prepared viruses. demonstrated a decrease in BW by 5.88C28.57%. Five out of six showed a decrease in BW by 2.17C10.51% (Supplementary Table 2), suggesting more severe loss of BW in than in and at 12 dpi. In particular, aged showed more interstitial patterning than young or adult (Fig. ?(Fig.1c).1c). Importantly, overall comparison of the chest radiographs showed that the pulmonary abnormalities were more severe in than in are more susceptible to SARS-CoV-2 infection than (HHH-13 and HHH-14) necropsied at the early stage of infection: the lung, trachea, bronchia, spleen, stomach, rectum, bladder, uterus, hilar lymph node, and mesenteric lymph node. However, at the late stage of infection, viral RNA was found in only the bronchus, laryngopharynx, and spleen of and (Fig. ?(Fig.2b).2b). Viral RNA and spike protein levels in respiratory tissue samples were confirmed by RNA hybridization with the RNAscope platform (Fig. ?(Fig.2c2c and Supplementary Table 3) and immunofluorescent assays (Supplementary Fig. 1). We did not find viral RNA in any of the 16C18 tissues collected from the RH2 and RH5 strains of and and that in addition to the respiratory route, the fecalCoral route may be involved in viral transmission. Open in a separate window Fig. 2 Viral shedding and the replication of SARS-CoV-2 in NHPs. a Every other day after virus inoculation, swab (nasal, throat, and anal), feces, and blood samples were collected from the monkeys for the quantification of virus genomic RNA via RT-qPCR. An X in the boxes indicates that the value was not determined. A heatmap was prepared via pheatmap in R package. b Tissue samples were harvested from eight necropsied animals at the indicated time points for the evaluation of viral load by RT-qPCR. c To determine viral localization in tissues, nucleic acid hybridization (RNAscope technology) was performed to detect viral RNA with a probe specific for SARS-CoV-2 Host responses to SARS-CoV-2 infection Finally, host responses to SARS-CoV-2 infection were evaluated by assessments of the production of virus-specific antibodies and secretion of inflammatory cytokines, hematology, and histopathology. Aceglutamide Aceglutamide We found that SARS-CoV-2 infection induced the production of virus-specific antibodies in the majority of monkeys, with these antibodies becoming detectable at 4 dpi (Fig. 3a, b). Overall, antibody levels in old and adult were higher than those in young at 21 dpi (Supplementary Table 4). Open PVRL1 in a separate window Fig. 3 Host responses to SARS-CoV-2 infection. The antibody responses of (a) and (b) to viral infection were evaluated with total virus-specific antibody ELISA kits. Inflammatory cytokines in serum samples from (c) and (d) were measured by Luminex multiplex assays as described in the Materials and methods section. All the figures here were prepared via GraphPad software Inflammatory responses to infection were determined via assays to detect multiple cytokines in serum samples. Eight cytokines (G-CSF, IL-1A, IL-8, IL-15, IL-18, MCP-1, MIP-1B, and sCD40-L) were detected in most of the infected animals. Notably, HHH-11 and SXH-6 showed high expression of the inflammatory cytokines IL-10, IL-1A, IL-8, IL-15, and MCP-1 (Fig. 3c, d). Interestingly, the nasal swabs, anal swabs and fecal samples from these two animals exhibited prolonged viral shedding (Fig. ?(Fig.2a),2a), indicating that these inflammatory cytokines were related to SARS-CoV-2 infection. High levels of IL-15, IL-18, and MCP-1 were observed in HHH-3, which showed no antibody induction after SARS-CoV-2 infection (Fig. ?(Fig.3c3c). Cellular responses to SARS-CoV-2 infection were analyzed by flow cytometry. Three major populations of immune cells (T cells, B cells, and monocytes) in red blood cell (RBC)-depleted peripheral blood were measured. Regardless of the age of or sex of and and but not showed pathological changes in all six examined tissues, whereas had four tissues (lung, trachea, bronchus, and kidney) with lesions and had pathological changes in three examined tissues (lung, liver, and Aceglutamide spleen). Furthermore, typical ultrastructural lesions were observed in the lung (increased numbers of type II pneumocytes, RBCs and macrophages in the alveolar airspace), mesenteric lymph node (intact structure with a large number of lymphocytes, secretory granules in the cytoplasm of cells) and spleen (increased number of granules in the macrophages) (Fig. ?(Fig.4b),4b), which was consistent with the results of microscopic inspection. These.