinocytosis and Clathrin-Mediated Endocytosis Dependent on pH and Dynamin.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.