Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.

SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: protection of African green monkeys from COVID-19 disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats.

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

Hepatic proinflammatory myeloid phenotypes are a hallmark of Ebola virus Kikwit pathogenesis in rhesus monkeys.

The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques (Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls (n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI (n = 3), 5 DPI (n = 3), 6 DPI (n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14-CD16+ to CD14+CD16- macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial M. avium spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12-16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12-16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection.

Quantification of Viral and Host Biomarkers in the Liver of Rhesus Macaques: A Longitudinal Study of Zaire Ebolavirus Strain Kikwit (EBOV/Kik).

Zaire ebolavirus (EBOV) causes Ebola virus disease (EVD), which carries a fatality rate between 25% and 90% in humans. Liver pathology is a hallmark of terminal EVD; however, little is known about temporal disease progression. We used multiplexed fluorescent immunohistochemistry and in situ hybridization in combination with whole slide imaging and image analysis (IA) to quantitatively characterize temporospatial signatures of viral and host factors as related to EBOV pathogenesis. Eighteen rhesus monkeys euthanized between 3 and 8 days post-infection, and 3 uninfected controls were enrolled in this study. Compared with semiquantitative histomorphologic ordinal scoring, quantitative IA detected subtle and progressive features of early and terminal EVD that was not feasible with routine approaches. Sinusoidal macrophages were the earliest cells to respond to infection, expressing proinflammatory cytokine interleukin 6 (IL6) mRNA, which was subsequently also observed in fibrovascular compartments. The mRNA of interferon-stimulated gene-15 (ISG-15), also known as ISG15 ubiquitin like modifier (ISG15), was observed early, with a progressive and ubiquitous hybridization signature involving mesenchymal and epithelial compartments. ISG-15 mRNA was prominent near infected cells, but not in infected cells, supporting the hypothesis that bystander cells produce a robust interferon gene response. This study contributes to our current understanding of early EVD progression and illustrates the value that digital pathology and quantitative IA serve in infectious disease research.

Association of vaping with decreased vascular endothelial growth factor expression and decreased microvessel density in cutaneous wound healing tissue in rats.

Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the molecular and histologic mechanisms underlying this postulation remain unknown. Our study sought to compare molecular and histologic changes in cutaneous flap and non-flap tissue between vaping, smoking and control cohorts. Animal study of 15 male Sprague-Dawley rats was randomized to three cohorts: negative control (n = 5), e-cigarette (n = 5) and cigarette (n = 5) and exposed to their respective treatments with serum cotinine monitoring. After 30 days, random pattern flaps were raised and healed for 2 weeks after which skin punch biopsies of flap and non-flap tissues were collected for quantitative-reverse transcription-polymerase chain reaction of three selected wound healing genes (transforming growth factor ß [TGF-ß], vascular endothelial growth factor [VEGF], matrix metalloproteinase-1 [MMP-1]); then, immunohistochemistry for CD68 expression, α-smooth muscle actin looking at microvessel density (MVD) and in situ hybridization to localize VEGF production were undertaken. In flap tissue, vaping (mean[SEM]) (0.61[0.07]) and smoking (0.70[0.04]) were associated with decreased fold change of VEGF expression compared with controls (0.91[0.03]) (p < 0.05, p < 0.05, respectively). In non-flap tissue, only vaping was associated with decreased VEGF expression (mean[SEM]) (0.81[0.07]), compared with controls (1.17[0.10]) (p < 0.05) with expression primarily localized to basal keratinocytes and dermal capillaries. Immunohistochemistry showed decreased MVD in smoking (0.27[0.06]) and vaping (0.26[0.04]) flap tissue compared to matched controls (0.65[0.14]) (p < 0.05, p < 0.05, respectively) and decreased areas of fibrosis compared with controls on gross histology. Vaping and smoking were similarly associated with decreased VEGF expression, MVD and fibrotic changes in flap tissue. The results suggest attenuated angiogenesis via decreased VEGF expression as a mechanism for poor wound healing in vaping-exposed rats.

Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats.

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

Late Disseminated Lyme Disease: Associated Pathology and Spirochete Persistence Posttreatment in Rhesus Macaques.

Nonhuman primates currently serve as the best experimental model for Lyme disease because of their close genetic homology with humans and demonstration of all three phases of disease after infection with Borrelia burgdorferi. We investigated the pathology associated with late disseminated Lyme disease (12 to 13 months after tick inoculation) in doxycycline-treated (28 days; 5 mg/kg, oral, twice daily) and untreated rhesus macaques. Minimal to moderate lymphoplasmacytic inflammation, with a predilection for perivascular spaces and collagenous tissues, was observed in multiple tissues, including the cerebral leptomeninges, brainstem, peripheral nerves from both fore and hind limbs, stifle synovium and perisynovial adipose tissue, urinary bladder, skeletal muscle, myocardium, and visceral pericardium. Indirect immunofluorescence assays that combined monoclonal (outer surface protein A) and polyclonal antibodies were performed on all tissue sections that contained inflammation. Rare morphologically intact spirochetes were observed in the brains of two treated rhesus macaques, the heart of one treated rhesus macaque, and adjacent to a peripheral nerve of an untreated animal. Borrelia antigen staining of probable spirochete cross sections was also observed in heart, skeletal muscle, and near peripheral nerves of treated and untreated animals. These findings support the notion that chronic Lyme disease symptoms can be attributable to residual inflammation in and around tissues that harbor a low burden of persistent host-adapted spirochetes and/or residual antigen.

Pneumonia in a Captive Central Bearded Dragon With Concurrent Detection of Helodermatid Adenovirus 2 and a Novel Mycoplasma Species.

A 4-year-old captive male central bearded dragon ( Pogona vitticeps) was presented for recurrent episodic dyspnea and anorexia with occasional expulsion of oral mucoid discharge. Despite empirical antimicrobial therapy and supportive care, the animal died and was submitted for autopsy. Defining histologic features included heterophilic and lymphocytic interstitial pneumonia, with occasional amphophilic intranuclear inclusions and prominent type II pneumocyte hyperplasia. Transmission electron microscopy revealed intranuclear 80-nm, nonenveloped, hexagonal viral particles within pneumocytes. Helodermatid adenovirus 2 (HeAdV2) was determined as the etiologic agent through pan-adenoviral consensus polymerase (PCR) chain reaction and sequencing. Nucleic acid from a novel Mycoplasma sp. (provisionally called Mycoplasma pogonae) was identified by pan-generic PCR targeting the mycoplasma 16S ribosomal RNA gene with sequencing and phylogenetic analysis. As bacteria morphologically consistent with Mycoplasma sp. were not observed by special stains and transmission electron microscopy, the detection of M. pogonae nucleic acid is of indeterminate significance; however, M. pogonae and HeAdV2 coinfection may have exacerbated disease.

Diabetes Mellitus With Concurrent Cerebellar Degeneration and Necrosis in a Domestic Goose ( Anser anser domesticus).

A 5-year-old sexually intact male Toulouse goose ( Anser anser domesticus) was presented for ataxia, polyuria, and polydipsia. The goose was cachectic and exhibited head tremors. Results of plasma biochemical analysis and point-of-care glucometry revealed persistent hyperglycemia. Despite supportive care and oral glipizide, the goose died within 48 hours of presentation. Necropsy revealed severe pancreatic atrophy and fibrosis with regionally extensive cerebellar encephalomalacia and generalized Purkinje cell degeneration and necrosis. On a wet basis, hepatic zinc concentration was determined to be twice the reference interval by atomic absorption spectroscopy. Based on these findings, the pancreatic insufficiency with secondary diabetes mellitus was attributed to chronic zinc toxicosis. Despite birds' relative resistance to high blood glucose concentrations, prolonged hyperglycemia is suspected to have caused selective Purkinje cell degeneration and necrosis by glial activation, mitochondrial dysfunction, and glutamate toxicity, which resulted in the clinically observed motor deficits. This is consistent with experimental diabetic rat models. This case highlights the need for further investigation of the complex pathophysiology of diabetes mellitus in birds.

EFFECTS OF PLANT PROTEIN DIETS ON THE HEALTH OF FARMED AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS).

The objective of this prospective, blinded study was to compare plasma biochemical values and gross and histologic evaluation of kidney and liver from American alligators ( Alligator mississippiensis ) fed extruded diets with protein derived from animal or plant sources. Alligators in two treatment groups were fed an extruded diet with protein derived primarily from plant products for 7 (n = 20) or 10 (n = 20) mo prior to harvest. A control group (n = 20) was fed a commercial diet with protein derived from animal products for the duration of the study. Plasma biochemistry panels were obtained and gross and histologic examination of kidney and liver tissues was conducted for each animal. No differences were found between alligators fed diets with animal or plant protein in terms of either biochemistry profiles or gross or histologic examination of kidney and liver. Plant-based diets, fed for up to 10 mo, do not appear to have any ill effects on the kidney or liver of American alligators.

Aging is associated with an insufficient early inflammatory response of lung endothelial cells in SARS-CoV-2 infection.

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses.

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patients on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Resolution of SARS-CoV-2 infection in human lung tissues is driven by extravascular CD163+ monocytes.

The lung-resident immune mechanisms driving resolution of SARS-CoV-2 infection in humans remain elusive. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication. Acute replication results in the emergence of cell subsets enriched in viral RNA, including extravascular inflammatory monocytes (iMO) and macrophage-like T-cells, which dissipate upon infection resolution. iMO display robust antiviral responses, are transcriptomically unique among myeloid lineages, and their emergence associates with the recruitment of circulating CD4+ monocytes. Consistently, mice depleted for human CD4+ cells but not CD3+ T-cells failed to robustly clear infectious viruses and displayed signatures of chronic infection. Our findings uncover the transient differentiation of extravascular iMO from CD4+ monocytes as a major hallmark of SARS-CoV-2 infection resolution and open avenues for unravelling viral and host adaptations defining persistently active SARS-CoV-2 infection.