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 metatranscriptomic 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 crossspecies transmission.

Delayed severe cytokine storm and immune cell infiltration in SARS-CoV-2-infected aged Chinese rhesus macaques

As of June 2020, Coronavirus Disease 2019(COVID-19) has killed an estimated 440 000 people worldwide, 74% of whom were aged ≥65 years,making age the most significant risk factor for death caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection. To examine the effect of age on death, we established a SARSCoV-2 infection model in Chinese rhesus macaques(Macaca mulatta) of varied ages. Results indicated that infected young macaques manifested impaired respiratory function, active viral replication, severe lung damage, and infiltration of CD11b^+ and CD8^+ cells in lungs at one-week post infection(wpi), but also recovered rapidly at 2 wpi. In contrast, aged macaques demonstrated delayed immune responses with a more severe cytokine storm, increased infiltration of CD11b^+ cells, and persistent infiltration of CD8^+ cells in the lungs at 2 wpi. In addition,peripheral blood T cells from aged macaques showed greater inflammation and chemotaxis, but weaker antiviral functions than that in cells from young macaques. Thus, the delayed but more severe cytokine storm and higher immune cell infiltration may explain the poorer prognosis of older aged patients suffering SARS-CoV-2 infection.

Cucurbitane triterpenoids from Hemsleya penxianensis

Two new cucurbitacins,jinfushanencins A(1)and B(2),seven new cucurbitane glycosides,jinfushanosides E-K(3-9),along with nine known analogues,were obtained from the tubers of Hemsleya penxianensis.Their structures were elucidated on the basis of extensive spectroscopic and chemical methods.Selected isolates were tested their anti-HIV-1 activities,and compound 5 showed weak anti-HIV-1 in C8166 cell(EC50=5.9μg/mL)with a selectivity index of 13.5.

Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque （Macaca leonina）

The northern pig-tailed macaque （Macaca leonina） has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 （HIV-1）, which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-INL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of v/f-substituted HIV-1 （HSIV） in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-lsv chimeras, two HIV-1Ni.4-3-derived viruses encoding the viral infectivity factor （Vif） protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.

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)-αin 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.

Lower respiratory tract samples are reliable for severe acute respiratory syndrome coronavirus 2 nucleic acid diagnosis and animal model study

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2) and coronavirus disease 2019(COVID-19) continue to impact countries worldwide.At present, inadequate diagnosis and unreliable evaluation systems hinder the implementation and development of effective prevention and treatment strategies. Here, we conducted a horizontal and longitudinal study comparing the detection rates of SARS-Co V-2 nucleic acid in different types of samples collected from COVID-19 patients and SARS-Co V-2-infected monkeys. We also detected anti-SARS-Co V-2 antibodies in the above clinical and animal model samples to identify a reliable approach for the accurate diagnosis of SARS-Co V-2 infection. Results showed that, regardless of clinical symptoms, the highest detection levels of viral nucleic acid were found in sputum and tracheal brush samples, resulting in a high and stable diagnosis rate. Anti-SARS-Co V-2 immunoglobulin M(Ig M) and G(Ig G) antibodies were not detected in6.90% of COVID-19 patients. Furthermore,integration of nucleic acid detection results from the various sample types did not improve the diagnosis rate. Moreover, dynamic changes in SARS-Co V-2 viral load were more obvious in sputum and tracheal brushes than in nasal and throat swabs. Thus,SARS-Co V-2 nucleic acid detection in sputum and tracheal brushes was the least affected by infection route, disease progression, and individual differences. Therefore, SARS-Co V-2 nucleic acid detection using lower respiratory tract samples alone is reliable for COVID-19 diagnosis and study.

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

DEAR EDITOR,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).

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

After the publication of Wang et al.(2020),we realized that there were some inappropriate statements in the content.Hereby,we correct them and apologize for any confusion this may have caused.

Stability of SARS-CoV-2 on the Surfaces of Three Meats in the Setting That Simulates the Cold Chain Transportation

Dear Editor,The coronavirus disease 2019(COVID-19)pandemic presents a severe threat to public health and the global economy and the direct origin of the causative agent severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)remains unresolved(WHO 2020).The outbreak in China was successfully contained by the implementation of a strict quarantine strategy,however sporadic outbreaks still occurred and were associated with trade markets in Beijing and Dalian cities.The COVID-19 outbreak in Beijing was traced to contaminated salmon meat sold at the Xinfadi market,Beijing,China(Chinese CDC 2020)。

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 syn drome coronavirus 2(SARS-CoV-2)infect io n in an animal model is crucial for the treatment of coronavirus disease 2019(COVID-19).Here,we compared imnnunopathological changes in young and old rhesus macaques(RMs)before and after SARS-CoV-2 infection at the tissue level.Quantitative analysis of multiplex immunofluoresce nee staining images of formali n-fixed paraffi n-embedded(FFPE)sections showed that SARS-CoV-2 infectio n specifically induced elevated levels of apoptosis,autophagy,and nuclear factor kappa-B(NF-kB)activation of angiotensirv convert!ng enzyme 2(ACE2)+cells,and increased interferon a(IFN-a)-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.

Longitudinal transcriptome analyses show robust T cell immunity during recovery from COVID-19

Understanding the processes of immune regulation in patients infected with the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is crucial for improving treatment.Here,we performed longitudinal whole-transcriptome RNA sequencing on peripheral blood mononuclear cell(PBMC)samples from 18 patients with coronavirus disease 2019(COVID-19)during their treatment,convalescence,and rehabilitation.After analyzing the regulatory networks of differentially expressed messenger RNAs(mRNAs),microRNAs(miRNAs)and long non-coding RNAs(lncRNAs)between the different clinical stages,we found that humoral immunity and type I interferon response were significantly downregulated,while robust T-cell activation and differentiation at the whole transcriptome level constituted the main events that occurred during recovery from COVID-19.The formation of this T cell immune response might be driven by the activation of activating protein-1(AP-1)related signaling pathway and was weakly affected by other clinical features.These findings uncovered the dynamic pattern of immune responses and indicated the key role of T cell immunity in the creation of immune protection against this disease.

HIV-1 can infect northern pig-tailed macaques(Macaca leonina) and form viral reservoirs in vivo

Viral reservoirs of HIV-1 are a major obstacle for curing AIDS. The novel animal models that can be directly infected with HIV-I will contribute to develop effective strategies for eradicating infections. Here, we inoculated 4 northern pig-tailed macaques （NPM） with the HIV-1 strain HIV-1NL4.3 and moni- tored the infection for approximately 3 years （150 weeks）. The HIV-l-infected NPMs showed transient viremia for about 10 weeks after infection. However, cell-associated proviral DNA and viral RNA persisted in the peripheral blood and lymphoid organs for about 3 years. Moreover, replication-competent HIV-1 could be successfully recovered from peripheral blood mononuclear cells （PBMCs） during long-term infection. The numbers of resting CD4~ T cells in HIV-1 infected NPMs harboring proviruses fell within a range of 2- to 3-1oglo per million cells, and these proviruses could be reactivated both ex vivo and in vivo in response to co-stimulation with the latency-reversing agents JQ1 and prostratin. Our results suggested that NPMs can be infected with HIV-1 and a long-term viral reservoir was formed in NPMs, which might serve as a potential model for HIV-1 reservoir research.

CD24Fc protects against viral pneumonia in simian immunodeficiency virus-infected Chinese rhesus monkeys

Viral pneumonia is a major cause of mortality caused by both systemic and respiratory infections.The danger-associated molecular patterns(DAMPs)released during cell death in viral infection may cause a self-propagating inflammatory response with lasting lung damage.The CD24–Siglec 10/G interaction is an emerging immune checkpoint that regulates inflammation caused by DAMPs.1,2,3 While we have demonstrated that fortifying this immune checkpoint can reduce inflammation in the colon,4 joints5 and central nervous system,6 it is unclear whether CD24Fc can protect against pneumonia.To address this issue,we evaluated the lung pathology of simian immunodeficiency virus(SIV)-infected rhesus monkeys that received either normal saline(NS)or CD24Fc.

Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2.Here we report that nelfinavir,an FDA approved drug for the treatment of HIV infection,is effective against SARS-CoV-2 and COVID-19.Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2(IC50=8.26μM),while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93μM(EC50).In comparison with vehicle-treated animals,rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals.At necropsy,nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude.A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center,which were randomized(1:1)to nelfinavir and control groups,showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days(9.0 vs.14.5 days,P=0.055)and the duration of fever time by 3.8 days(2.8 vs.6.6 days,P=0.014)in mild/moderate COVID-19 patients.The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients,together with its well-established good safety profile in almost all ages and during pregnancy,indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

Longitudinal transcriptome analyses show robust T cellimmunity during recovery from COVID-19

Understanding the processes of immune regulation in patients infected with the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is crucial for improving treatment.Here,we performed longitudinal whole-transcriptome RNA sequencing on peripheral blood mononuclear cell(PBMC)samples from 18 patients with coronavirus disease 2019(COVID-19)during their treatment,convalescence,and rehabilitation.After analyzing the regulatory networks of differentially expressed messenger RNAs(mRNAs),microRNAs(miRNAs)and long non-coding RNAs(lncRNAs)between the different clinical stages,we found that humoral immunity and type I interferon response were significantly downregulated,while robust T-cell activation and differentiation at the whole transcriptome level constituted the main events that occurred during recovery from COVID-19.The formation of this T cell immune response might be driven by the activation of activating protein-1(AP-1)related signaling pathway and was weakly affected by other clinical features.These findings uncovered the dynamic pattern of immune responses and indicated the key role of T cell immunity in the creation of immune protection against this disease.