Control mice were injected with DMEM by the same route, with 5C10 mice in each group
Control mice were injected with DMEM by the same route, with 5C10 mice in each group. Quantification of viral loads The mice Fudosteine were sacrificed 14 days post-infection, after which tissues (brain, lungs and skeletal muscles of hind limbs) were dissected, and immediately immersed in RNAlater stabilization reagent (Takara, Shiga, Japan) for virus load quantification. epidemic disease in the Asia-Pacific region. It often exhibits seasonal increases in morbidity and mortality, especially in children under the age of five . Enterovirus A71 (EV-A71), inclusive of coxsackievirus A10 (CVA10) and coxsackievirus A16 (CVA16), are the major etiological agents which belong to members of Enterovirus A species of the Enterovirus genus under the family [2C4]. EV-A71 can cause severe neurological complications such as central nervous system symptoms due to its intrinsic neurotropism , whereby severe cases may result in convulsions, shock and even death . In China, it was listed as a class C infectious disease by the ministry of health for management in 2008 , and the incidence of HFMD was ranked first [8,9]. Because there is still no specific therapeutic drug, vaccination is the only way to prevent and control this disease . The successful development of a vaccine has played a great role in the control of HFMD caused by EV-A71, reducing the number of deaths from 142 in 2016 to 14 in 2019 . At present, three inactivated EV-A71 vaccines are available in China, and the annual authorization quantity exceeds 10 million [12C15]. Some companies and academic institutions are still active in the research and development of novel types of vaccines [16C18] or polyvalent vaccines . The protection efficacy of vaccines is of great concern to manufacturers, Fudosteine regulators, and consumers. Animal models that are susceptible to the virus are essential for assessing the protective effect of a vaccine [20,21]. Both non-human primate and mouse Fudosteine models of EV-A71 infection have been developed [2,22C24]. Due to economic and ethical considerations, mouse models are more widely used, but it was reported that wild-type inbred mice are optimally susceptible to EV-A71 only within 7 days of age, and mice older than 2 weeks were no longer susceptible [25,26]. Consequently, suckling mice under 5 days of age are usually selected for testing . However, neonatal mice generally have a poor antibody response, and only animals aged 4C6 weeks can be selected for vaccination. Moreover, the production of neutralizing antibodies requires 2C3 weeks after primary immunization. This means that mice will be as old as 6C9 weeks of age when enough neutralizing antibodies were elicited, at which point they are no longer susceptible for enterovirus infection, and therefore no longer suited to evaluate the efficacy of HFMD vaccines . To overcome this dilemma, an evaluation assay depending on maternally transferred antibodies was developed [28,29]. After immunizing the dams prior to gestation, the antibodies produced by CDKN2AIP the mother are transmitted to the suckling mice through the placenta, and the suckling mice are then challenged with EV-A71. The protective efficacy of the vaccine was calculated by comparing the clinical symptoms with those of suckling mice from unimmunized dams . This assay process is tedious, and each suckling mouse may obtain a different amount of transferred maternal antibodies, resulting in irreproducible results. Human scavenger receptor class B member 2 (hSCARB2) is widely expressed in many human tissues and cell types, such as lungs, Fudosteine muscles, and neurons [31,32]. Several studies have shown that hSCARB2 is the key receptor for EV-A71 infection in humans, both and [33C35]. Although the scavenger receptor class B member 2 of mice share significant sequence similarity with its human ortholog, it cannot mediate EV-A71 infection . Early studies relied on transgenic mice expressing the hSCARB2 receptor [37C39]. A knock-in mouse model with a pure C57BL/6 background was constructed using embryonic stem-cell targeting technology in our own laboratory, and named the challenge experiments. Virus and challenge Human rhabdomyosarcoma (RD) cells were obtained from the Center for Cell Resource Conservation Research, NIFDC. The cells were Fudosteine maintained in Dulbeccos modified Eagles medium (DMEM, Gibco) supplemented with 10% faetal bovine serum (FBS, Gibco) and 1% penicillinCstreptomycin solution (PS, Gibco). Since our previous study had shown that EV-A71 Isehara strain (CDV-Isehara/Japan/99, GenBank NO. “type”:”entrez-nucleotide”,”attrs”:”text”:”AB811772″,”term_id”:”548960215″,”term_text”:”AB811772″AB811772, kindly provided by Prof. Satoshi Koike, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan)  could infect hSCARB2 mice , it was amplified in.