柯萨奇病毒A10型（Coxsackievirus A10, CVA10）是手足口病的主要病原之一。目前尚无控制CVA10感染的疫苗和抗病毒药物。CVA10反向遗传学工具将有助于其机制研究及其疫苗和抗病毒药物研发。本研究中，我们构建了CVA10原型和带Myc标签的感染性克隆。将病毒mRNA转染人横纹肌肉瘤（RD）细胞，获得存活的CVA10病毒。二代测序技术和病毒学实验进一步证实了拯救的CVA10特性。进而构建带有荧光素酶报告基因的CVA10亚基因组复制子和带有EGFP报告基因的病毒衣壳蛋白表达载体。在人胚肾293T细胞（HEK293T）中，共转染病毒复制子RNA和衣壳蛋白表达载体可获得单轮感染性颗粒（Single round infectious particles, SRIPs）。基于CVA10复制子RNA，制备带有肠道病毒A71（EVA71）衣壳或CVA10衣壳的SRIPs。EVA71型SRIPs感染依赖SCARB2，而CVA10型SRIPs则不需要。发现在5'-非翻译区（UTR）之前插入cis-active hammerhead ribozyme（HHRib）可以显著提高复制子的活性和SRIPs的生产。综上所述，我们成功建立并优化了CVA10原型毒株的反向遗传学工具，包括感染性克隆和SRIPs系统，这些工具将有助于CVA10的基础和转化研究。

Flaviviruses are a genus of mostly arthropod-borne RNA viruses that cause a range of pathologies in humans. Basic knowledge on flaviviruses is rapidly expanding, partly due to their status as frequent emerging or re-emerging pathogens. Flaviviruses include the dengue, Zika, West Nile, tick-borne encephalitis and yellow fever viruses (DENV, ZIKV, WNV, TBEV and YFV, respectively). As is the case with other families of viruses, the success of productive infection of human cells by flaviviruses depends in part on the antiviral activity of a heterogeneous group of cellular antiviral proteins called restriction factors. Restriction factors are the effector proteins of the cell-autonomous innate response against viruses, an immune pathway that also includes virus sensors as well as intracellular and extracellular signal mediators such as type Ⅰ interferons (IFN-I). In this review, I summarize recent progress toward the identification and characterization of flavivirus restriction factors. In particular, I focus on IFI6, Schlafen 11, FMRP, OAS-RNase L, RyDEN, members of the TRIM family of proteins (TRIM5α, TRIM19, TRIM56, TRIM69 and TRIM79α) and a new mechanism of action proposed for viperin. Recent and future studies on this topic will lead to a more complete picture of the flavivirus restrictome, defined as the ensemble of cellular factors with demonstrated anti-flaviviral activity.

Children with Coronavirus Disease 2019 (COVID-19) were reported to show milder symptoms and better prognosis than their adult counterparts, but the difference of immune response against SARS-CoV-2 between children and adults hasn’t been reported. Therefore we initiated this study to figure out the features of immune response in children with COVID-19. Sera and whole blood cells from 19 children with COVID-19 during different phases after disease onset were collected. The cytokine concentrations, SARS-CoV-2 S-RBD or N-specific antibodies and T cell immune responses were detected respectively. In children with COVID-19, only 3 of 12 cytokines were increased in acute sera, including interferon (IFN)-γ-induced protein 10 (IP10), interleukin (IL)-10 and IL-16. We observed an increase in T helper (Th)-2 cells and a suppression in regulatory T cells (Treg) in patients during acute phase, but no significant response was found in the IFN-γ-producing or tumor necrosis factor (TNF)-α-producing CD8⁺ T cells in patients. S-RBD and N IgM showed an early induction, while S-RBD and N IgG were prominently induced later in convalescent phase. Potent S-RBD IgA response was observed but N IgA seemed to be inconspicuous. Children with COVID-19 displayed an immunophenotype that is less inflammatory than adults, including unremarkable cytokine elevation, moderate CD4⁺ T cell response and inactive CD8⁺ T cell response, but their humoral immunity against SARS-CoV-2 were as strong as adults. Our finding presented immunological characteristics of children with COVID-19 and might give some clues as to why children develop less severe disease than adults.

我们的最新研究发现人二氢乳清酸脱氢酶（DHODH）的抑制剂具有广谱抗病毒活性，在感染细胞中可以有效抑制新型冠状病毒的复制。然而,还未有临床数据证明DHODH抑制剂在新型冠状病毒病感染（COVID-19）的患者中具有治疗效果。本研究中，我们评估了一种已经上市并广泛用应用于自身免疫病治疗的DHODH抑制剂，来氟米特 (Leflunomide)，在小范围COVID-19患者中同情用药的效果。研究招募了10例实验室确诊的普通型COVID-19患者，其中5名患者接受了来氟米特治疗，另外5名患者为空白对照组（没有使用安慰剂），所有患者都接受了新型冠状病毒感染的标准支持治疗方案。结果表明，服用来氟米特的患者比对照组的排毒时间明显缩短，用药组排毒时间中位数为5天，而对照组排毒时间中位数为11天（P=0.046）。服用来氟米特的患者在用药后C反应蛋白水平也显著下降，表明炎症反应得到了很好的控制。在来氟米特治疗的患者中，没有观察到明显的不良反应，能在短期康复出院。这项小规模的同情用药初步研究为进一步了解来氟米特作为抗新型冠状病毒感染的潜在药物提供了线索。

抗体在中和病毒感染中发挥关键作用，并越来越多地被用作治疗药物和诊断工具。病毒-抗体免疫复合物的结构研究对于更好地理解抗体所介导中和反应的分子机制具有重要意义，也为以结构为基础的疫苗设计提供了有价值的信息。冷冻电镜技术是近几年成熟的一种用于研究生物大分子复合物结构的强大手段，当与X射线晶体学结合时，可以常规研究正二十面体病毒与抗体所形成的免疫复合物。本文阐述了正二十面体病毒在与抗体相互作用时的结构变化，重点讨论了不同功能状态下病毒衣壳的动态变化，同时也简要讨论了多形性嚢膜病毒的糖蛋白与其抗体所形成的免疫复合物结构。病毒作为展示平台，可以用来研究病毒抗原表位的结构，为未来疫苗研发提供重要的依据。

Hand, foot, and mouth disease (HFMD) recently emerged as a global public threat. The licensure of inactivated enterovirus A71 (EV-A71) vaccine was the first step in using a vaccine to control HFMD. New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage. The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines. The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes. The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a cross-protective antibody response.

Human parainfluenza virus type 3 (HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein (P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein (L). P connects nucleocapsid protein (N) with L to initiate genome transcription and replication. Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine (P_K492R/K532R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that P_K492R/K532R had no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies. Finally, we found that incorporation of K492R/K532R into a recombinant HPIV3 (rHPIV3-P_K492R/K532R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.

The temporal change patterns of laboratory data may provide insightful clues into the whole course of COVID-19. This study aimed to evaluate longitudinal change patterns of key laboratory tests in patients with COVID-19, and identify independent prognostic factors by examining the associations between laboratory findings and outcomes of patients. This multicenter study included 56 patients with COVID-19 treated in Jilin Province, China, from January 21, 2020 to March 5, 2020. The laboratory findings, epidemiological characteristics and demographic data were extracted from electronic medical records. The average value of eosinophils and carbon dioxide combining power continued to significantly increase, while the average value of cardiac troponin I and mean platelet volume decreased throughout the course of the disease. The average value of lymphocytes approached the lower limit of the reference interval for the first 5 days and then rose slowly thereafter. The average value of thrombocytocrit peaked on day 7 and slowly declined thereafter. The average value of mean corpuscular volume and serum sodium showed an upward trend from day 8 and day 15, respectively. Age, sex, lactate dehydrogenase, platelet count and globulin level were included in the final model to predict the probability of recovery. The above parameters were verified in 24 patients with COVID-19 in another area of Jilin Province. The risk stratification and management of patients with COVID-19 could be improved according to the temporal trajectories of laboratory tests.

The coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has spread around the world with high mortality. To diagnose promptly and accurately is the vital step to effectively control its pandemic. Dynamic characteristics of SARS-CoV-2-specific antibodies which are important for diagnosis of infection have not been fully demonstrated. In this retrospective, single-center, observational study, we enrolled the initial 131 confirmed cases of COVID-19 at Jin-Yin-Tan Hospital who had at least one-time antibody tested during their hospitalization. The dynamic changes of IgM and IgG antibodies to SARS-CoV-2 nucleocapsid protein in 226 serum samples were detected by ELISA. The sensitivities of IgM and IgG ELISA detection were analyzed. Result showed that the sensitivity of the IgG ELISA detection (92.5%) was significantly higher than that of the IgM (70.8%) (P < 0.001). The meantimes of seroconversion for IgM and IgG were 6 days and 3 days, respectively. The IgM and IgG antibody levels peaked at around 18 days and 23 days, and then IgM fell to below the baseline level at about day 36, whereas IgG maintained at a relatively high level. In conclusion, antibodies should be detected to aid in diagnosis of COVID-19 infection. IgG could be a sensitive indicator for retrospective diagnosis and contact tracing, while IgM could be an indicator of early infection.

To understand the epidemiological and clinical features of the symptomatic and asymptomatic pediatric cases of COVID-19, we carried out a prospective study in Shanghai during the period of January 19 to April 30, 2020. A total of 49 children (mean age 11.5 ± 5.12 years) confirmed with SARS-CoV-2 infection were enrolled in the study, including 11 (22.4%) domestic cases and 38 (77.6%) imported cases. Nine (81.8%) local cases and 12 (31.6%) imported cases had a definitive epidemiological exposure. Twenty-eight (57.1%) were symptomatic and 21 (42.9%) were asymptomatic. Neither asymptomatic nor symptomatic cases progressed to severe diseases. The mean duration of viral shedding for SARS-CoV-2 in upper respiratory tract was 14.1 ± 6.4 days in asymptomatic cases and 14.8 ± 8.4 days in symptomatic cases (P > 0.05). Forty-five (91.8%) cases had viral RNA detected in stool. The mean duration of viral shedding in stool was 28.1 ± 13.3 days in asymptomatic cases and 30.8 ± 18.6 days in symptomatic participants (P > 0.05). Children < 7 years shed viral RNA in stool for a longer duration than school-aged children (P < 0.05). Forty-three (87.8%) cases had seropositivity for antibodies against SARS-CoV-2 within 1–3 weeks after confirmation with infection. In conclusion, asymptomatic SARS-CoV-2 infection may be common in children in the community during the COVID-19 pandemic wave. Asymptomatic cases shed viral RNA in a similar pattern as symptomatic cases do. It is of particular concern that asymptomatic individuals are potentially seed transmission of SARS-CoV-2 and pose a challenge to disease control.

The immense patient number caused by coronavirus disease 2019 (COVID-19) global pandemic brings the urge for more knowledge about its immunological features, including the profile of basic immune parameters. In this study, eighty-eight reported COVID-19 patients in Wuhan were recruited from January to February, 2020, including 32 severe/critical cases and 56 mild/moderate cases. Their mean age was 56.43 years (range 17–83) and gender ratio (male/female) was 43:45. We tested SARS-CoV-2 RNA with commercial kits, investigated the level of serologic IgM and IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using magnetic particle chemiluminescence immunoassays, and compared the results of serologic tests and nucleic acid test (NAT). Among 88 patients, 95.45% were confirmed as positive by the combination of NAT and antibody test, which was significantly higher (P < 0.001) than by single nucleic acid test (73.86%) or serologic test (65.91%). Then the correlation between temporal profile and the level of antibody response was analyzed. It showed that seroconversion started on day 5 after disease onset and IgG level was rose earlier than IgM. Comparison between patients with different disease severity suggested early seroconversion and high antibody titer were linked with less severe clinical symptoms. These results supported the combination of serologic testing and NAT in routine COVID-19 diagnosis and provided evidence on the temporal profile of antibody response in patients with different disease severity.

COVID-19 patients can recover with a median SARS-CoV-2 clearance of 20 days post initial symptoms (PIS). However, we observed some COVID-19 patients with existing SARS-CoV-2 for more than 50 days PIS. This study aimed to investigate the cause of viral clearance delay and the infectivity in these patients. Demographic data and clinical characteristics of 22 long-term COVID-19 patients were collected. The median age of the studied cohort was 59.83 ± 12.94 years. All patients were clinically cured after long-term SARS-CoV-2 infection ranging from 53 to 112 days PIS. Peripheral lymphocytes counts were normal. The ratios of interferon gamma (IFN-γ)-secreting cells to total CD4⁺ and CD8⁺ cells were normal as 24.68% ± 9.60% and 66.41% ± 14.87% respectively. However, the number of IFN-γ-secreting NK cells diminished (58.03% ± 11.78%). All patients presented detectable IgG, which positively correlated with mild neutralizing activity (Mean value neutralisation antibodies titers = 157.2, P = 0.05). No SARS-CoV-2 virus was isolated in Vero E6 cells inoculated with nasopharyngeal swab samples from all patients 50 days PIS, and the cytopathic effect was lacking. But one sample was positive for SARS-CoV-2 nucleic acid test in cell supernatants after two passages. Genome sequencing revealed that only three synonymous variants were identified in spike protein coding regions. In conclusion, decreased IFN-γ production by NK cells and low neutralizing antibodies might favor SARS-CoV-2 long-term existence. Further, low viral load and weak viral pathogenicity were observed in COVID-19 patients with long-term SARS-CoV-2 infection.

African swine fever (ASF) is an infectious transboundary disease of domestic pigs and wild boar and spreading throughout Eurasia. There is no vaccine and treatment available. Complex immune escape strategies of African swine fever virus (ASFV) are crucial factors affecting immune prevention and vaccine development. MGF360 genes have been implicated in the modulation of the IFN-I response. The molecular mechanisms contributing to innate immunity are poorly understood. In this study, we demonstrated that ASFV MGF360-12L (MGF360 families 12L protein) significantly inhibited the mRNA transcription and promoter activity of IFN-β and NF-кB, accompanied by decreases of IRF3, STING, TBK1, ISG54, ISG56 and AP-1 mRNA transcription. Also, MGF360-12L might suppress the nuclear localization of p50 and p65 mediated by classical nuclear localization signal (NLS). Additionally, MGF360-12L could interact with KPNA2, KPNA3, and KPNA4, which interrupted the interaction between p65 and KPNA2, KPNA3, KPNA4. We further found that MGF360-12L could interfere with the NF-кB nuclear translocation by competitively inhibiting the interaction between NF-кB and nuclear transport proteins. These findings suggested that MGF360-12L could inhibit the IFN-I production by blocking the interaction of importin a and NF-кB signaling pathway, which might reveal a novel strategy for ASFV to escape the host innate immune response.