Identification of novel mammalian viruses in tree shrews ( Tupaia belangeri chinensis).

The Chinese tree shrew ( Tupaia belangeri chinensis), a member of the mammalian order Scandentia, exhibits considerable similarities with primates, including humans, in aspects of its nervous, immune, and metabolic systems. These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer, infectious diseases, metabolic disorders, and mental health conditions. Herein, we used meta-transcriptomic sequencing to analyze plasma, as well as oral and anal swab samples, from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses. In total, eight mammalian viruses with complete genomes were identified, belonging to six viral families, including Flaviviridae, Hepeviridae, Parvovirinae, Picornaviridae, Sedoreoviridae, and Spinareoviridae. Notably, the presence of rotavirus was recorded in tree shrews for the first time. Three viruses - hepacivirus 1, parvovirus, and picornavirus - exhibited low genetic similarity (<70%) with previously reported viruses at the whole-genome scale, indicating novelty. Conversely, three other viruses - hepacivirus 2, hepatovirus A and hepevirus - exhibited high similarity (>94%) to known viral strains. Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants. These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews, highlighting the necessity for further research into their potential for cross-species transmission.

Increased cAMP-PKA signaling pathway activation is involved in up-regulation of CTLA-4 expression in CD4+ T cells in acute SIVmac239-infected Chinese rhesus macaques.

Human immunodeficiency virus-1 (HIV-1) infection can cause chronic activation, exhaustion, and anergy of the immune system. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint molecule, which plays an important role in immune homeostasis and disease. CTLA-4 expression is elevated in HIV-1-infected patients and is associated with disease progression. However, the mechanism controlling expression of CTLA-4 in HIV-1 infection is poorly characterized. In this study, we used a SIV-infected Chinese rhesus macaque (ChRM) model to explore CTLA-4 expression in SIV infection. Results showed that SIV infection significantly increased CTLA-4 expression in all T cell subsets, especially central memory T cells. CTLA-4+CD4+ T cell frequency was significantly associated with disease progression markers. Activation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway regulated CTLA-4 expression in CD4+T cells, as confirmed by stimulation with dibutyryl cyclic adenosine monophosphate, forskolin, and 3-isobutyl-1-methylxanthine, and inhibition with H-89 ex vivo. Simultaneously, cAMP concentration in PBMCs and PKA activity in both PBMCs and CD4+ T cells were increased in acute SIV-infected ChRMs, accompanied by an increase in adenylate cyclase 6 expression and a decrease in cAMP-phosphodiesterase 3A (PDE3A), PDE4B, and PDE5A expression in PBMCs. In addition, selective inhibition of PDE4B and PDE5A activity enhanced CTLA-4 expression in CD4+ T cells. These results suggest that SIV infection alters cAMP metabolism and increases cAMP-PKA signaling pathway activation, which up-regulates the expression of CTLA-4 in acute SIVmac239-infected ChRMs. Thus, regulation of the cAMP-PKA signaling pathway may be a potential strategy for the restoration of T cell function and therapy for AIDS.

Severe fever with thrombocytopenia syndrome virus induces platelet activation and apoptosis via a reactive oxygen species-dependent pathway.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV) and with a high fatality rate. Thrombocytopenia is a major clinical manifestation observed in SFTS patients, but the underlying mechanism remains largely unclear. Here, we explored the effects of SFTSV infection on platelet function in vivo in severely infected SFTSV IFNar-/- mice and on mouse and human platelet function in vitro. Results showed that SFTSV-induced platelet clearance acceleration may be the main reason for thrombocytopenia. SFTSV-potentiated platelet activation and apoptosis were also observed in infected mice. Further investigation showed that SFTSV infection induced platelet reactive oxygen species (ROS) production and mitochondrial dysfunction. In vitro experiments revealed that administration of SFTSV or SFTSV glycoprotein (Gn) increased activation, apoptosis, ROS production, and mitochondrial dysfunction in separated mouse platelets, which could be effectively ameliorated by the application of antioxidants (NAC (N-acetyl-l-cysteine), SKQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) and resveratrol). In vivo experiments showed that the antioxidants partially rescued SFTSV infection-induced thrombocytopenia by improving excessive ROS production and mitochondrial dysfunction and down-regulating platelet apoptosis and activation. Furthermore, while SFTSV and Gn directly potentiated human platelet activation, it was completely abolished by antioxidants. This study revealed that SFTSV and Gn can directly trigger platelet activation and apoptosis in an ROS-MAPK-dependent manner, which may contribute to thrombocytopenia and hemorrhage during infection, but can be abolished by antioxidants.

Bromodomain and Extra-Terminal Inhibitor BMS-986158 Reverses Latent HIV-1 Infection In Vitro and Ex Vivo by Increasing CDK9 Phosphorylation and Recruitment.

Latent reservoir persistence remains a major obstacle for curing human immunodeficiency virus type 1 (HIV-1) infection. Thus, strategies for the elimination of latent HIV-1 are urgently needed. As a bromodomain and extra-terminal (BET) inhibitor, BMS-986158 has been used in clinical trials for advanced solid tumors and hematological malignancies. Here, we found that BMS-986158 reactivated latent HIV-1 in three types of HIV-1 latency cells in vitro, and in combination antiretroviral therapy (cART)-treated patient-derived peripheral blood mononuclear cells ex vivo, without influencing global immune cell activation. BMS-986158 reactivated latent HIV-1 by increasing phosphorylation of CDK9 at Thr186 and promoting recruitment of CDK9 and RNA polymerase II to the HIV-1 long terminal repeat in J-Lat cells. Furthermore, BMS-986158 exerted strong synergism in reactivating latent HIV-1 when combined with prostratin and vorinostat and enhanced the antiviral activity of anti-HIV-1 drugs. Finally, BMS-986158 showed antiviral activity in an HIV-1 acute infection model, possibly by arresting the cell cycle in infected cells. Thus, these results suggest that BMS-986158 is a potential candidate for AIDS/HIV-1 therapy.

Glucose Metabolism Disorder Induces Spermatogenic Dysfunction in Northern Pig-Tailed Macaques (Macaca leonina) With Long-Term SIVmac239 Infection.

Although spermatogenic dysfunction is widely found in patients with human immunodeficiency virus (HIV), the underlying reasons remain unclear. Thus far, potential hypotheses involving viral reservoirs, testicular inflammation, hormone imbalance, and cachexia show inconsistent correlation with spermatogenic dysfunction. Here, northern pig-tailed macaques (NPMs) exhibited marked spermatogenic dysfunction after long-term infection with simian immunodeficiency virus (SIVmac239), with significant decreases in Johnsen scores, differentiated spermatogonial stem cells, and testicular proliferating cells. The above hypotheses were also evaluated. Results showed no differences between SIV- and SIV+ NPMs, except for an increase in follicle stimulating hormone (FSH) during SIV infection, which had no direct effect on the testes. However, long-term SIVmac239 infection undermined pancreatic islet ß cell function, partly represented by significant reductions in cellular counts and autophagy levels. Pancreatic islet ß cell dysfunction led to glucose metabolism disorder at the whole-body level, which inhibited lactate production by Sertoli cells in testicular tissue. As lactate is the main energy substrate for developing germ cells, its decrease was strongly correlated with spermatogenic dysfunction. Therefore, glucose metabolism disorder appears to be a primary cause of spermatogenic dysfunction in NPMs with long-term SIVmac239 infection.

Pro-inflammatory microenvironment and systemic accumulation of CXCR3+ cell exacerbate lung pathology of old rhesus macaques infected with SARS-CoV-2.

Understanding the pathological features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an animal model is crucial for the treatment of coronavirus disease 2019 (COVID-19). Here, we compared immunopathological changes in young and old rhesus macaques (RMs) before and after SARS-CoV-2 infection at the tissue level. Quantitative analysis of multiplex immunofluorescence staining images of formalin-fixed paraffin-embedded (FFPE) sections showed that SARS-CoV-2 infection specifically induced elevated levels of apoptosis, autophagy, and nuclear factor kappa-B (NF-κB) activation of angiotensin-converting enzyme 2 (ACE2)+ cells, and increased interferon α (IFN-α)- and interleukin 6 (IL-6)-secreting cells and C-X-C motif chemokine receptor 3 (CXCR3)+ cells in lung tissue of old RMs. This pathological pattern, which may be related to the age-related pro-inflammatory microenvironment in both lungs and spleens, was significantly correlated with the systemic accumulation of CXCR3+ cells in lungs, spleens, and peripheral blood. Furthermore, the ratio of CXCR3+ to T-box protein expression in T cell (T-bet)+ (CXCR3+/T-bet+ ratio) in CD8+ cells may be used as a predictor of severe COVID-19. These findings uncovered the impact of aging on the immunopathology of early SARS-CoV-2 infection and demonstrated the potential application of CXCR3+ cells in predicting severe COVID-19.

Northern pig-tailed macaques ( Macaca leonina) infected with SARS-CoV-2 show rapid viral clearance and persistent immune response.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has become an unprecedented global health emergency. At present, SARS-CoV-2-infected nonhuman primates are considered the gold standard animal model for COVID-19 research. Here, we showed that northern pig-tailed macaques ( Macaca leonina, NPMs) supported SARS-CoV-2 replication. Furthermore, compared with rhesus macaques, NPMs showed rapid viral clearance in lung tissues, nose swabs, throat swabs, and rectal swabs, which may be due to higher expression of interferon (IFN)-α in lung tissue. However, the rapid viral clearance was not associated with good outcome. In the second week post infection, NPMs developed persistent or even more severe inflammation and body injury compared with rhesus macaques. These results suggest that viral clearance may have no relationship with COVID-19 progression and SARS-CoV-2-infected NPMs could be considered as a critically ill animal model in COVID-19 research.

Successful implementation of intestinal resection and anastomosis in non-human primates suggests the possibility of longitudinal intestinal research.

Intestinal biopsy is a basic experimental method for studying pathological changes in the intestinal tract during human immunodeficiency virus (HIV) infection. In this study, jejunal resection and anastomosis were successfully performed in 12 Chinese rhesus macaques ( Macaca mulatta). The sampled gut tissues were then examined by hematoxylin and eosin (H&E) staining, electron microscopy, flow cytometry, immunofluorescence detection, and RNA quality analysis to ensure suitability for histological, physiological, pathological, and immunological detection, as well as mechanistic analysis at the cellular and molecular level. Importantly, the surgery did not affect the ratio or number of immune cells in peripheral blood or the concentration of lipids, proteins, and vitamins in plasma, which are important indicators of nutritional status. Our results thus indicated that jejunal resection and anastomosis are feasible, and that immune homeostasis and intestinal barrier integrity are not altered by surgery. All macaques recovered well (except for one), with no postoperative complications. Therefore, this animal surgery may be applicable for longitudinal intestinal research related to diseases such as acquired immunodeficiency syndrome (AIDS).

Translocation of microbes and changes of immunocytes in the gut of rapid- and slow-progressor Chinese rhesus macaques infected with SIVmac239.

Human/simian immunodeficiency virus (HIV/SIV) infection can cause severe depletion of CD4(+) T cells in both plasma and mucosa; it also results in damage to the gut mucosa barrier, which makes the condition more conducive to microbial translocation. In this study, we used SIV-infected Chinese rhesus macaques to quantify the extent of microbial translocation and the function of immune cells in the entire gastrointestinal tract and to compare their differences between rapid and slow progressors. The results showed that in the slow progressors, microbial products translocated considerably and deeply into the lamina propria of the gut; the tissue macrophages had no significant differences compared with the rapid progressors, but there was a slightly higher percentage of mucosal CD8(+) T cells and a large amount of extracellular microbial products in the lamina propria of the intestinal mucosa of the slow progressors. The data suggested that although microbial translocation increased markedly, the mucosal macrophages and CD8(+) T cells were insufficient to clear the infiltrated microbes in the slow progressors. Also, therapies aimed at suppressing the translocation of microbial products in the mucosa could help to delay the progression of SIV disease.

Lipopolysaccharide Increases Immune Activation and Alters T Cell Homeostasis in SHIVB'WHU Chronically Infected Chinese Rhesus Macaque.

Immune activation plays a significant role in the disease progression of HIV. Microbial products, especially bacterial lipopolysaccharide (LPS), contribute to immune activation. Increasing evidence indicates that T lymphocyte homeostasis disruptions are associated with immune activation. However, the mechanism by which LPS affects disruption of immune response is still not fully understood. Chronically SHIVB'WHU-infected Chinese rhesus macaques received 50 µg/kg body weight LPS in this study. LPS administration affected the virus/host equilibrium by elevating the levels of viral replication and activating T lymphocytes. LPS induced upregulation of CD8(+) naïve T cells and downregulated the number of CD4(+) and CD8(+) T effector memory cells. The downregulated effector memory cells are associated with a lower frequency of monofunctional and polyfunctional cells, and an upregulated programmed cell death-1 (PD-1) expression on CD4(+) and CD8(+) T cells was observed in monkeys after LPS stimulation. Our data provide new insights into the function of LPS in the immune activation in SHIV/HIV infection.