COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2

Thecoronavirusdisease2019(COVID-19)pandemic continues to pose a global threat to the human population. Identifying animal species susceptible to infection with the SARS-CoV-2/HCoV-19 pathogen is essential for controlling the outbreak and for testing valid prophylactics or therapeutics based on animal model studies. Here,different aged Chinese tree shrews(adult group, 1 year old;old group, 5–6 years old), which are close relatives to primates, were infected with SARS-CoV-2. X-ray, viral shedding, laboratory, and histological analyses were performed on different days postinoculation(dpi). Results showed that Chinese tree shrews could be infected by SARS-CoV-2. Lung infiltrates were visible in X-ray radiographs in most infected animals. Viral RNA was consistently detected in lung tissues from infected animals at 3,5, and 7 dpi, along with alterations in related parameters from routine blood tests and serum biochemistry, including increased levels of aspartate aminotransferase(AST) and blood urea nitrogen(BUN). Histological analysis of lung tissues from animals at 3 dpi(adult group) and 7 dpi(old group) showed thickened alveolar septa and interstitial hemorrhage. Several differences were found between the two different aged groups in regard to viral shedding peak. Our results indicate that Chinese tree shrews have the potential to be used as animal models for SARS-CoV-2 infection.

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.

Single-nucleus transcriptomic profiling of multiple organs in a rhesus macaque model of SARS-CoV-2 infection

Infection with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) causes diverse clinical manifestations and tissue injuries in multiple organs.However, cellular and molecular understanding of SARS-CoV-2 infection-associated pathology and immune defense features in different organs remains incomplete. Here, we profiled approximately 77 000single-nucleus transcriptomes of the lung, liver,kidney, and cerebral cortex in rhesus macaques(Macaca mulatta) infected with SARS-CoV-2 and healthy controls. Integrated analysis of the multiorgan dataset suggested that the liver harbored the strongest global transcriptional alterations. We observed prominent impairment in lung epithelial cells, especially in AT2 and ciliated cells, and evident signs of fibrosis in fibroblasts. These lung injury characteristics are similar to those reported in patients with coronavirus disease 2019(COVID-19).Furthermore, we found suppressed MHC class I/II molecular activity in the lung, inflammatory response in the liver, and activation of the kynurenine pathway,which induced the development of an immunosuppressive microenvironment. Analysis of the kidney dataset highlighted tropism of tubule cells to SARS-CoV-2, and we found membranous nephropathy(an autoimmune disease) caused by podocyte dysregulation. In addition, we identified the pathological states of astrocytes and oligodendrocytes in the cerebral cortex, providing molecular insights into COVID-19-related neurological implications. Overall, our multi-organ single-nucleus transcriptomic survey of SARS-CoV-2-infected rhesus macaques broadens our understanding of disease features and antiviral immune defects caused by SARS-CoV-2 infection,which may facilitate the development of therapeutic interventions for 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)-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.

Emerging SARS-CoV-2 B.1.621/Mu variant is prominently resistant to inactivated vaccine-elicited antibodies

Although it first appeared almost two years ago,the COVID-19 pandemic continues to have an impact on a global scale,in part due to newly emerging SARS-CoV-2 variants such as Delta and Lambda.The B.1.621 variant,first identified in Colombia in January 2021,was classified as a variant of interest(VOI)and designated as Mu by the World Health Organization(WHO)in August 2021.However,its infectivity and resistance to neutralizing antibodies remain largely unknown.Here,in comparison to Delta,the Mu variant showed an unexpectedly enhanced immune resistance to inactivated vaccine-elicited antibodies.Nevertheless,Mu demonstrated less infectivity than Delta,implying a biological trade-off between viral transmission and immune escape.This study strongly calls for urgent evaluation of the protective efficacy of current COVID-19 vaccines against the Mu variant.Variants of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)are of concern regarding control of the global COVID-19 pandemic(Wang et al.,2021).The SARS-CoV-2 B.1.621 variant was first identified in Colombia in January 2021.Considering its epidemiological prevalence,the WHO defined B.1.621(named Mu)as a VOI on 30 August 2021.As of September 2021,the WHO has classified four variants of concern(VOC),i.e.,Alpha(B.1.1.7),Beta(B.1.351),Gamma(P.1),and Delta(B.1.617.2),and two VOI,i.e.,Lambda(C.37)and Mu(B.1.621)(Supplementary Figure S1A).

Infectivity of SARS-CoV-2 and protection against reinfection in rats

DEAR EDITOR,The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) pandemic remains an important global public health issue. In this study, we unexpectedly found that wildtype Sprague-Dawley(SD) rats can be infected with the SARS-CoV-2 prototype. Our results showed direct experimental evidence of the infectivity of SARS-CoV-2 infection, subsequent pathogenicity, and protection against reinfection in rats.

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.

Inhibitory effects of chloroquine on the activation of plasmacytoid dendritic cells in SIVmac239-infected Chinese rhesus macaques

It is currently widely accepted that immune activation in HIV-infected individuals leads to a severe loss of CD4＋ T cells and the progression to AIDS. However, the underlying mechanism of this immune activation remains unclear. Experimental data suggest that the activation of plasmacytoid dendritic cells （pDCs） by plasma viremia may play a critical role in HIV-induced immune activation. In this study, we found that the level of immune activation was higher in the late phase of SIVmac239 infection compared with chronic infection, which suggests that immune activation might be related to disease progression in SIVmac239-infected non-human primate models. Our work also showed that chloroquine could effectively inhibit the activation of pDCs in vitro and in vivo. However, chloroquine treatment of SIVmac239-infected macaques had no significant influence on the Cellular composition of peripheral blood in these animals.

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally,from B.1.17(Alpha), B.1.351(Beta), P.1(Gamma), B.1.617.2(Delta) to B.1.1.529(Omicron). The emerging or reemerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics,pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 cells. We utilized both animal models of human ACE2(hACE2)transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.

Tissue distribution of SARS-CoV-2 in non-human primates after combined intratracheal and intranasal inoculation

Dear Editor,As of 5 July,2020,approximately 20,162,000 confirmed cases of coronavirus disease 2019(COVID-19),which is caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),have been reported globally,including an estimated 740,000 deaths.Although the clinical manifestations of COVID-19 are heterogeneous,viral infection and pathological changes in the respiratory system,especially the lungs,still represent common characteristics of COVID-19.The nose,served as the entrance to the respiratory system,seems to produce absent or minimal symptoms of nasal congestion or rhinorrhea during SRAS-CoV-2 infection,while other viral rhinitis(i.e.,adenovirus,rhinovirus,influenza virus,etc.)are characterized by typical nasal symptoms,including nasal congestion,rhinorrhea,and sneezing.Recent studies suggest that,despite a lack of other nasal symptoms,anosmia is an important sign of early-stage COVID-19(Lechien et al.,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.

A recombinant receptor-binding domain in trimeric form generates protective immunity against SARSCoV-2 infection in nonhuman primates

A safe and effective vaccine is critical to combat the COVID-19 pandemic.Here,we developed a trimeric SARS-CoV-2 receptor-binding domain(RBD)subunit vaccine candidate that simulates the natural structure of the spike(S)trimer glycoprotein.Immunization with the RBD trimer-induced robust humoral and cellular immune responses,and a high level of neutralizing antibodies was maintained for at least 4.5 months.Moreover,the antibodies that were produced in response to the vaccine effectively cross-neutralized the SARS-CoV-2501Y.V2 variant(B.1.351).Of note,when the vaccine-induced antibodies dropped to a sufficiently low level,only one boost quickly activated the anamnestic immune response,conferring full protection against a SARSCoV-2 challenge in rhesus macaques without typical histopathological changes in the lung tissues.These results demonstrated that the SARS-CoV-2 RBD trimer vaccine candidate is highly immunogenic and safe,providing long-lasting,broad,and significant immunity protection in nonhuman primates,thereby offering an optimal vaccination strategy against COVID-19.