Gastrointestinal (GI) infection is evidenced with involvement in COVID-19 pathogenesis caused by SARS-CoV-2. However, the correlation between GI microbiota and the distinct pathogenicity of SARS-CoV-2 Proto and its emerging variants remains unclear. In this study, we aimed to determine if GI microbiota impacted COVID-19 pathogenesis and if the effect varied between SARS-CoV-2 Proto and its variants. We performed an integrative analysis of histopathology, microbiomics, and transcriptomics on the GI tract fragments from rhesus monkeys infected with SARS-CoV-2 proto or its variants. Based on the degree of pathological damage and microbiota profile in the GI tract, five of SARS-CoV-2 strains were classified into two distinct clusters, namely, the clusters of Alpha, Beta and Delta (ABD), and Proto and Omicron (PO). Notably, the abundance of potentially pathogenic microorganisms increased in ABD but not in the PO-infected rhesus monkeys. Specifically, the high abundance of UCG-002, UCG-005, and Treponema in ABD virus-infected animals positively correlated with interleukin, integrins, and antiviral genes. Overall, this study revealed that infection-induced alteration of GI microbiota and metabolites could increase the systemic burdens of inflammation or pathological injury in infected animals, especially in those infected with ABD viruses. Distinct GI microbiota and metabolite profiles may be responsible for the differential pathological phenotypes of PO and ABD virus-infected animals. These findings improve our understanding the roles of the GI microbiota in SARS-CoV-2 infection and provide important information for the precise prevention, control, and treatment of COVID-19.

Objective To obtain the non-structure protein 14 (Nsp14) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) with higher purity and enzymatic activity. Methods This study firstly analyzed the rare codons in the gene of nsp14 according to the codon usage bias of E. coli, followed by codon optimization. The optimized nucleotide fragment of nsp14 was cloned into four kinds of expressing vectors respectively. Comparative analysis of yield and solubility was performed among these expressed four fusion proteins. The best one was chosen for further optimization of expressing conditions. After the fusion protein was purified by glutathione affinity column, the fusion tag was removed by 3C protease, and then the protein was purified by glutathione affinity column and molecular sieve column for further analysis of enzymatic activity through urea polyacrylamide gel electrophoresis. Results Many rare codons were found in expression of SARS-CoV-2 nsp14 in E. coli, among which some rare codons were distributed in close range and tandem. The best recombinant plasmid for expressing the fusion protein was pGEX6P1-GST-OPTI-Nsp14, which gave an eptimal expression in 30 ℃ with high yield and solubility. After purification, a higher purity of Nsp14 with nuclease activity was obtained. Conclusions This study shows that the SARS-CoV-2 Nsp14 protein with nuclease activity is successfully prepared, which lays a foundation for the further research on the structure and function of SARS-CoV-2 Nsp14, and provides favorable conditions for screening antiviral drugs targeting at Nsp14 of SARS-CoV-2.

Relapsing fever due to Borrelia hermsii is characterized by recurrent bacteremia episodes. However, infection of B. hermsii, if not treated early, can spread to various organs including the central nervous system (CNS). CNS disease manifestations are commonly referred to as relapsing fever neuroborreliosis (RFNB). In the mouse model of B. hermsii infection, we have previously shown that the development of RFNB requires innate immune cells as well as T cells. Here, we found that prior to the onset of RFNB, an increase in the systemic proinflammatory cytokine response followed by sustained levels of IP-10 concurrent with the CNS disease phase. RNA sequencing analysis of the spinal cord tissue during the disease phase revealed an association of the interleukin (IL)-17 signaling pathway in RFNB. To test a possible role for IL-17 in RFNB, we compared B. hermsii infection in wild-type and IL-17A-/- mice. Although the onset of bacteremia and protective anti-B. hermsii antibody responses occurred similarly, the blood-brain barrier permeability, proinflammatory cytokine levels, immune cell infiltration in the spinal cord, and RFNB manifestations were significantly diminished in IL-17A-/- mice compared to wild-type mice. Treatment of B. hermsii-infected wild-type mice with anti-IL-17A antibody ameliorated the severity of spinal cord inflammation, microglial cell activation, and RFNB. These data suggest that the IL-17 signaling pathway plays a major role in the pathogenesis of RFNB, and IL-17A blockade may be a therapeutic modality for controlling neuroborreliosis.

Chikungunya virus (CHIKV), a highly infectious and rapidly spread viral pathogen, is classified as a pathogenic agent at the biosafety level 3. Operation of live authentic CHIKV needs a specific laboratory with the P3 or above containment, which greatly confines the CHIKV-associated studies. To establish an evaluation system of CHIKV that can be utilized in a BSL2 laboratory, we constructed a pseudovirus (PsV) system of CHIKV containing double reporter genes (ZsGreen1 and luciferase). The fluorescent ZsGreen1 is a convenient and cheap reporter for monitoring the efficiency of transfection and titration of PsV. The enzyme luciferase is a sensitive reporter for the application of PsV to neutralization assay or drug screening. The CHIKV PsV produced in this study, with a titer of up to 3.16 × 106 TU/ml, was confirmed by Western blotting and transmission electronic microscopy (TEM). Finally, we developed a microneutralization assay with the CHIKV PsV produced in this study, which was successfully applied to evaluate neutralizing activities of convalescent sera from CHIKV-infected patients. In summary, we have established a convenient and sensitive double-reporter CHIKV pseudovirus system, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.

Neurological manifestations are frequently reported in the COVID-19 patients. Neuromechanism of SARS-CoV-2 remains to be elucidated. In this study, we explored the mechanisms of SARS-CoV-2 neurotropism via our established non-human primate model of COVID-19. In rhesus monkey, SARS-CoV-2 invades the CNS primarily via the olfactory bulb. Thereafter, viruses rapidly spread to functional areas of the central nervous system, such as hippocampus, thalamus, and medulla oblongata. The infection of SARS-CoV-2 induces the inflammation possibly by targeting neurons, microglia, and astrocytes in the CNS. Consistently, SARS-CoV-2 infects neuro-derived SK-N-SH, glial-derived U251, and brain microvascular endothelial cells in vitro. To our knowledge, this is the first experimental evidence of SARS-CoV-2 neuroinvasion in the NHP model, which provides important insights into the CNS-related pathogenesis of SARS-CoV-2.

目的 对Sabin株脊髓灰质炎灭活疫苗(Sabin inactivated poliovirus vaccine,sIPV)在生产过程中的灭活效果进行验证及评价.方法 分别制备Sabin株Ⅰ、Ⅱ及Ⅲ型脊髓灰质炎病毒连续5批纯化液,加入终浓度为92.5 μg/mL的甲醛,37℃灭活12d.收集灭活前(0 d)及灭活1～6、9和12 d样品,采用人喉癌上皮细胞(Hep-2)通过细胞病变法检测0～6 d样品的病毒滴度,ELISA法检测不同灭活时间样品的D抗原含量,并验证9及12d样品的灭活效果.结果 Sabin株Ⅰ、Ⅱ及Ⅲ型脊髓灰质炎病毒均在灭活第3天下降至0.00 lgCCID50/mL,3种型别病毒同日的灭活速率差异无统计学意义(P＞0.05).灭活动力学曲线显示,0～3d样品的病毒滴度呈直线下降,病毒灭活效果与灭活时间密切相关(Ⅰ、Ⅱ及Ⅲ型相关系数r分别为-0.995、-0.976及-0.996,R2分别为0.991、0.952及0.992).Ⅰ、Ⅱ及Ⅲ型病毒12d样品D抗原平均回收率分别为80％、85％和83％,抗原有一定的损失,但差异无统计学意义(P＞0.05).灭活9及12 d样品灭活验证结果均为阴性.结论 Sabin株脊髓灰质炎病毒在目前的灭活工艺条件下,均可稳定地被灭活,且能收获含量较高的D抗原,保证了sIPV疫苗的稳定性及安全性.

目的 研究白细胞介素(IL)-17信号通路在回归热神经性疏螺旋体病中的作用.方法 将14只C57BL/6野生型小鼠分为两组,S组为6只,E组为8只;8只IL-17信号通路缺陷(IL-17-/-)小鼠为IL-17-/-组.三组小鼠通过腹腔注射感染回归热疏螺旋体DAH菌株后,观察小鼠体内菌血症、神经系统发病情况及特异性抗体的发生情况.比较E组和IL-17-/-组小鼠脊髓的病理变化.结果 S组小鼠体内有效地复制扩增并引起菌血症,产生螺旋体特异性IgG和IgM,中枢神经系统疾病症状发病率达到100％.E组和IL-17-/-组小鼠体内的菌血症变化水平基本一致,且均能诱导E组和IL-17-/-组小鼠螺旋体特异性IgG和IgM产生.感染9～14 d IL-17-/-组感染回归热疏螺旋体后的中枢神经系统疾病评分低于E组,差异有统计学意义(P<0.05或P<0.01).IL-17-/-组小鼠脊髓组织中小胶质细胞的数量少于E组小鼠.结论 IL-17信号通路在回归热疏螺旋体感染诱导的中枢神经系统疾病中起重要作用.

BACKGROUND & AIMS:Gastrointestinal (GI) manifestations have been increasingly reported in patients with coronavirus disease 2019 (COVID-19). However, the roles of the GI tract in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We investigated how the GI tract is involved in SARS-CoV-2 infection to elucidate the pathogenesis of COVID-19. METHODS:Our previously established nonhuman primate (NHP) model of COVID-19 was modified in this study to test our hypothesis. Rhesus monkeys were infected with an intragastric or intranasal challenge with SARS-CoV-2. Clinical signs were recorded after infection. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction. Host responses to SARS-CoV-2 infection were evaluated by examining inflammatory cytokines, macrophages, histopathology, and mucin barrier integrity. RESULTS:Intranasal inoculation with SARS-CoV-2 led to infections and pathologic changes not only in respiratory tissues but also in digestive tissues. Expectedly, intragastric inoculation with SARS-CoV-2 resulted in the productive infection of digestive tissues and inflammation in both the lung and digestive tissues. Inflammatory cytokines were induced by both types of inoculation with SARS-CoV-2, consistent with the increased expression of CD68. Immunohistochemistry and Alcian blue/periodic acid-Schiff staining showed decreased Ki67, increased cleaved caspase 3, and decreased numbers of mucin-containing goblet cells, suggesting that the inflammation induced by these 2 types of inoculation with SARS-CoV-2 impaired the GI barrier and caused severe infections. CONCLUSIONS:Both intranasal and intragastric inoculation with SARS-CoV-2 caused pneumonia and GI dysfunction in our rhesus monkey model. Inflammatory cytokines are possible connections for the pathogenesis of SARS-CoV-2 between the respiratory and digestive systems.

Neurological manifestations are frequently reported in the COVID-19 patients. Neuromechanism of SARS-CoV-2 remains to be elucidated. In this study, we explored the mechanisms of SARS-CoV-2 neurotropism via our established non-human primate model of COVID-19. In rhesus monkey, SARS-CoV-2 invades the CNS primarily via the olfactory bulb. Thereafter, viruses rapidly spread to functional areas of the central nervous system such as hippocampus, thalamus and medulla oblongata. The infection of SARS-CoV-2 induces the inflammation possibly by targeting neurons, microglia and astrocytes in the CNS. Consistently, SARS-CoV-2 infects neuro-derived SK-N-SH, glial-derived U251 and brain microvascular endothelial cells in vitro. To our knowledge, this is the first experimental evidence of SARS-CoV-2 neuroinvasion in the NHP model, which provides important insights into the CNS-related pathogenesis of SARS-CoV-2.Funding Statement: This study was supported by the National Research and Development Project of 5 China (2020YFC0841100, 2020YFC0846400, 2020YFA0707600), CAMS Innovation Fund for Medical Sciences (2016-I2M-2-006, 2020-I2M-CoV19-012) and Major special Projects in Yunnan Province (Establishment of animal model of novel coronavirus infection and development of emergency vaccine).Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All animal procedures were approved by the Institutional Animal Care and Use Committee of Institute of Medical Biology, Chinese Academy of Medical Science (ethics number: DWSP202002001).

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and Coronavirus disease (COVID-19) were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature particularly in female animals. Low levels of virus shedding and replication in tissues occurred in all three age groups. Notably, young tree shrews (6 months to 12 months) showed virus shedding at the earlier stage of infection than adult (2 years to 4 years) and old (5 years to 7 years) animals that had longer duration of virus shedding comparatively. Histopathological examine revealed that pulmonary abnormalities were the main changes but mild although slight lesions were also observed in other tissues. In summary, tree shrew is less susceptible to SARS-CoV-2 infection compared with the reported animal models and may not be a suitable animal for COVID-19 related researches. However, tree shrew may be a potential intermediate host of SARS-CoV-2 as an asymptomatic carrier.

Identification of a suitable nonhuman primate (NHP) model of COVID-19 remains challenging. Here, we characterized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in three NHP species: Old World monkeys Macaca mulatta (M. mulatta) and Macaca fascicularis (M. fascicularis) and New World monkey Callithrix jacchus (C. jacchus). Infected M. mulatta and M. fascicularis showed abnormal chest radiographs, an increased body temperature and a decreased body weight. Viral genomes were detected in swab and blood samples from all animals. Viral load was detected in the pulmonary tissues of M. mulatta and M. fascicularis but not C. jacchus. Furthermore, among the three animal species, M. mulatta showed the strongest response to SARS-CoV-2, including increased inflammatory cytokine expression and pathological changes in the pulmonary tissues. Collectively, these data revealed the different susceptibilities of Old World and New World monkeys to SARS-CoV-2 and identified M. mulatta as the most suitable for modeling COVID-19.

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta , 6 Macaca fascicularis ) and New world monkeys (6 Callithrix jacchus ), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta , 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis , the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence Summary M. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus .

Background: COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Remarkable achievements have been made in diagnosis, prevention and treatment of COVID-19 from clinical studies. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. Methods: Two families of three species of non-human primates (NHP), old world monkeys (Macaca mulatta, Macaca fascicularis) and new world monkeys (Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and evaluation of host responses to SARS-CoV-2 infection. Findings: Increased body temperature was observed in M. mulatta and M. fascicularis, but not C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in swab samples and blood from all 3 species of monkeys. Viral shedding from upper respiratory reached the peak between day 6 and day 8 post inoculation. Viruses were mainly detected in lung, weasand, bronchus and spleen of M. mulatta and M. fascicularis, but not C. jacchus. M. mulatta showed stronger cytokine responses to SARS-CoV-2 infection than M. fascicularis. Severe gross lesions and histopathological changes were observed mainly in lung and secondary lymphoid of SARS-CoV-2 infected M. mulatta and M. fascicularis. The susceptibility to SARS-CoV2 infection is M. mulatta > M. fascicularis > C. jacchus. Interpretations: We have established a NHP model for COVID-19, which recapitulates several important aspects of COVID-19 patients and could be used to evaluate anti-viral drugs and vaccines, and investigate viral pathogenesis.Funding Statement: This study was supported by 2020YFC0841100 and 2020YFC0846400. Declaration of Interests: None.Ethics Approval Statement: All animal procedures were approved by the Institutional Animal Care and Use Committee of Institute of Medical Biology, Chinese Academy of Medical Science (Ethics number: DWSP202002 001), and performed in the ABSL-4 facility of National Kunming High-level Biosafety Primate Research Center, Yunnan China.