Human respiratory syncytial virus (RSV) remains a leading cause of severe lower respiratory tract infections in infants and immunocompromised populations, causing approximately 160,000 annual deaths globally. Despite recent approvals of prefusion F (pre-F) protein-based vaccines (Arexvy, Abrysvo) for older adults and pregnant women, pediatric vaccine development faces unique challenges including enhanced respiratory disease (ERD) risks, maternal antibody interference, and immature infant immune responses. Meanwhile, G protein glycosylation variability and NS1/NS2-mediated interferon suppression remain the outstanding difficulties in structure-based vaccine design. Additionally, current animal models demonstrate notable constraints in virus replication, host susceptibility, immune responses, clinical symptoms, and ERD phenomena. This review synthesizes current obstacles and innovative strategies, highlighting that the selection of multi-antigen strategies, appropriate adjuvants, and the development of more precise preclinical animal models are critical elements that will determine the efficacy and safety of future RSV vaccines.

Dengue virus (DENV) infection remains a critical global health threat, with DENV-2 being the most virulent serotype capable of causing lethal dengue hemorrhagic fever (DHF), a severe complication characterized by plasma leakage and hemorrhagic manifestations. While the search for viral receptors and immunocompetent animal models has persisted since the first recorded outbreak in 1779, significant gaps remain. Here, we establish the first immunocompetent murine model of DHF with intact innate/adaptive immunity by generating hTim4-transgenic C57BL/6J mice. This model recapitulates fatal DHF complications seen in humans, including systemic hemorrhage, dengue encephalitis and intestinal ischemia/gangrene. Integrated single-cell RNA sequencing and spatial transcriptomics analysis of hemorrhagic gut lesions demonstrated that DENV-2 infection induces Syk protein overexpression, leading to enhanced Th2 cytokine secretion and impaired hemostatic regulation. This cascade enhances vascular permeability, promotes plasma leakage, and drives multiorgan hemorrhage, a mechanism corroborated by parallel analyses of human DHF tissues. Critically, Th2-biased cytokine storm mirrors clinical findings in severe human dengue cases. Our work not only identifies hTim4 as a functional DENV-2 receptor but also provides a mechanistically grounded platform for DHF pathogenesis studies, bridging critical gaps between preclinical models and human immunopathology.

Enteroviral 2A proteinase (2Apro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2Apro ; however, the mechanism underlying the regulation of 2Apro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2Apro or Thr125 of coxsackievirus B3 2Apro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2Apro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2Apro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2Apro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2Apro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2Apro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

Intensive efforts have been made to develop models of hRSV infection or disease using various animals. However, the limitations such as semi-permissiveness and short duration of infection have impeded their applications in both the pathogenesis of hRSV and therapeutics development. Here, we present a mouse model based on a Rag2 gene knockout using CRISPR/Cas9 technology. Rag2−/− mice sustained high viral loads upon intranasal inoculation with hRSV. The average peak titer rapidly reached 1 × 109.8 copies/g and 1c106 TCID50 in nasal cavity, as well as 1 × 108 copies/g and 1 × 105 TCID50 in the lungs up to 5 weeks. Mild interstitial pneumonia, severe bronchopneumonia, elevated cytokines and NK cells were seen in Rag2−/− mice. A humanized monoclonal antibody showed strong antiviral activity in this animal model, implying that Rag2−/− mice that support long-term stable infection are a useful tool for studying the transmission and pathogenesis of human RSV, as well as evaluating therapeutics.

COVID-19, caused by SARS-CoV-2, is the most consequential pandemic of this century. Since the outbreak in late 2019, animal models have been playing crucial roles in aiding the rapid development of vaccines/drugs for prevention and therapy, as well as understanding the pathogenesis of SARS-CoV-2 infection and immune responses of hosts. However, the current animal models have some deficits and there is an urgent need for novel models to evaluate the virulence of variants of concerns (VOC), antibody-dependent enhancement (ADE), and various comorbidities of COVID-19. This review summarizes the clinical features of COVID-19 in different populations, and the characteristics of the major animal models of SARS-CoV-2, including those naturally susceptible animals, such as non-human primates, Syrian hamster, ferret, minks, poultry, livestock, and mouse models sensitized by genetically modified, AAV/adenoviral transduced, mouse-adapted strain of SARS-CoV-2, and by engraftment of human tissues or cells. Since understanding the host receptors and proteases is essential for designing advanced genetically modified animal models, successful studies on receptors and proteases are also reviewed. Several improved alternatives for future mouse models are proposed, including the reselection of alternative receptor genes or multiple gene combinations, the use of transgenic or knock-in method, and different strains for establishing the next generation of genetically modified mice.

人肠道病毒71型(enterovirus 71,EV71)能够引起儿童的手足口病,并且具有嗜神经毒性.该病症在全球范围内的传播逐渐引起科研和医务人员的重视.动物模型是研究传染病的重要手段,现阶段对EV71易感动物模型研究有了多方面成果.本文对EV71动物模型近期的研究进行整理总结,并对后续研究方向进行展望.

目的测定R26-hSCARB2小鼠的血液生理生化指标,分析性别和周龄对上述指标的影响。方法分别取20只4周龄和8周龄的R26-hSCARB2小鼠,雌雄各半。取静脉血后,测定22项血液生理指标和12项血液生化指标,使用统计学方法分析各指标在性别和周龄间的差异。结果检测的22项生理指标和12项生化指标中,4周龄的R26-hSCARB2小鼠雌雄之间在淋巴细胞绝对值（LYM）、中性粒细胞百分率（NEUT%）及尿素（BUN）指标上具有显著差异（P ＜0.05）,白细胞计数（WBC）、总胆固醇（CHO）及甘油三酯（TG）指标上具有极显著差异（P ＜0.01）。8周龄的R26-hSCARB2小鼠雌雄之间在WBC、LYM、单核细胞百分率（MON%）、嗜碱性粒细胞百分率（BAS%）、总蛋白（TP）、磷（P）及CHO指标上具有极显著差异（P ＜0.01）;在血小板计数（PLT）、血小板分布宽度（PDW）、中性粒细胞绝对值（NEUT）、嗜酸性粒细胞百分率（EOS%）、白蛋白（ALB）、碱性磷酸酶（ALP）、葡萄糖（GLU）及TG指标上,具有显著差异（P ＜0.05）。不同周龄的R26-hSCARB2小鼠在WBC、红细胞分布宽度（RDW）、PLT、LYM、单核细胞绝对值（MON）、淋巴细胞百分率（LYM%）、MON%、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、TP、ALB、ALP、BUN、肌酐（CREA）、葡萄糖（GLU）、钙（Ca）、P、CHO及TG指标上具有极显著差异（P ＜0.01）,在血小板压积（PCT）、平均血小板体积（MPV）、NEUT及NEUT%指标上具有显著差异（P ＜0.05）。结论性别和周龄都会影响R26-hSCARB2小鼠的血液生理生化指标,但周龄对该小鼠的血液生理生化指标的影响更显著。本实验为R26-hSCARB2小鼠的研究应用提供了基础的数据。

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5–6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

目的 对不同性别的4周龄、8周龄C57BL/6-Kdrem1 (hKDR)/NIFDC(以下简称hVEGFR2-KI)小鼠的血液参数进行测定,分析性别以及年龄对于该品系小鼠血液参数的影响.方法分别取20只4周龄、8周龄小鼠雌雄各半,采静脉血.用日本光电Mek7222血细胞分析仪测定hVEGFR2-KI小鼠的生理指标,奥林巴斯AU400全自动生化分析仪测定生化指标.结果在所测得的生理生化指标中,4周龄的hVEGFR2-KI小鼠雌雄之间于血红蛋白浓度(HGB)、总蛋白(TP)、总胆固醇(CHO)3项指标有显著差异(P＜0.05),在红细胞计数(RBC)、红细胞压积(HCT)、平均红细胞血红蛋白(MCH)、平均红细胞血红蛋白浓度(MCHC)4项指标上有极显著差异(P＜0.01).8周龄的hVEGFR2-KI小鼠雌雄之间共有11项生理生化指标有极显著差异(P＜0.01),分别是RBC、HCT、HGB、血小板计数(PLT)、血小板压积(PCT)、中性粒细胞绝对值(NEUT)、单核细胞百分率(MON％)7项血液生理指标和CHO、碱性磷酸酶(ALP)、葡萄糖(GLU)、甘油三酯(TG)4项血液生化指标;有5项具有显著差异(P＜0.05)的指标,包括淋巴细胞百分率(LYM％)、中性粒细胞百分率(NEUT％)、TP、尿素(BUN)和磷(P).不同周龄hVEGFR2-KI小鼠之间的差异分布更广,其中具有极显著差异(P＜0.01)的有RBC、HCT、MCH、HGB、白细胞计数(WBC)、平均红细胞体积(MCV)、红细胞分布宽度(RDW)、平均血小板体积(MPV)、血小板分布宽度(PDW)、淋巴细胞绝对值(LYM)、单核细胞百分率(MON％)、TP、CHO、白蛋白(ALB)、ALP、肌酐(CREA)、GLU、P共18项指标;具有显著差异的为PLT、LYM％、NEUT％、丙氨酸氨基转移酶(ALT)、TG共5项指标.结论本次研究测定了不同性别的4周龄、8周龄的hVEGFR2-KI小鼠的生理生化指标,为hVEGFR2-KI小鼠的应用以及正常血液参数标准提供一定参考.

目的 测定自主建立的转基因小鼠hTIM4-EGFP的血液生理生化指标.方法 分别测定12周龄各8只雌性和10只雄性的同窝阴性小鼠与转基因hTIM4-EGFP小鼠的22项血液生理指标和12项血液生化指标,利用统计学方法分析组间差异.结果 转基因组与野生组在10项生理指标(WBC、RBC、HGB、HCT、MCH、RDW、PLT、NEUT、LYM％、NEUT％)和4项生化指标(ALT、AST、TG、P)间存在差异;转基因组在MCV、MCH、MCHC、RDW、PLT、NEUT、NEUT％、ALP、CHO指标中存在雌雄差异.对照组在RBC、HCT、MCV、LYM、NEUT、LYM％、NEUT％、AST、ALT、P指标中存在雌雄差异.结论 人源化Tim4基因敲入的C57BL/6小鼠的部分血液生理生化指标与野生型存在差异,该测定为hTIM4-EGFP小鼠的研究应用提供了基础数据.

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.

建立高致病性冠状病毒动物模型对疫苗、抗体、药物、病毒致病机制研究意义重大.非人灵长类、雪貂、叙利亚仓鼠等动物均可用于建立冠状病毒的感染和疾病动物模型.不同动物模型能从不同层面模拟重现临床感染症状.然而,从适用性、经济性、易于获取等角度综合考虑,建立冠状病毒易感的小鼠模型,并快速提供数量充足的动物,对于冠状病毒疫情防控更有现实意义.本文简述了MERS-CoV、SARS-CoV以及新型冠状病毒(2019-nCoV)3种高致病性冠状病毒小鼠模型的研究进展.基于近日发表在Nature的研究成果,即SARS-CoV和2019-nCoV均利用hACE2受体感染宿主细胞推测,我们于2018年构建的hACE2-KI/NIFDC人源化小鼠模型有望用于2019-nCoV的相关研究.

中东呼吸系统综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)是2012年发现的新型高致病性冠状病毒,其病死率高达34.4％.人二肽基肽酶-4(human dipeptidyl peptidase-4,hDPP4)分子是MERS-CoV进入宿主细胞的受体.为了获得稳定的MERS-CoV易感小鼠模型,本文分别利用Tol2转基因技术和CRISPR/Cas9技术将hDPP4受体导入C57BL/6小鼠,筛选获得转基因小鼠hDPP4-Tg、定点敲入小鼠hDPP4-KI;通过Q-PCR、Western Blot及活体成像技术,分析了hDPP4基因的表达特征.结果 显示,在hDPP4-Tg小鼠中,其表达水平较低,在脑中高表达;而在hDPP4-KI中整体表达水平高,表达优势脏器为肺.假病毒感染实验表明,hDPP4-Tg几乎不易感,而hDPP4-KI小鼠对假病毒高度易感.进而,对假病毒感染hDPP4-KI小鼠的感染途径、感染剂量、是否剃除被毛等条件进行优化,获得了稳定易感的MERS-CoV假病毒感染模型,并利用该模型初步评价了单克隆抗体hMS-1的体内活性.本研究显示,即使相同的受体基因,不同策略构建的遗传修饰小鼠模型的效果差异较大;受体分子表达水平与模型的易感性高度相关;本研究获得的高效而稳定的MERS-CoV假病毒感染模型可望在抗体评价、疫苗研制、药物筛选等领域提供模型工具,提示将受体分子定点插入小鼠基因组、高表达受体基因是成功构建病毒易感模型首选策略.

组织切片多光谱定量分析技术是近年建立起来的新的组织病理学分析方法,由三大技术环节组成,即多重免疫荧光标记、多光谱成像设备与光谱拆分算法、定量组织切片图像分析软件.该分析技术可以获得传统病理学、免疫组化、多标记流式细胞术无法获得的信息.我们简述了该方法的原理,及其在肿瘤免疫和其他疾病诊断、预后效果研判、疾病发生机制等领域的应用进展.