JP6772155B2 - Enterovirus 71 animal model - Google Patents

Description

Cross-reference to related applications
[0001] This application relates to US Provisional Patent Application No. 62 / 114,880 filed February 11, 2015 and US Provisional Patent Application No. 62 / 108,828 filed January 28, 2015. And they claim priority. Each application is incorporated herein by reference in its entirety.

Submission of sequence listing
[0002] This application has been submitted with an electronic sequence listing. The sequence listing is 2577243PCTSequenceListing. Titled txt, created on December 29, 2015, and is 32 kb in size. The entire information in the sequence listing in electronic form is incorporated herein by reference.

[0003] The present invention relates to enterovirus 71 (EV71), the development of animal models, and the screening of candidate anti-EV71 compounds.
[0004] Publications and other materials used herein to clarify the background of the invention, and in particular examples that provide further details regarding practice, are incorporated herein by reference and, for convenience, the following: In the text of, mentioned by author name and date, and listed in alphabetical order by author in the accompanying bibliography.

[0005] Enterovirus 71 (EV71) is a small non-enveloped virus with a diameter of approximately 30 nm. Viral capsids exhibit icosahedron symmetry and are composed of 60 identical units (protomers), each consisting of four viral structural proteins VP1 to VP4. The capsid surrounds the core of the 7,450 nucleotide (nt) long single-strand plus sense RNA genome. The genome encodes a polyprotein of 2193 amino acids (aa) and has a shorter 3'UTR of 85 nt, including a long 5'untranslated region (UTR) of 745 nt and a variable length poly A tract at the 3'end. Adjacent. The polyprotein is divided into three regions, namely P1, P2 and P3. P1 encodes four viral structural proteins 1A-1D (VP4, VP2, VP3 and VP1); P2 and P3 encode seven non-structural proteins 2A-2C and 3A-3D [1-3]. By phylogenetic analysis of the major capsid protein (VP1) gene, EV71 has six genotypes (referred to as A to F) [66], and genotypes B and C have further subgenotypes B1 to B5. And C1 to C5 [3].

[0006] EV71 causes a number of clinical disorders, including hand-foot-and-mouth disease (HFMD), aseptic meningitis, encephalitis and polio-like paralysis, primarily in infants and young children [4, 5]. The virus was first isolated from a child with acute encephalitis in California, USA in 1969, and was subsequently characterized as a novel serotype of the genus Enterovirus in 1974 [6]. Outbreaks of HFMD with or without neurological complications and death have been reported in various parts of the world [7-20]. Since 1997, EV71 infection has been a major public health burden and is a constant epidemiological concern in the Asia-Pacific region. A highly neurotoxic EV71 outbreak of HFMD occurred in Malaysia, killing 48 people in 1997 [21, 22], followed by a larger outbreak in Taiwan in 1998. In particular, HFMD exceeded 129,000 cases, 405 cases were severely infected, and there were 78 deaths due to acute brainstem encephalomyelitis with neurological heart failure and pulmonary edema [23-26]. ]. In the People's Republic of China, 488,955 HFMD cases were recorded in 2008, including 126 deaths, and increased to 1,155,525 cases in 2009, including 353 deaths [28]. ]. In 2010, China experienced the largest HFMD pandemic ever, with more than 1.7 million cases, 27,000 patients with severe neurological complications, and 905 deaths [29]. ].

[0007] Like other human enteroviruses, EV71 is unable to infect non-human animals, but rhesus monkeys and cynomolgus monkeys are experimentally infectious [30-32]. ]. This is primarily due to the inability of EV71 to efficiently bind to its receptor due to decapsulation and entry into mouse cells, which have recently been scavengered. It has been identified as a receptor class B member 2 (SCARB2) protein [47]. The human and murine SCARB2 proteins show only 84% amino acid sequence identity and therefore show significant structural differences [49,67] and thus underlie the natural resistance of non-human cells to EV71 infection, the virus. -Causes receptor incompatibility. When the virus successfully binds to the SCARB2 receptor on the cell surface and decapsulates into the cytoplasm, the viral RNA is translated, resulting in the expression of various viral non-structural proteins. Viral RNA is subsequently replicated, packaged within the capsid, and freely released into the environment to reinfect healthy cells.

[0008] The inability of EV71 to infect small rodents by nature has led to the lack of a suitable small animal model, which has hampered understanding of its pathogenesis and the development of specific therapies for the virus. Attempts to establish a mouse model of EV71 infection and disease have been made mostly through viral adaptation by continuous passage in young suckling mice [33-39]. Although some models were able to reproduce the symptoms of clinical disease, none have been reported to cause disease in immunocompetent mice 2 weeks of age or older. Moreover, the clinical features of the disease and pathology of EV71 infection in humans and experimental monkeys could not be replicated in mice, with the exception of the susceptible interferon receptor deficient AG129 mice [35]. More recently, transgenic mice expressing human PSGL-1 [68] and human SCARB2 [69,70] proteins have become available, but these have only minimal improvements in susceptibility to EV71 infection. Only show.

[0009] Due to its error-prone replication and high mutation rate [40-42], RNA viruses are replicated as a swarm of related mutant sequences known as pseudospecies [43,44]. It consists of a master species that best fits in a particular environment, and a mutation spectrum consisting of a collection of closely related mutant sequences with a particular probability distribution [44, 45]. These confer genomic plasticity on RNA viruses, which is reflected in their ability to adapt quickly to changing environments.

[0010] The mechanism by which EV71 infection causes lethal neurological disease is not fully understood, and therefore several research groups have found rhesus monkeys and cynomolgus monkeys [114-120], experimental mammals [112, 121- 129], and experimental animals, including other mammals [130-133], have been attempted to reproduce the pathology of human infections. Unfortunately, none of these models show the full spectrum of neurological features observed in human cases and may contribute, in particular, to acute brainstem encephalitis with fulminant neurogenic pulmonary edema (NPE). No features are shown [21, 22, 134-137]. In fact, even in experimental systems that more accurately replicate the signs and symptoms of EV71 infection in humans, the underlying mechanisms are substantially different from those that cause disease in human patients. To date, no single animal model has definitively replicated EV71-induced NPE. An important difference is that EV71 is limited to CNS tissue in human patients [92-94, 111], whereas in animal models the virus is also skeletal muscle [33-36, 38, 98] and liver [119]. ] Can also be detected in non-nervous tissues. Efforts to generate transgenic mice expressing the human EV71 receptor protein PSGL-1 (P-selectin glycoprotein ligand-1) and SCARB2 (scavenger receptor class B, member 2) [68-70, 47, 138] Although done, none of these models show NPE and therefore their usefulness for identifying new interventions for human patients is limited.

[0011] There is no suitable animal model that can be used to study infection and disease progression in EV71-infected animals, or to screen for antiviral compounds or antiviral vaccines. It is desirable to develop animal models for these purposes.

[0012] The present invention relates to enterovirus 71 (EV71), the development of animal models, and the screening of candidate anti-EV71 compounds. More specifically, the present invention comprises enterovirus 71 (EV71) strains that are suitable for rodent cell line infection, or cloning-derived viruses that contain mutations in VP1 that are immunoeligible rodents Regarding the discovery that it is possible to cause disease in easily infectious rodents.

[0013] Furthermore, the present invention relates to an EV71-induced neurological disorder by infecting BALB / c mice with a modified strain (eg, EV71: TLLmv) adapted to infect NIH / 3T3 mouse fibroblasts. Regarding the development of clinical authenticity models. Using this approach, modified EV71 is used to induce acute encephalomyelitis associated with neurogenic pulmonary edema in mice compared to pseudoinfected lung, which is characterized by lung swelling and increased organ weight. Despite lung or heart tissue inflammation disease is not present, localized bleeding and proteinaceous liquid in the alveoli, high serum levels of catecholamines, and brain stem, thorough tissue damage particularly in the medulla oblongata observed. These data demonstrate that the model accurately reproduces the signs and symptoms of human EV71-induced neurogenic pulmonary edema.

[0014] Thus, in one aspect, the invention relates to an animal model comprising a rodent infected with enterovirus 71 capable of infecting rodents, sometimes referred to herein as modified enterovirus 71. .. In one embodiment, such enterovirus 71 is a rodent cell line compatible enterovirus 71. In another embodiment, such enterovirus 71 is a clone-derived virus (CDV) containing a mutation in VP1. In some embodiments, mutations in VP1 allow CDV to infect rodent cells with the rodent SCARB2 protein. In one embodiment, the rodent is an immunoeligible rodent. In another embodiment, the rodent is a immunocompromised rodent. Suitable animals for use as a model are preferably mammals, most preferably suitable laboratory animals such as rabbits, rats, mice and the like. In one embodiment, the animal is a mouse. In some embodiments, the mouse is a BALB / c mouse. In another embodiment, the rodent cell line is a mouse cell line. In a further embodiment, the mouse cell line is a mouse NIH / 3T3 cell line. In another embodiment, the mouse cell line is a mouse Neuro-2a cell line. In one embodiment, the rodent cell line compatible enterovirus 71 is EV71: TLLm. In another embodiment, the rodent cell line compatible enterovirus 71 is EV71: TLLmv. In one embodiment, the clone-derived virus containing the mutation in VP1 is CDV: BS _VP1 [K98E / E145A / L169F]. Animal models are useful for studying systemic transmission of viruses and human disease spectra in animal models. Animal models are also useful for screening antiviral drugs and vaccines.

[0015] In another aspect, the invention provides a method for preparing an animal model that includes the entire spectrum of EV71-induced neurological infections, diseases and pathologies observed in humans. In some embodiments, the method comprises infecting the rodents described herein with the modified enterovirus 71 described herein and rearing the infected rodents for up to about 4 weeks. In some embodiments, the age of the rodent to be infected is between about 1 week and about 4 weeks. In another embodiment, infected rodents are bred for about 1 to about 4 weeks. In some embodiments, the rodent is a mouse as described herein. In another embodiment, rodents are infected by inoculating rodents with modified enterovirus 71. In one embodiment, the inoculation is intraperitoneal (IP). In another embodiment, the inoculation is intramuscular (IM). In some embodiments, the virus dose inoculated into rodents is the median cell culture infective dose of between about ¹⁰³ to about 10 ⁷ (CCID _50).

[0016] In a further aspect, the invention provides a method of screening for antiviral agents. According to this aspect, the method comprises the following steps: providing a test group of animals and a control group of animals, where each group of animals is an animal of the animal model described herein; to the test group. Administer antiviral drug candidates; monitor disease progression in test and control groups; compare disease progression in test group to disease progression in control group; and reduce disease progression in test group compared to control group The process of selecting antiviral drug candidates to be made. In one embodiment, the antiviral agent is first screened in a test rodent cell line infected with a rodent cell line compatible enterovirus 71 prior to screening in the animal. In another embodiment, the antiviral agent is first screened in a test rodent cell line infected with a clonal virus (CDV) containing a mutation in VP1 prior to screening in animals.

[0017] In a further aspect, the invention provides a method of screening for an effective antiviral vaccine. According to this aspect, the method comprises the following steps: providing a test group of animals and a control group of animals, where each group of animals is an animal of the animal model described herein; to the test group. Antiviral vaccine candidates were administered; disease progression was monitored in the study and control groups; disease progression in the study group was compared to disease progression in the control group; and disease progression was reduced in the study group compared to the control group. The process of selecting anti-virus vaccine candidates to be allowed. In one embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a rodent cell line compatible enterovirus 71 prior to screening in animals. In another embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a clone-derived virus (CDV) containing a mutation in VP1 prior to screening in animals.

[0018] FIGS. 1A-1O show the cytopathic effect (CPE) observed after viral infection of various primate cell lines. 1 MOI EV71: BS (FIGS. 1A, 1D, 1G, 1J and 1M), EV71: TLLm (FIGS. 1B, 1E, 1H, 1K and 1N), or EV71: TLLmv (FIGS. 1C, 1F, 1I, 1L and 1O). Primal cells infected with any of the viruses: RD cells (FIGS. A1-1C), HeLa cells (FIGS. 1D-1F), HEp-2 cells (FIGS. 1G-1l), Vero cells (FIGS. 1J-1L), And COS-7 cells (FIGS. 1M-1O) were observed at 48 hpi for cell degeneration effects or cell death of cell monolayers. The image is representative of the results of three independent experiments. [0019] FIGS. 2A-2O show viral antigen detection in cell lines infected with EV71: BS, EV71: TLLm and EV71: TLLmv. Mammalian cell lines planted overnight: HeLa (FIGS. 2A-2C), HEp-2 (FIGS. 2D-2F), CHO-K1 (FIGS. 2G-2I), NRK (FIGS. 2J-2L), and TCKM (FIGS. 2M). ~ 2O) was infected with each virus of 1MOI. Cells were harvested at 48 hpi, coated on Teflon slides and fixed in cold acetone. Cells were explored with pan-enterovirus antibody and stained with FITC-conjugated anti-mouse IgG. The image is representative of two independent experiments. [0020] Figures 3A-3D show the proliferative kinetics of EV71: BS, EV71: TLLm and EV71: TLLmv determined in NIH / 3T3 and Vero cells. Supernatants from various mammalian cells infected with each virus of 1 MOI were collected at various time points and subjected to titration and counted using the Red and Muench methods. FIG. 3A: EV71: BS virus titer determined in Vero cells. FIG. 3B: EV71: TLLmv virus titer determined in NIH / 3T3 cells. EV71: TLLm virus titers determined in 3C and 3D: NIH / 3T3 cells. No growth curve from a cell line showing no proliferative infection is shown. [0021] FIGS. 4A-4O show the cytopathic effect (CPE) observed after viral infection of various rodent cell lines. 1 MOI EV71: BS (Fig. 4A, 4D, 4G, 4J and 4M), EV71: TLLm (4B, 4E, 4H, 4K and 4N), or EV71: TLLmv (Fig. 4C, 4F, 4I, 4L and 4O) virus Rodent cells infected with any of: NIH / 3T3 cells (FIGS. A4-4C), Neuro-2A cells (FIGS. 4D-4F), TCKM cells (FIGS. 4G-4I), CHO-K1 cells (FIGS. 4J). ~ 4L), and NRK cells (FIGS. 4M-4O) were observed at 48 hpi for cell degeneration effects or cell death of cell monolayers. The image is representative of the results derived from three independent experiments. [0022] FIGS. 5A-5D show the viral compatibility assessment of EV71: BS, EV71: TLLm and EV71: TLLmv in NIH / 3T3, as determined by the titer ratio. Viral compatibility was calculated as log [(titer in NIH / 3T3 cells) / (titer in Vero cells)] using virus titers determined separately in NIH / 3T3 and Vero cells. From the viral potency values shown in FIGS. 3A-3D, the virus compatibility of (FIG. 5A) EV71: BS, (FIG. 5B) EV71: TLLmv and (FIGS. 5C and 5D) EV71: TLLm was calculated. Virus compatibility assays obtained from cell lines showing no proliferative infection are not shown. [0023] Figures 6A and 6B show that transfection of NIH / 3T3 with EV71: BS viral RNA induces proliferative infections. NIH / 3T3 and Vero cells planted overnight were inoculated with virus supernatants collected from NIH / 3T3 cells pre-transfected with viral RNA extracted from EV71: BS, EV71: TLLm, and EV71: TLLmv. FIG. 6A: Cells were imaged at 24 hp i using an inverted light microscope to observe the induced CPE. FIG. 6B: Cells were harvested at 7 dpi, coated on Teflon slides, explored with pan-enterovirus antibody, and stained with anti-mouse FITC conjugated antibody. [0024] FIGS. 7A and 7B show virus compatibility assessments of EV71: BS, EV71: TLLm, and EV71: TLLmv in NIH / 3T3 and Vero cells at 30 ° C. Infected with EV71: BS (panels a, d, g), EV71: TLLm (panels b, e, h), or EV71: TLLmv (panels c, f, i), planted overnight (FIG. 7A) NIH / 3T3 and (FIG. 7B) Vero cells were incubated at 30 ° C. and observed under a phase difference light microscope at 24 hpi (panels a-c), 48 hpi (panels d-f), and 72 hpi (panels g-i). .. The images taken represent two independent experiments. [0025] FIGS. 8A and 8B show virus compatibility assessments of EV71: BS, EV71: TLLm, and EV71: TLLmv in NIH / 3T3 and Vero cells at 37 ° C. Infected with EV71: BS (panels a, d, g), EV71: TLLm (panels b, e, h), or EV71: TLLmv (panels c, f, i), planted overnight (FIG. 8A) NIH / 3T3 and (FIG. 8B) Vero cells were incubated at 37 ° C. and observed under phase difference light microscopy at 24 hpi (panels a-c), 48 hpi (panels d-f), and 72 hpi (panels g-i). .. The images taken represent two independent experiments. [0026] FIGS. 9A and 9B show virus compatibility assessments of EV71: BS, EV71: TLLm, and EV71: TLLmv in NIH / 3T3 and Vero cells at 39 ° C. Infected with EV71: BS (panels a, d, g), EV71: TLLm (panels b, e, h), or EV71: TLLmv (panels c, f, i), planted overnight (FIG. 9A) NIH / 3T3 and (FIG. 9B) Vero cells were incubated at 39 ° C. and observed under phase difference light microscopy at 24 hpi (panels a-c), 48 hpi (panels d-f), and 72 hpi (panels g-i). .. The images taken represent two independent experiments. [0027] FIGS. 10A-10L show transfection of murine cell lines NIH / 3T3, Neuro-2A, and TCKM with EV71: BS viral RNA for evidence of viral replication. NIH / 3T3, Neuro-2A, and TCKM cells planted overnight are infected with 1000 CCID ₅₀ EV71: BS virus (FIGS. 10A, 10C, and 10E) or transfected with equal amounts of viral RNA (FIG. 10A, 10C, and 10E). 10B, 10D, and 10F), and collected at 48 hpi for detection of viral antigens. The virus in the supernatant was collected at 7 dpi and passaged on fresh Vero (FIGS. 10G, 10I, and 10K) and NIH / 3T3 cells (FIG. 10H, 10J, and 10L). Cells were harvested at 48 hpi and stained for viral antigens. [0028] FIGS. 11A-11D show the positions of matching mutations in the genomes of EV71: TLLm and EV71: TLLmv in VP1 and VP2. VP1 (FIGS. 11B and 11D) of EV71: TLLm (FIGS. 11A and 11C) and EV71: TLLmv (FIGS. 11B and 11D) using the 3D structure (PDB ID 4AED) of DeepView / SwissPDBviewer v3.7 and EV71 capsid P1 region. And the matching mutations observed in the VP2 (FIGS. 11C and 11D) regions were modeled. Mutations were observed to be most localized in the surface exposure loop of the proteins shown. [0029] FIG. 12 shows the force in NIH / 3T3 cells compared to the titer in Vero cells of the virus supernatant collected from cells either transfected with EV71: BS virus RNA or infected with viable virus. Shows the price ratio. Supernatants from either NIH / 3T3, Neuro-2A, Vero, and TCKM, either transfected with viral RNA or infected with live virus, were collected and subjected to virus counting by the Red and Muench methods. The ratio of the log (titer) determined in NIH / 3T3 cells is shown as compared to the titer determined in Vero cells. 3T3-TRANS: RNA-transfected NIH / 3T3 cells; 3T3-INF: virus-infected NIH / 3T3 cells. The asterisk indicates a student's t-test with a p-value <0.05. [0030] Figures 13A and 13B show survival analysis of infected animals. Infected animals were observed daily and weighed. FIG. 13A: Kaplan-Meier plot of infected animals showing the number of deaths in days after various infections. FIG. 13B: Weight changes were plotted to determine the general health of the animal. [0031] FIGS. 14A-14D show the symptoms and pathology of infected animals. The majority of infected animals showed symptoms of the disease. FIG. 14A: Paralysis of hind limbs (arrow). FIG. 14B: Macroscopic anatomical findings of swollen lungs after necrosis (arrows). Tissue sections were also stained with hematoxylin and eosin stains (10x Figure 14C and 20x Figure 14D). Black arrows point to mucous substances that infiltrate the alveolar space. [0032] Figures 15A-15E show that transfection of viral genomic RNA into both primate and rodent cells yields a viable virus. Genomic RNA extracted from either EV71: BS, EV71: TLLm, or EV71: TLLmv was individually transfected into Vero, NIH / 3T3, and Neuro-2a cells (P0). Transfection supernatants were harvested and inoculated onto Vero or NIH / 3T3 cells (P1) and evaluated for viability of viral offspring. Infection of P0 cells was evaluated by observation of cytopathic effect (CPE) (FIG. 15B) and immunofluorescence detection of viral antigens (FIG. 15C). Similarly, infection of P1 cells from EV71: BS RNA transfected cells was assessed by CPE induction (FIG. 15D) and immunofluorescence detection of the expressed viral antigen (FIG. 15E). [0033] Figures 16A-16F show that the capsid coding region of mouse cell-compatible EV71: TLLm drives proliferative infection of mouse cells in EV71: BS. FIG. 16A: An infectious cDNA clone of the entire genome of EV71: BS was generated, and the P1 region was replaced with a sequence derived from EV71: TLLm capsid to generate a chimeric virus, EV71: BS [MP1]. FIG. 16B: Cells are infected with clonal virus (CDV) from either EV71: BS or EV71: BS [MP1], and soluble cytopathic effect (CPE) (FIG. 16C) and viral antigen expression (FIG. 16C) Infection was assessed by induction in FIG. 16D). Supernatants were re-inoculated onto fresh cells and from passages (P1 and P2) obtained from infected Vero (FIG. 16E), and passages of NIH / 3T3 (3T3) and Neuro-2A (N2a) cells P1. (Fig. 16F), the virus titer was measured. Error bars indicate SD. ^* P <0.05. [0034] FIGS. 17A-17G show that VP1-L169F amino acid substitutions in capsids are sufficient to allow EV71: BS entry into murine cells. FIG. 17A: Amino acid substitutions at VP1: K98E, E145A, and L169F; and amino acid substitutions at VP2: S144T and K149I were incorporated into the full-length EV71: BS genome to generate a variety of mutant cDNA clones. Mutations corresponding to amino acid substitutions are listed in parentheses. Infection of various cell lines with clonal virus (CDV) was monitored by assessing the induction of soluble cytopathic effect (CPE) (FIGS. 17B and 17C) and expression of viral antigens (FIGS. 17D and 17E). .. Viral titers from infected Vero cells (FIG. 17F) and NIH / 3T3 and Neuro-2a cells (FIG. 17G) were determined to assess the production of viable virus offspring. Error bars indicate SD. [0035] FIGS. 18A-18E show that EV71: BS virus containing combined VP1 amino acid substitutions in capsids shows amelioration of mouse cell infection. FIG. 18A: Full length EV71: Various mutant cDNA clones were generated by incorporating a combination of amino acid substitutions at VP1 and VP2 into the BS genome. Mutations corresponding to amino acid substitutions are listed in parentheses. Assess the cytopathic effect (FIG. 18B), viral antigen expression (FIG. 18C), and viral yields obtained from infected Vero (FIG. 18D) and NIH / 3T3 (3T3) and Neuro-2a (N2A) cells (FIG. 18E). By monitoring the infection of various cell lines with clone-derived virus (CDV). No other clones that do not produce the virus are shown. Error bars indicate SD. [0036] FIGS. 19A-19C show that the EV71: BS virus containing the combined VP1-K98E, E145A, L169F amino acid substitutions in the capsid can be stably passaged in the mouse neuron cell line Neuro-2a. Clone-derived virus was passaged twice in Neuro-2a and NIH / 3T3 cells. At each passage, infection was monitored by evaluation of viral antigen expression (FIG. 19A) and viral titers measured in Vero cells (FIG. 19B) (FIG. 19B). Error bars indicate SD. ^* P <0.05; ^** p <0.005; ^*** p <0.0005. FIG. 19C: Representative CDV: BS _VP1 [K98E / E145A / L169F] genome sequence was determined and evidence of amino acid substitutions K98E (A2734G), E145A (A2876C), and L169F (C2947T) was evaluated. Mark the mutation site with an asterisk. [0037] FIGS. 20A-20F show that EV71: TLLmv utilizes SCARB2 to infect both primate and murine cells. Preincubation of NIH / 3T3 cells (FIG. 20A) and Vero cells (FIG. 20B) immobilized on Teflon slides with murine SCARB2 (mSCARB2) antiserum is determined by reduced fluorescence signal EV71: TLLmv. Inhibits binding. Fluorescence intensity on the membrane was measured using Imalis imaging software (n = 100). NSP (non-specific rabbit serum). Prior to inoculation on NIH / 3T3 cells, preincubation of EV71: TLLmv from either mSCARB2 (FIG. 20C) or human SCARB2 (hSCARB2) (FIG. 20D) with recombinant soluble protein is assessed by immunofluorescence assay. As such, the severity of viral infection is reduced. EV71: Preincubation of live NIH / 3T3 cells with either hSCARB2 (FIG. 20E) or mSCARB2 (FIG. 20F) prior to infection with TLLmv reduces the viral titer in the culture supernatant. ^* P <0.05; ^** p <0.005; ^*** p <0.0005. [0038] Figures 21A-21D show that incubation of murine SCARB2 rabbit antisera with Neuro-2A cells reduced the severity of infection with the CDV mutant. Infection in cells preincubated with rabbit mSCARB2 antiserum prior to infection with CDV: BS _VP1 [K98E / E145A / L169F] (FIGS. 21A and 21B) or CDV: BS [MP1] (FIGS. 21C and 21D). Severity was monitored by assessing induction of cytopathic effect (CPE) (FIGS. 21A and 21C) and virus yield 7 days after infection (FIGS. 21B and 21D). Error bars indicate SD; ^* p <0.05; ^** p <0.005. Degree of CPE: 1 (0-25% cell death), 2 (25-50%), 3 (50-75%), 4 (75-100%). [0039] Figures 22a-22c show that EV71: TLLmv is the most malignant of the three modified virus strains, as evidenced by the induction of severe disease in 1-week-old BALB / c mice. FIG. 22a: Mice (intraperitoneal) infected with either EV71: BS (n = 6), EV71: TLLm (n = 5) or EV71: TLLmv (n = 7) when evaluated at the end of the observation period. Neutralizing antibody titer in serum collected from IP). 22b and 22c: I.I. P. 10 ⁶ CCID ₅₀ (median cell culture infectious dose) of EV71: BS, EV71: TLLm, or EV71: TLLmv through either the pathway (FIG. 22b) or the intramuscular (IM) pathway (FIG. 22c). Kaplan-Meier survival curve of mice inoculated with. Statistical significance was determined using a t-test (FIG. 22a) with Welch correction for unequal variations, or a Mantel-Cox log rank test (FIGS. 22b and 22c). ^* P <0.05; ^** p <0.005; ^*** p <0.0005. [0040] Figures 23a-23h show that EV71: TLLmv infection in mice is characterized by acute severe disease similar to the human disease spectrum. Figures 23a and 23b: Dose-dependent lethality of EV71: TLLmv infection in 1-week-old mice. FIG. 23a: Various doses of EV71: TLLmv in I. P. Kaplan-Meier survival curve of injected 6-day-old pups. FIG. 23b: Median humanitarian endpoint (HD50) was equivalent to a viral dose of 3.98x10 ³ CCID ₅₀ . FIG. 23c: I. P. Or I. M. Kaplan-Meier survival curve of 1-week-old mice inoculated with EV71: TLLmv through the pathway. Figure ^23d: the 10 6 CCID ₅₀ virus dose mice inoculated with the, EV71: TLLmv age and route dependent lethality induced by infection. Figures 23e and 23f: Clinical signs observed in terminally ill mice, some of which showed paralysis of the hind limbs (gray arrows) and / or forelimbs. Others also showed small hairless lesions on the torso (black arrows). 23g and 23h: I.I. P. Route (Fig. 23g) or I. M. Disease classification of 1-week-old mice inoculated with EV71: TLLmv through the pathway (Fig. 23h). [0041] Figures 24a-24e show that the severity of EV71: TLLmv infection in BALB / c mice depends on the age of the host, the viral dose, and the route of administration. FIGS. 24a and 24b: In a group of 8-10 mice, I. P. Route (Fig. 24a) or I. M. By the path (FIG. 24b), were inoculated with ¹⁰ 6 CCID ₅₀ of virus and for different age groups of animals was determined Kaplan-Meier survival curves. FIGS. 24c and 24d: I. P. Route (Fig. 24c) or I. M. Neutralizing antibody titers in sera collected at the end of the observation period from mice of various ages inoculated through any of the routes (FIG. 24d); 1 week (IP n = 7; IM) . N = 4), 2 weeks (n = 5, n = 7), 3 weeks (n = 4; n = 8), and 4 weeks (n = 4; n = 6). FIG. 24e: Various doses of EV71: TLLmv; CCID50 102 (n = 5), 103 (n = 4), 104 (n = 4), 105 (n = 5), or 106 (n = 7) inoculated. Neutralizing antibody titer in serum collected from (IP) mice. Statistical significance was determined by the Mantel-Cox log rank test (FIGS. 24a and 24b) or the t-test with Welch correction for unequal dispersion (FIGS. 24c, 24d and 24e). ^* P <0.05; ^** p <0.005; ^*** p <0.0005. [0042] Figures 25a-25k show signs of EV71-induced neurogenic pulmonary edema (NPE) in class IA mice. 25a-25d: Pseudo-infected mice (FIG. 25a) or lungs obtained from EV71: TLLmv-infected mice showing signs of disease class IA (FIG. 25b), class IB (FIG. 25c), or class II (FIG. 25d). Typical gross pathology. The image shows a top view and a side view. Note the incomplete crushing of the lungs that appears in Figure 25b (white arrow). FIG. 25e: taken from pseudo-infected mice (n = 4) or EV71: TLLmv-infected mice showing signs of disease class IA (n = 8), class IB (n = 9), or class II (n = 4). Wet weight comparison of lungs (FIGS. 25f-25i: Representative image of lung tissue section (5 μm) stained with hematoxylin & eosin (H & E). Shown are pseudo-infected mice (FIG. 25f) or disease class IA (FIG. 25f). 25 g), low-magnification and high-magnification images of lungs from EV71: TLLmv-infected mice showing signs of class IB (FIG. 25h), or class II (FIG. 25i). Pink proteinaceous fluid in alveolar space. Note the presence of (Fig. 25 g, high magnification asterisk), some of which are also filled with red blood cells (Fig. 25 g, high magnification gray arrow). FIGS. 25j and 25k: In pseudo-infected mice (n = 9), or EV71: TLLmv-infected mice showing signs of disease class IA (n = 8), class IB (n = 9), or class II (n = 3). Serum levels of adrenaline / epinephrine (Fig. 25j) and noradrenaline / norepinephrine (Fig. 25k) as determined. Error bars indicate SEM. Statistical significance was determined by the Mann-Whitney test (FIG. 25e) or the t-test with Welch correction for unequal dispersion (FIGS. 25j and 25k). ^* P <0.05; ^** p <0.005. [0043] Figures 26a and 26b show the lack of viral replication or inflammation in the lung and heart tissue of Class IA mice. FIG. 26a: EV71: derived from a diverse group of mice infected with TLLmv and stained with hematoxylin & eodin for histopathological examination (H & E) or with rabbit serum against EV71 antigen for viral antigen localization. Representative image of labeled (EV71 IHC), lung tissue section (5 μm). FIG. 26b: Representative images of cardiac tissue sections (5 μm) processed for H & E and EV71 IHC. [0044] Figures 27a-27d show representative maps showing the localization and distribution of EV71 antigen and virus-induced lesions in different regions of class IA and class IB mouse brains. Cerebellar cortex (CTX) (FIGS. 27a, 27b and 27c); hypothalamus (HY) (FIGS. 27a and 27b); hippocampus (HP) (FIGS. 27b and 27c); thalamus (TH) (FIG. 27b); midbrain (MB) ) And the pons (P) (Fig. 27c); and the cerebellum (CBX) and medulla oblongata (MY) (Fig. 27d). Areas where viral antigens and lesions are detected are detected and indicated as appropriate. Larger dots indicate stronger signal / lesion size. The template image is from brainstars. It was downloaded from org1 and licensed under Creative Commons Japan. A coronal section map of brain tissue was obtained from an interactive mouse brain atlas (http://mouse.brain-map/org/static/atlas) [113]. [0045] Figures 28a-28n show that EV71: TLLmv infection in mice is associated with neural tissue destruction and large-scale viral replication. Figures 28a-28l: Representative images of brain tissue sections (5 μm) stained with hematoxylin and eosin (H & E) or immunostained with rabbit serum against EV71 antigen (EV71 IHC). Sections were obtained from mice showing signs of disease class IA (left panel) or class IB (right panel). Intracerebral lesions include edema (dotted box), infiltrating cells (diagonal region of the upper left quadrant and diagonal region of the lower right quadrant of the left panel of Figure 28k), and neurodegeneration (in the left panel of Figures 28a-28c). , Neurodegeneration (black asterisk), and quadrant cell degeneration (gray asterisk). 28m and 28n: Representative maps of spinal coronal sections from mice in disease class IA (FIG. 28m) or class IB (FIG. 28n). Emphasize areas where viral antigens and lesions have been detected. Larger dot sizes indicate larger signals / lesions. V, dorsal; D, ventral. [0046] Figures 29a-29c show EV71 antigen and virus-induced lesions in other regions of the posterior brain of Class IA mice. FIG. 29a: Representative image of dentate nucleus stained with either hematoxylin and eosin (H & E) for histopathological examination or rabbit serum (EV71 IHC) against EV71 antigen for viral antigen localization. .. The enclosed area is enlarged and shown in the inset. FIG. 29b: Representative image of the caudal brainstem from Class I mice. The image shows the cerebellar cortex (CBX) and medulla oblongata (MY). The last field (AP: asterisk) and solitary tract nucleus (NTS; dotted circle) are also shown for reference. Indicates areas where viral antigens and lesions are detected. Larger dots indicate stronger signal / lesion size. The template image is from brainstars. It was downloaded from org1 and licensed under Creative Commons Japan. A coronal section map of brain tissue was obtained from a mouse brain atlas (http://mouse.brain-map/org/static/atlas) [113]. FIG. 29c: Representative images of medulla from class IA mice showing H & E and EV71 IHC staining patterns in AP and NTS. [0047] Figures 30a-30f show histological sections of neural tissue from pseudo-infected mice (healthy controls). Representative of normal mouse tissue sections (5 μm) stained with either hematoxylin and eosin (H & E) for histopathological examination or rabbit serum (EV71 IHC) against EV71 antigen for viral antigen localization. image. Brain sections are CA3 pyramidal neurons in the hippocampus (Fig. 30a); reticular neurons in the hypothalamus (Fig. 30b) and thalamus (Fig. 30c); neurons in the periaqueductal gray (Fig. 30d); Purkinier cell layer in the cerebellar cortex. (Fig. 30e) is shown. Note the normal Purkinje cell morphology (black asterisk in the left panel of FIG. 30e); and reticular neurons in the medulla oblongata (FIG. 30f). [0048] Figures 31a-31e show EV71: TLLmv-induced pathology and viral antigen distribution in other neural tissues. Representative images of mouse tissue sections (5 μm) stained with either hematoxylin & eosin (H & E) for histopathological examination or rabbit serum (EV71 IHC) against EV71 antigen for immunohistochemical analysis. EV71: Brain sections were obtained from TLLmv-infected or pseudo-infected mice. The cortical cross section is a spinal cortical cross section of motor cortex pyramidal neurons (Fig. 31a); pontine gray matter neurons (Fig. 31b); Shown. Note the cell infiltrates found in the motor cortex (lower right quadrant of the left panel of FIG. 31a) and nerve necrosis (black asterisk of the second panel of FIG. 31). The enclosed areas of FIGS. 31c to 31e are enlarged and shown in each inset.

The present invention relates to enterovirus 71 (EV71), the development of animal models, and the screening of candidate anti-EV71 compounds. More specifically, in the present invention, enterovirus 71 (EV71) strains that are adapted to infect rodent cell lines or clone-derived viruses containing mutations in VP1 are immunoeligible rodents. And with respect to the discovery that it is possible to cause disease in susceptible rodents. These EV71 strains are sometimes referred to herein as modified enterovirus 71.

[0050] Furthermore, the present invention relates to EV71-induced neurological disorders by infecting BALB / c mice with a modified strain (eg, EV71: TLLmv) adapted to infect NIH / 3T3 mouse fibroblasts. Regarding the development of clinical authenticity models. Using this approach, modified EV71 is used to induce acute encephalomyelitis associated with neurogenic pulmonary edema in mice compared to pseudoinfected lung, which is characterized by lung swelling and increased organ weight. Despite lung or heart tissue inflammation disease is not present, localized bleeding and proteinaceous liquid in the alveoli, high serum levels of catecholamines, and brain stem, thorough tissue damage particularly in the medulla oblongata observed. These data demonstrate that the model accurately reproduces the signs and symptoms of human EV71-induced neurogenic pulmonary edema.

[0051] Thus, in one aspect, the invention relates to an animal model comprising a rodent infected with enterovirus 71 capable of infecting rodents, sometimes referred to herein as modified enterovirus 71. .. In one embodiment, such modified enterovirus 71 is rodent cell line compatible enterovirus 71. In another embodiment, such modified enterovirus 71 is a clone-derived virus (CDV) containing a mutation in VP1. In some embodiments, mutations in VP1 allow the CDV to infect rodent cells with the rodent SCARB2 protein. In one embodiment, the rodent is an immunoeligible rodent. In another embodiment, the rodent is a immunocompromised rodent. Suitable animals for use as a model are preferably mammals, most preferably suitable laboratory animals such as rabbits, rats, mice and the like. In one embodiment, the animal is a mouse. In another embodiment, the rodent cell line is a mouse cell line. In a further embodiment, the mouse cell line is a mouse NIH / 3T3 cell line. In another embodiment, the mouse cell line is a mouse Neuro-2a cell line. In one embodiment, the rodent cell line compatible enterovirus 71 is EV71: TLLm. In another embodiment, the rodent cell line compatible enterovirus 71 is EV71: TLLmv. In one embodiment, the clone-derived virus containing the mutation in VP1 is CDV: BS _VP1 [K98E / E145A / L169F]. Animal models are useful for studying systemic transmission of viruses and human disease spectra in animal models. Animal models are also useful for screening antiviral drugs and vaccines.

[0052] Prepare animal models as needed. A large standardized stock of rodent cell line compatible EV71 strains is prepared, titrated, and maintained in a deep freezer (-80 ° C). Several "standardized" (based on statistical calculations) rodents, such as BALB / c mice or NSG mice, are infected with standardized titer virus strains to produce animal models. The animal models of the present invention develop neurological symptoms (similar to those that can develop in humans) upon infection. As shown herein, modified enterovirus 71, such as rodent cell line-matched EV71 virus lines, affects a variety of neurological disorders that appear in the brain and mice.

[0053] In some embodiments, the rodent cell line compatible enterovirus 71 is EV71: TLLm. EV71: TLLm was obtained after continuous passage of human EV71 BS strain in NIH / 3T3 mouse cell lines for a minimum of 60 cycles. In one embodiment, EV71: TLLm was deposited on January 12, 2015 at the Type Culture Collection China Center located at Wuhan University, Wuhan 430072, People's Republic of China, and the deposit number CCTCC V201437 was assigned. In another embodiment, when viral RNA is synthesized using a viral RNA sequence (GenBank Deposit No. KF514879; SEQ ID NO: 1), EV71: TLLm can be recovered using advanced reverse genetics. The technique of advanced reverse genetics is well known in the art [84-87].

[0054] In another embodiment, the rodent cell line compatible enterovirus 71 is EV71: TLLmv. EV71: TLLmv was obtained from further passage of EV71: TLLm in NIH / 3T3 mouse cell lines for an additional 40 cycles. In one embodiment, EV71: TLLmv was deposited on January 12, 2015 at the Type Culture Collection China Center located in Wuhan University, Wuhan 430072, People's Republic of China, and with a deposit number CCTCC V201438 was assigned. In another embodiment, when viral RNA is synthesized using a viral RNA sequence (GenBank Deposit No. KF514880; SEQ ID NO: 2), EV71: TLLmv can be recovered using advanced reverse genetics. The technology of advanced reverse genetics is well known in the art.

[0055] In a further embodiment, the modified enterovirus 71 is a clone having a mutation in the capsid protein VP1 that allows the modified enterovirus 71 to use the rodent SCARB2 protein to infect rodent cells. It is a derived virus (CDV). Modified enterovirus 71 with a mutation in VP1 is made using techniques known to those of skill in the art or by preparing full-length genomic cDNA clones as described herein. Mutations in VP1 or other proteins of enterovirus 71 are made using site-specific mutagenesis or CRISPR techniques (see, eg, PCT Publication No. WO2014 / 127287). Live viruses (clone-derived viruses (CDVs)) are prepared from cDNA clones using techniques known to those of skill in the art or as described herein. CDVs with different mutations or collections of mutations are then tested for their ability to infect rodent cells. Alternatively, CDVs with different mutations or collections of mutations are then tested for their ability to bind rodent SCARB2 protein as initial screening. It is also possible to develop a CDV with a mutation in VP1 using any suitable enterovirus 71 strain. The number of mutations and the particular mutation can vary for each strain as it produces enough CDV in the target rodent cells to cause a full-blown infection. Thus, it is possible to produce a large number of rodent pathogenic EV71 strains that can infect some, many or all rodents, such as mouse cell lines. In one embodiment, the EV71 strain used to produce a CDV with a mutation in VP1 is an enterovirus 71 BS strain. In one embodiment, the modified enterovirus 71 is CDV: BS _VP1 [K98E / E145A / L169F].

[0056] In another aspect, the invention provides a method for preparing an animal model that includes the entire spectrum of E71-induced neurological infections, diseases and pathologies observed in humans. In some embodiments, the method comprises infecting the rodents described herein with the modified enterovirus 71 described herein and rearing the infected rodents for up to about 4 weeks. In one embodiment, the modified enterovirus 71 is EV71: TLLmv. In another embodiment, the modified enterovirus 71 is EV71: TLLm. In a further embodiment, the modified enterovirus 71 is CDV: BS _VP1 [K98E / E145A / L169F]. In some embodiments, the age of the rodent to be infected is between about 1 week and about 4 weeks. In other embodiments, the age of the rodent to be infected is between about 1 week and about 3 weeks. In other embodiments, the age of the rodent to be infected is between about 1 week and about 2 weeks. In one embodiment, the age of the rodent to be infected is about 1 week. In another embodiment, the age of the rodent to be infected is about 2 weeks. In a further embodiment, the age of the rodent to be infected is about 3 weeks. In some embodiments, infected rodents are bred for about 1 to about 4 weeks. In another embodiment, infected rodents are bred for about 1 to about 3 weeks. In another embodiment, infected rodents are bred for about 1 to about 2 weeks. In one embodiment, infected rodents are bred for about 1 week. In another embodiment, the infected rodents are bred for about 2 weeks. In a further embodiment, the infected rodents are bred for about 3 weeks. In a further embodiment, the infected rodents are bred for about 4 weeks. In some embodiments, the rodent is an immunoeligible rodent. In some embodiments, the rodent is a mouse as described herein. In one embodiment, the immunoqualified mouse is a Balb / c mouse. In some embodiments, the rodent is a immunocompromised rodent. In some embodiments, the rodent is a mouse as described herein. In one embodiment, the immunocompromised mouse is an NSG mouse. In another embodiment, rodents are infected by inoculating rodents with modified enterovirus 71. In one embodiment, the inoculation is intraperitoneal (IP). In another embodiment, the inoculation is intramuscular (IM). In some embodiments, the virus dose inoculated into rodents is the median cell culture infective dose of between about ¹⁰³ to about 10 ⁷ (CCID _50). In another embodiment, the viral dose inoculated into rodents is the CCID ₅₀ of between about 10 ³ to about 10 ^6. In one embodiment, the viral dose inoculated into rodents is the CCID ₅₀ of between about 4x10 ³ ~ about ^106. In another embodiment, the virus dose inoculated into rodents is the CCID ₅₀ of between about 10 ⁴ to about 10 ^6. In further embodiments, the virus dose inoculated into rodents is the CCID ₅₀ between about 10 ⁵ to about 10 ^6. In one embodiment, the viral dose inoculated into rodents is the CCID ₅₀ of about ^106.

Animal models prepared in this manner show superficial validity, i.e., these animals are observed over the entire spectrum of neurological disease induced by EV71 infection in human patients, including NPE. A true mouse model of EV71 neuroinfection showing the full range of possible clinical signs. The animal model also demonstrates construct validity with respect to the gross and histopathological features of the disease, which are very similar to those reported in lethal human cases. This novel in vivo model identifies key events in EV71 neuropathogenesis, dissects the mechanism of EV71-induced NPE, develops new therapies and potential antiviral therapies, and preclinically evaluates new vaccines. Equivalent to a powerful tool for doing.

[0058] In a further aspect, the invention provides a method of screening for antiviral agents. According to this aspect, the method comprises the following steps: providing a test group of animals and a control group of animals, where each group of animals is an animal of the animal model described herein; to the test group. Administer antiviral drug candidates; monitor disease progression in test and control groups; compare disease progression in test group to disease progression in control group; and reduce disease progression in test group compared to control group The process of selecting antiviral drug candidates to be made. In one embodiment, the antiviral agent is first screened in a test rodent cell line infected with a rodent cell line compatible enterovirus 71 prior to screening in the animal. In another embodiment, the antiviral agent is first screened in a test rodent cell line infected with a clonal virus (CDV) containing a mutation in VP1 prior to screening in animals.

[0059] In a further aspect, the present invention provides a method of screening for an effective antiviral vaccine. According to this aspect, the method comprises the following steps: providing a test group of animals and a control group of animals, where each group of animals is an animal of the animal model described herein; to the test group. Antiviral vaccine candidates were administered; disease progression was monitored in the study and control groups; disease progression in the study group was compared to disease progression in the control group; and disease progression was reduced in the study group compared to the control group. The process of selecting anti-virus vaccine candidates to be allowed. In one embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a rodent cell line compatible enterovirus 71 prior to screening in animals. In another embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a clone-derived virus (CDV) containing a mutation in VP1 prior to screening in animals.

[0060] A large standardized stock of rodent cell line compatible EV71 strains is prepared, titrated, and maintained in a deep freezer (-80 ° C) according to the methods of the invention. Alternatively, a large standardized stock of clone-derived virus (CDV) containing the mutation in the VP1 strain is prepared, titrated, and maintained in a deep freezer (-80 ° C). Several "standardized" (based on statistical calculations) animals, such as Balb / c and NSG mice, are infected with a standardized titer of virus strain. Candidate antiviral drugs or antiviral vaccines are applied to infected rodents before the onset of the disease (for assaying the prophylactic effect) or at the onset of the disease (for assaying the therapeutic effect of the drug) at various standardized dosages. Administer at. In one embodiment, using a tissue culture cell line that is susceptible to cytolytic infection by a rodent cell line compatible EV71 virus strain, such as those described herein, including those described in the Examples. , High throughput in vitro screening of anti-EV71 compounds. In another embodiment, a tissue culture cell line that is susceptible to cytolytic infection by a clone-derived virus (CDV) containing a mutation in the VP1 strain, such as those described herein, including examples. Will be used for high throughput in vitro screening of anti-EV71 compounds. In vitro screening is performed using techniques well known in the art. Promising compounds selected from in vitro screening are then screened in vivo in the animal models described herein.

[0061] As shown in Examples 2-8, continuous passage of human EV71 isolates (EV71: BS) produced viral strains that were capable of infecting in vitro cultured rodent cell lines. The mouse-adhesive fibroblast cell line NIH / 3T3 [46] obtained from NIH / Swiss mouse embryos was used to adapt the human EV71 strain to infect rodent cells. Two such NIH / 3T3 compatible strains are described as EV71: TLLm and EV71: TLLmv, where EV71: TLLm corresponds to the early stages of the conforming process (passage 60), and EV71: TLLmv is late. Corresponds to (100 passages). Based on the appearance of virus-induced CPE, measurement of high force value, and positive detection of virus antigens through immunostaining, we present virus-induced infections in cells, either proliferative or non-proliferative. It was classified into. Proliferative infections show positive viral antigen detection, as well as high viral titers, regardless of CPE observations. Non-proliferative infections, on the other hand, are characterized by unmeasurable viral titers at the cutoff assay limit, despite viral antigen detection and / or observation of CPE.

[0062] Clinical isolate EV71: BS infects only primate cell lines, whereas EV71: TLLm proliferatively infects both primate and rodent cell lines. It is important to note that the EV71: TLLm virus has successfully achieved its ability to infect rodent cells, but its degree of compatibility with NIH / 3T3 cells is less pronounced. Viral titers determined using Vero cells are much higher than those determined in NIH / 3T3 cells, as indicated by negative values in the relative replication rate (RRR) assay (FIGS. 5C and 5D). In addition, EV71: TLLm successfully infects Vero cells and leads to complete CPE at various incubation temperatures, while in infected NIH / 3T3, complete CPE can only be achieved at 37 ° C. (Table 1). Further adaptation in mouse cells, which yields EV71: TLLmv virus, shows a higher degree of adaptation to mouse cells (FIG. 5B), but in exchange for a narrowed permissible host cell spectrum. EV71: TLLmv does not infect primate cell lines as efficiently as mouse cells, but shows successful infection and leads to complete CPE of NIH / 3T3 cells incubated at a wider temperature range (Table 1). However, compared to the ancestral EV71: TLLm, EV71: TLLmv appears to have invaded monkey kidney COS-7 cells, as well as human HeLa and Hep-2 cells, and lost the ability to replicate within these cells. (FIGS. 3B-3C; FIGS. 2B-2C, 2E-2F, and 2H-2I). The ability to replicate efficiently within hamster CHO-K1 and rat NRK cells was also lost (FIGS. 3B-3D; FIGS. 2K-2L and 2N-2O). These observations indicate that further passage of the virus in NIH / 3T3 cells increases the degree of fit in mouse cells in exchange for loss of infectivity in cell lines of other origin.

[0063] Although it is not possible to point out which amino acid substitutions are compatible with mouse NIH / 3T3 host cells, viral whole genome sequencing may also shed light on potential matching mechanisms. .. Most of the amino acid substitutions identified are in the P1 (capsid) and RNA polymerase (3D region) proteins (Tables 2 and 3), suggesting possible altered viral protein activity in host cell entry and replication. Accumulation of mutations in the P1 region is expected from the acquisition of the ability of EV71: TLLm and EV71: TLLmv strains to infect new host cells. Capsid proteins form a structural background in which the virus initiates interaction with tolerant host cells through the viral receptor, which has recently been identified as scavenger receptor class B member 2 (SCARB2). [47], and later characterized as the major viral decoupling receptor for EV71 [48], which has also been utilized by some members of human enterovirus A (HEV-A) species. There is. The human SCRB2 protein shares approximately 99% sequence identity with that of other primates. Mouse SCARB2 protein, on the other hand, showed 15% sequence dissimilarity compared to primate protein [49], showing a significant structural deviation from primate SCARB2, and perhaps this is a rodent against native EV71 infection. It contributes to cell refractory. Compatible mutations in the viral capsid make the virus eligible for binding to mouse cell receptors and result in successful entry and novel host infection.

[0064] Capsid protein mutation mapping shows that most of the amino acid substitutions identified in the viral P1 region are exposed regions of the protein (FIGS. 11A-11D), especially BC, DE on the surface of VP1. , EF, and GH loops. VP1 residues 150-180 harbor a viral capsid canyon that binds to the SCARB2 protein. This region, centered on Gln-172, contains the major VP1 neutralizing epitopes at amino acids 163 to 177 [32]. Both EV71: TLLm and EV71: TLLmv show substitutions E167D and L169F in the EF loop of the VP1 canyon (FIGS. 11A-11B), and this locus has not been previously reported. Other important amino acid substitutions near the SCARB2 docking site include N104D in the BC loop and S241L in the GH loop, which are located within a 20 Å radius of Gln-172. It has been previously reported that the VP1 S241L mutation resulted from a mouse passage of CHO cell line compatible EV71 in association with K244E [50]. This mutation was found to be associated with a non-pathogenic phenotype in 5-day-old mouse pups in combination with VP2 K149I. However, adaptation in NOD / SCID mouse brain tissue has been reported to result in a reverse mutation of VP1 241 from Leu to Ser [51], and the mutation has been found to be associated with a mouse pathogenic phenotype. It was issued. The VP1 E145A mutation, located away from the SCARB2 docking site and in the DE loop, is another candidate that imparts the ability to infect murine cells. VP1 145 mutations have been previously reported [34, 37], and single E145A mutations lead to pathogenicity in NOD / SCID mice [51]. Another mutation in VP1 of C4 genotype EV71, Q145E, is associated with pathogenicity in 5-day-old mice [52]. Mouse cell-compatible mutations in VP2, especially those within neutralizing epitopes of residues 136-150 [53], can also contribute to the ability of the virus to infect rodent cells. Two substitutions in the VP2 EF loop were observed at EV71: TLLm (FIG. 11C), while there were three substitutions at EV71: TLLmv (FIG. 11D). None of these mutations have been previously described, but the nearby loci of VP2 149 in the EF loop have been mentioned in the literature [34, 50, 54], and passage in PSGL-1 overexpressing cells. It has been described as a compatible mutation in the generation [55].

To investigate the possible role of P1 region mutations in binding to viral receptors for host cell entry, EV71: BS viral RNA was transfected into murine cells. EV71: Direct introduction of BS RNA into mouse cytoplasm, NIH / 3T3 cells, as suggested by observation of virus-inducible CPE and measurable virus titers in culture supernatant when assayed in Vero cells. Proliferative infection occurs in (Fig. 12). However, reinoculation of the virus supernatant on fresh NIH / 3T3 cells failed to induce proliferative infection (Fig. 6A), and no viral antigen was detected (Fig. S4H). Similarly, transfection of EV71: BS RNA into Neuro-2A cells results in positive antigen staining (FIGS. 10C and 10D) and measurable viral titers (FIG. 12) in Neuro-2A cells, but directly to the virus. The infection did not give rise to these. Viral supernatants passaged on fresh Vero and NIH / 3T3 cells produced positive antigen staining in Vero (FIG. 10I) but not in NIH / 3T3 cells (FIG. 10J). These data indicate that avoiding the need for receptor binding for entry into host cells led to successful infection and viral progeny production in NIH3T3 and Neuro-2A cells. These data also support the inability of human EV71: BS to successfully enter mouse NIH / 3T3 and Neuro-2A cells through murine cell receptors. In addition, these data show that mutations within the P1 region of the EV71: TLLm and EV71: TLLmv genomes give the virus the ability for efficient receptor binding and, as a result, allow host cell entry. Suggest to do.

[0066] Several amino acid mutations have also been observed in the P2 and P3 regions, which are definitive for viral replication and hijacking of host cell protein translational mechanisms [56]. These matching mutations may function in optimizing EV71 genome replication and translation within mouse cells. The viral 3D protein alone accumulates eight amino acid substitutions in EV71: TLLmv and four substitutions in EV71: TLLm, and both strains have one mutation each in 3B and two suddenly each in 3C. It showed a mutation. Direct inoculation of viral RNA into TCKM cells provides insight into the possible role of compatible mutations in viral unstructured proteins. TCKM cells have been shown to be tolerant of EV71: TLLm and EV71: TLLmv infections (FIGS. 3B and 3D; FIGS. 4Q and 4R), but of EV71: BS viral RNA into TCKM cells. Transfection did not result in successful infection. Viral antigen signals were not detected in infected and transfected cells (FIGS. 10E-10F), and passage of viral supernatants on fresh NIH / 3T3 and Vero cells did not result in positive viral antigen detection. (FIGS. 10K and 10L). Moreover, there were no assayable viral titers in both infected and transfected cells (Fig. 12). These data indicate that, apart from mutations in the capsid region, mutations in the P2 and P3 regions found in EV71: TLLm and EV71: TLLmv are required to successfully infect TCKM cells. Suggest.

[0067] It is originally derived from human clinical samples that have successfully acquired the ability to proliferately infect several rodent cell lines after continuous passage in mouse cell lines. It is believed to be the first report of the EV71 strain. A relatively high mutation rate during RNA replication results in a mutant genome that acts as a genetic reservoir of phenotypic traits for future suitability, and the resulting replication as a pseudospecies distribution [57,58]. Guides the dynamic plasticity of the RNA virus genome, giving it adaptability to changing environments [59, 60]. EV71-infected suckling mice [34, 37, 38, 51] and other rodents (eg, gerbils) [39] have previously been reported to proliferate in vitro cultured rodent cells. There was nothing. This may be due to the high genetic barriers associated with major changes in phenotype and host range [58]. Interestingly, this is the first report of a large number of matching mutations within the EV71 consensus whole genome sequence. Previously documented mouse-matched EV71s reported less than 10 amino acid substitutions in the genome [34, 38, 51], but we found 21 in EV71: TLLm and 36 in EV71: TLLmv. Was reported, which was still higher than the maximum number of previously identified matching mutations [37]. These may not be sufficient for several passages of the virus in mouse tissue [34, 36, 37] to successfully adapt the virus to break genetic barriers and infect cultured mouse cells. Suggests that there is sex. Instead, hundreds of consecutive passages may be required, as observed in this study.

Host cell restriction of EV71 is that in recent years EV71 has scavenger receptor class B member 2 (SCARB2) as a functional receptor for host cell entry [47] and viral decoupling in endosomes [72]. Has been proven and explained to use. The human and murine SCARB2 proteins show only 84% amino acid sequence identity [67], suggesting structural differences between the two proteins. In this scenario, it is generally not possible for EV71 clinical isolates to infect mouse-derived cells, while mice and other rodents are generally resistant to EV71 experimental infection. Explaining that, this makes attempts to develop small animal models of EV71 infection difficult. However, despite this viral-receptor incompatibility, we find that the first step in infection, receptor-mediated cell entry, is bypassed through intracellular viral RNA transfection. It has been shown that some murine cells support EV71 infection. EV71: BS does not infect Neuro-2a and NIH / 3T3 cells [71], but transfection of viral genomic RNA results in expression of EV71 protein, induction of lytic cytopathic effect (CPE), and production of viable viral progeny. Produces. Similarly, live virus was produced [73-75] after previously transfecting poliovirus RNA into mammalian cells, but the cells used (HeLa) were tolerant of poliovirus infection. It was known that there was. In our experiments, non-tolerant mouse neurons Neuro-2a and fibroblast NIH / 3T3 cells support viral replication and generate live viral progeny upon transfection of EV71: BS RNA into the cytoplasm. It has been demonstrated that the internal environment of murine cells contains the host factors required for EV71 infection and supports the completion of the viral infection cycle, suggesting that the EV71 protein is functional in the murine cytoplasmic sol. Was done. These findings also indicate that the absence of NIH / 3T3 and Neuro-2a cellular infections when inoculated with the virus may be due to defects in receptor-mediated host cell entry and decapsulation. , This is primarily a function of the capsid protein. These results are similar to our previous observations (or above or [71]), and we allow certain amino acid substitutions in the EV71: BS capsid protein to bind to the receptor. And therefore leads to the hypothesis that virus entry and decapsulation can be induced, and subsequently infect mouse cells. Two unique tools, a mouse cell line (NIH / 3T3) compatible EV71 strain (EV71: TLLm and EV71: TLLmv) and two mouse cell lines (Neur-2a and NIH / 3T3) provide an opportunity to test this hypothesis. did.

[0069] The infection phenotype of a chimeric clone-derived virus (CDV: BS [MP1]) expressing EV71: TLLm capsid protein, but expressing EV71: BS unstructured protein is "mouse cell entry phenotype". Was confirmed to be conferred by a capsid protein from a mouse cell line compatible EV71 strain. These chimeric CDVs behave similarly to mouse cell-matched EV71 strains and induce proliferative infections in Vero, as well as NIH / 3T3 and Neuro-2a cells, which complete the viral infection cycle, i.e. the generation of viable viral offspring. Accompanied by direct evidence of. In addition, mutagenesis to introduce amino acid substitutions K98E, E145A, and L169F into EV71: BS VP1, and S144T and K149I in VP2 led to limited infection of murine cells. Only EV71: CDV with TLLm P1 region substitution (CDV: BS [M-P1]) and CDV with combined amino acid substitution VP1-K98E / E145A / L169F (CDV: BS _VP1 [K98E / E145A / L169F]) It was successfully passaged in Neuro-2a and NIH / 3T3 cells to produce viable virus progeny in the resulting culture supernatant. The rest of the CDV containing other amino acid substitutions, either individually or in various combinations, has limited entry into murine cells, as evidenced by observation of CPE and detection of viral antigens in inoculated cells. Only showed, leading to only one cycle of replication. Live virus progeny were not present in the infected cell culture supernatant because reinoculation on healthy mouse cells failed to induce infection. It is not clear why these CDV-infected cells caused a failure in viable virus progeny production in culture supernatants, but one possibility is that the capsids produced after mutation are structurally unstable. That is. We speculate that the introduced amino acids are incompatible with the other amino acids that make up the protein and therefore may affect the overall capsid protein fold and may alter the capsid structure. This assumption underscores the complexity of capsid protein assembly, where the four viral proteins (VP1-4) interact cooperatively to produce functional capsids. Therefore, amino acid substitutions that can allow the virus to bind to novel receptors can also have catastrophic consequences, especially because of their incompatibility with other amino acid residues in the protein. This is possible if the complex is inconveniently destabilized.

[0070] The results shown in Examples 10-17 show that three VP1 substitutions: K98E, E145A, and L169F are required and sufficient for binding to receptors on mouse cells, and allow EV71 entry. Clarify what to do. VP1-169 residues have not been previously mentioned in the literature, but VP1-98 and VP1-145 residues have previously been shown as markers for binding to human PSGL-1 receptor protein on leukocytes. It is coming [76]. Analysis of the 1,702 VP1 sequences of EV71 clinical isolates published in Genbank confirmed that the mutant combination, VP1 98E / 145A, was rare but viable, and these were only 5 Found in one isolate (0.3% of database). On the other hand, the VP1-169F mutant was not observed in the survey database of the EV71 VP1 sequence and was shown to be extremely rare. CDV: BS _VP1 [K98E / E145A / L169F] is stably passaged on Neuro-2a cells, and the introduced mutations are retained in the viral genome for three passages while the live viral offspring. Consistently produced, which suggested that the virus was viable and stable, at least in Neuro-2a cells. Therefore, introduction of these three residues into other EV71 clinical isolates: VP198E, 145A, and 169F may allow the isolate to infect murine cells, but this is still the case. Must be proved.

[0071] Similar to previous findings that EV71 utilizes scavenger receptor class B member 2 (SCARB2) protein as a receptor for host cell entry and escape to the cytosol [47, 72, 77]. ], Our data also demonstrate that mouse cell-compatible EV71: TLLmv utilizes mouse SCARB2 (mSCARB2) to infect murine cells. The virus bound directly to recombinant soluble mSCARB2 in vitro, and preincubation of the live virus with the protein prior to inoculation on healthy cells reduced the severity of cell infection. Furthermore, blocking the free surface of mSCARB2 on host cells by binding with a polyclonal antibody against mSCARB2 reduced viral binding on fixed cells and also inhibited live cell infection. The results also show EV71: TLLmv binding to the human SCARB2 (hSCARB2) protein, which the virus uses to infect primates. Binding and infection of primate cells at EV71: TLLmv was also blocked by antibodies to hSCARB2. The data presented herein show only EV71: TLLmv, but the results are also extensible to EV71: TLLm. EV71: TLLmv was obtained from further passages of EV71: TLLm in NIH / 3T3c cells, and only slight amino acid substitutions were observed between the two mouse cell line-matched EV71 strains. Thus, they indicate that both EV71: TLLm and EV71: TLLmv utilize cellular mSCARB2 for rodent cell infection.

[0072] Similarly, both CDV: BS [MP1] and CDV: BS _VP1 [K98E / E145A / L169F] utilize mSCARB2 to infect murine cells. Preincubation of Neuro-2a cells with mSCARB2 antiserum blocked cell infection with the virus, as evidenced by reduced CPE induction and lower viral titers compared to controls. Recent data show that SCRAB2 binding to the EV71 canyon is a lipid within the capsid canyon, stabilizes the mature virion, and induces the release of a "pocket factor" that is predicted to be sphingosine in the EV71 [ 78], a series of events in viral decapsulation: capsid dilation (forming 135-S or A particles), VP1 N-terminal and VP4 protrusion from the 5-fold capsid junction into the endosome membrane, and within the host cytoplasm. It has been shown to cause the release of viral genomic RNA into [78-81]. Recent human SCARB2 crystal structure data also reveal a lipid tunnel across the protein [67], which is not relevant in the context of the SCARB2 function of delivering β-glucocerebrosidase to lysosomes, although In contrast, Dang et al. [72] proposed that it acts as a conduit for the removal and transport of sphingosine from capsid canyons during SCARB2 binding. SCARB2 amino acid residues 140-151 are very different in sequence between human and murine proteins and are the major binding sites for EV71 [49]. This same region acts as a gate that controls the opening and closing of the SCARB2 lipid tunnel, and this event is triggered by the acidic pH in the viral shedding. The VP1-169 residue is in the capsid canyon and probably has a direct function in SCARB2 binding. The dramatic change from leucine to phenylalanine at this position modifies the canyon structure, resulting in better compatibility with the murine SCARB2 protein. VP1 98 and 145 residues, on the other hand, are on the fringes surrounding the capsid canyon and may have functions other than SCARB2 binding. These are merely hypotheses, and to elucidate the exact mechanism by which the three VP1 amino acid substitutions can confer a "mouse cell entry phenotype", analyze the structure of the murine SCARB2 protein and with the viral capsid. You will need to map the interactions.

[0073] This is believed to be the first scientific exploration of EV71 capsid site-specific mutations that induce proliferative infections of murine cells, which are naturally unacceptable. Previous studies have overcome viral host restrictions and generate mutants with improved infection profiles in mice through selective viral adaptation, as in passages in live animals [34, 37, 38]. , 82] or have attempted to modify cells by ectopic expression of the human SCARB2 protein [47, 69, 70, 83]. The incompatibility of EV71 for murine SCARB2 is generally due to the resistance of mice to EV71 infection, thus hindering the development of mouse models of EV71 infection. This probably also explains why most mouse models of EV71 infection with either wild-type or mouse-compatible virus strains fail to reproduce the disease spectrum observed in severe human infections [] 34, 37, 38]. On the other hand, transgenic mice expressing human SCARB2 showed broader EV71 infection characteristics without the need for viral mouse adaptation, but the entire spectrum of human disease could not be clearly reproduced. [69, 70]. Mouse cell line-matched EV71: TLLm and EV71: TLLmv efficiently infect cultured rodent cells, as it has been demonstrated herein that specific amino acid substitutions in EV71 allow EV71 to infect murine cells. It provides the first step in understanding the molecular mechanism that can be achieved. In addition, this may provide another approach to generate a mouse model of EV71 infection, study detailed pathogenesis, and reproduce the full spectrum of neurological disease found in human infections.

In Examples 22-25, young BALB / c mice inoculated with mouse cell-compatible enterovirus 71 (EV71) had neurogenic pulmonary edema (NPE) that closely resembled the pathology observed in infected human patients. It is demonstrated to show acute encephalomyelitis associated with. Compatible virus strain EV71: Animals exposed to TLLmv are presented as paralysis, ataxia and tremor, and are consistent with CNS localized pathology identified in lethal cases of EV71 infection, pyramidal and extrapyramidal of the brain. It showed varying levels of virus-induced tissue damage in both areas [91-95]. In addition, some mice showed dyspnea consistent with autonomic dysfunction. Based on the disease presentation at the time of euthanasia, the animals could be easily divided into four groups: class IA, class IB, class II and survivors. Survivors show no signs of disease, class II mice show persistent flaccid paralysis and severe weight loss, while class IA and class IB mice also suffer from acute neurological disease, which is It was universally lethal within 3-7 DPI. Class IA mice also showed overt severe dyspnea, which was not due to either congestive heart failure or pneumonia. Instead, Class IA animals show extensive tissue damage in the caudal brainstem, especially in the medulla, which is associated with high serum levels of catecholamines, and the respiratory signs observed in these mice are neurogenic pulmonary edema ( It was strongly suggested that it was the result of NPE). Gross pathological comparison of lungs from class IA mice with those from other groups revealed incomplete lung crushing at autopsy, as well as a significant increase in lung wet weight, probably bleeding and alveoli. It was due to liquid leakage into the space. These features were very similar to those observed in laboratory animal models of lethal human cases with non-virus-induced NPE and fulminant NPE [96, 97]. In fact, histopathological analysis of class IA animals further revealed localized regions of the alveolar space filled with proteinaceous and erythrocyte-filled exudates, consistent with observations in lethal human cases [21. , 98-100].

[0075] Pulmonary edema (PE) is typically defined as extravasation in the water content of the lung, 48, and can be subdivided based on cardiogenic or neurogenic origin. Since class IA mouse heart tissue showed normal tissue and lacked overt signs of disease, we are able to eliminate cardiogenic PE, and in the lung parenchyma, viral replication or Direct virus-induced lung injury was ruled out because of the absence of inflammation. Instead, we detected high serum levels of catecholamines in class IA mice, which groups have previously been demonstrated as a result of severe sympathetic discharge-induced catecholamine storms. , Strongly shown to exhibit neurogenic PE (NPE) [96, 101]. In this scenario, autonomic dysfunction induces a catecholamine storm, resulting in systemic and pulmonary vasoconstriction. This leads to a shift in blood volume from the whole body to the pulmonary circulation, which results in plasma leakage and bleeding into the alveolar space through the hydrostatic mechanism or directly by pulmonary endothelial injury [101-104]. .. In recent years, several laboratory animal models of non-virus-induced NPE have been developed (see Sedy [103] and Davison [104] for an overview), but our model is EV71 infected. It is the first to successfully induce the classical signs of pulmonary edema using only. Therefore, our novel model represents an important advance in pursuing antiviral therapies and therapeutic measures that can limit EV71 infection and prevent the onset of fulminant NPE in human patients. To do. Pulmonary edema may also be induced by host cytokine storms in response to EV71 infection [105-107], but data show that the major mechanism of EV71-induced NPE is accompanied by a large-scale inflammatory response. However, and instead, it strongly suggests that it is associated with tissue damage in specific areas of the brain.

[0076] The brain region associated with NPE induction has been referred to as the trigger zone, which includes the periventricular and dorsomedial nuclei [101, 103] of the hypothalamus, as well as the ventrolateral and dorsal medulla, NTS. And the AP region [96, 104, 108-110]. EV71-induced NPEs have previously been shown to contribute to extensive damage to brainstem tissue [21, 22, 93, 111], and in our novel murine model, we , Both viral antigens and extensive damage were detected in the brainstem and spinal cord. Class IA and class IB mice exhibit similar distribution of lesions and viral antigens in the brainstem and spinal cord, as well as comparable upregulation of serum catecholamines, while lack of NPE in class IB animals is NTS, medullary reticular formation. It can be explained by the reduced number and severity of brain lesions and / or lower viral antigen intensity in the reticular formation (MdRN), and the AP region of the spinal cord, the dentate nucleus of the cerebellum, and the dorsomedial nucleus of the hypothalamic anterior area. .. We therefore found that acute and severe destruction of brainstem tissue in EV71-infected hosts, especially those associated with the vasomotor region, led to catecholamine storms, and known trigger zones were sufficiently damaged. If so, suggest that this can proceed to the NPE.

[0077] In summary, the present invention has shown superficial validity [112], i.e., these animals have been observed over the entire spectrum of neurological disorders induced by EV71 infection in human patients, including NPE. A true mouse model of EV71 neuroinfection showing the full range of possible clinical signs. Observations of salient features in EV71: TLLmv-infected mice that present with Class IA signs of disease were produced by video containing two video clips of two different Class IA mice. Both animals were unable to maintain their posture (self-right) and were in a coma. Severe dyspnea, presented as tachypnea with subcostal retraction, was evident in the first mice. Pant, sub-rib retraction, and foamy fluid coming out of the nostrils were seen in the second mouse. Observations of prominent features in EV71: TLLmv-infected mice presenting with Class IB signs of disease were made by video of one Class IB mouse. He was unable to maintain his posture and was in a coma. Ipsilateral paralysis of the right limb and persistent tremor of the left hind limb were also observed. The model also showed construct validity with respect to the gross and histopathological features of the disease [112], which are very similar to those reported in lethal human cases. This novel in vivo model identifies key events in EV71 neuropathogenesis, dissects the mechanism of EV71-induced NPE, develops new therapies and potential antiviral therapies, and preclinically evaluates new vaccines. Equivalent to a powerful tool for doing.

[0078] Unless otherwise indicated, the practice of the present invention is within the technical scope of the art, such as chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and Use conventional techniques of transgenic biology. For example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook et al., 1989, Molecular Cloning, Second Edition (Cold Spring Harbor, Cold Spring Harbor), Cold Spring Harbor, Cold Spring Harbor, Cold Spring Harbor. Russel, 2001, Molecular Cloning, 3rd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Green and Sambrook, 2012, Molecular Cloning, 4th Edition (Cold Spring Harbor, Cold Spring Harbor) Cold Spring Harbor, Cold Spring Harbor Ausube et al., 1992, Cold Spring Harbor Molecular Biology (John Willey & Sons, including regular revisions); Grover, 1985, DNA Cloning (IRL Press, Molecular Solar, Oxford); cold spring Harbor Laboratory Press, cold spring Harbor, NY); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991 ); Harlow and Lane, 1988, Antibodies, (Cold Spring) Harbor Laboratory Press, Cold Spring Harbor, NY); Nucleic Acid Hybridization (B.). D. Hames and S.M. J. Higgins supervision 1984); Transcription And Translation (BD Hames and S. J. Higgins supervision 1984); Culture Of Animal Cells (R. I. Frishney, Alan R. Ellis, Alan R. (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Papers, Methods In Enzymology (Academic Press, Inc., NY); Methods In Enzymol 154 and 155 (supervised by Wu et al.), Immunochemical Methods In Cell And Molecular Biology (supervised by Mayer and Walker, Academic Press, London, 1987); Handbox, Volume I . Blackwell supervision, 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al., RNA Interference Technology: From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), See Human Press, Totowa, New Jersey, 2004; Sohair, Gene Silking by RNA Interference: Technology and Application, CRC, 2004.

[0079] The invention is described by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention in any way. Standard techniques well known in the art, or techniques specifically described below, were used.

Example 1
Materials and methods for Examples 2-8.
[0080] Cell Lines and Virus Strains: All cell lines used in this study were purchased from the American Tissue Type Culture Collection (ATCC, USA). Diverse mammalian cell lines; human adenocarcinoma cell lines, HeLa (CCL-2) and HEp-2 (CCL-23), and lamellar myoma RD (CCL-136); African green monkey kidney Vero (CCL-81), and savanna monkey (Vero vet monkey) kidney fibroblast COS-7 (CRL-1651); mouse neuroblastoma Neuro2A (CCL-131), embryonic fibroblast NIH / 3T3 (CRL-1658) , And kidney epithelial TCKM (CCL-139); hamster ovary epithelial-like CHO-K1 (CCL-61), and normal rat kidney epithelium NRK (CRL-6509).

[0081] The human EV71 BS strain (EV71: BS) was previously isolated from the brainstem of a dead patient infected with EV71. The virus was passaged in Vero cells for 4 cycles before storage at −80 ° C. for further use. Mouse cell (NIH / 3T3) compatible EV71: TLLm strains were obtained from the EV71: BS strain in mouse NIH / 3T3 cells through continuous passage (> 60 cycles). The EV71: TLLm strain was further subcultured in NIH / 3T3 cells (40 cycles) to generate a mouse cell-compatible pathogenic strain (EV71: TLLmv).

[0082] Cell line maintenance and viral infection: 10% (v / v) fetal bovine serum (FBS, i-DNA Singapore) and 0.22% (w / v) sodium bicarbonate (NaHCO ₃ , Sigma Aldrich) , USA) supplemented Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) with all cell lines grown and incubated at 37 ° C. and 5% CO ₂ unless otherwise stated. All infected cells were incubated in maintenance medium (DMEM supplemented with 1% FBS and 0.22% NaHCO ₃ ).

[0083] cells (per well 2.5～5.0X10 ⁵ cells) tissue culture treated 6-well plates (Nunc, Fisher Scientific) in planting overnight infected with 500μl of the virus suspension (MOI 1), Then, the cells were incubated at 30 ° C., 37 ° C., or 39 ° C. for 2 hours. Cells were washed twice in sterile phosphate buffered saline (PBS, pH 7.4) and then added to fresh maintenance medium (DMEM, 1% FBS). Infected cells were observed daily for the appearance of a clear lytic cytopathic effect (CPE).

[0084] For viral replication kinetics studies, plates containing infected cells were frozen at -80 ° C at various time points: 0, 6, 12, 24, 36, 48, and 54 hours (hpi) after infection. did. The plate was subjected to 3 cycles of freezing and thawing, and the lysate was harvested and clarified by thorough vortexing and then centrifuged at 1,500 xg for 10 minutes. The clarified supernatant was stored at −80 ° C. in a frozen vial (Nunc, Fisher Scientific) until further use.

[0085] For temperature matching assays, inoculated Vero and NIH / 3T3 cells were incubated at 30 ° C., 37 ° C., and 39 ° C. and observed daily for the appearance of CPE. Each culture supernatant was collected at 48 hpi and stored in frozen vials at -80 ° C until further use.

[0086] Diverse mammalian cell lines, namely RD, HeLa, and HEp-2 (human), Vero and COS-7 (monkey), NIH / 3T3, Neuro-2A, and TCKM (mouse), CHO-K1 ( Hamsters), as well as NRK cells (rats), were infected with parent EV71: BS or the resulting NIH / 3T3 compatible EV71 strain at 1 MOI (multiplicity of infection) and incubated at 37 ° C. for 10 days. Cultures were observed daily for the appearance of CPE.

Determination of virus titers and relative replication rate (RRR): Virus supernatants were subjected to endpoint titer determination and assayed in both NIH / 3T3 and Vero cells. Virus titers were counted using the Red and Muench method [61] and the Red and Muench calculator [62]. Briefly, NIH / 3T3 (1x10 ⁴ cells per well) and Vero cells (4x10 ³ cells per well) were planted overnight in 96-well plates. Frozen virus thawed to room temperature was diluted in sterile 1% aqueous sodium deoxycholate (Sigma Aldrich, USA) ( ^10-1 ) and mixed vigorously for 15 minutes to disaggregate the virus. The deaggregated virus was subjected to 10-fold serial dilutions in maintenance medium, and 100 μl diluted virus from ^10-3 dilutions was added onto each well of the cells. The plate was incubated at 37 ° C. and observed daily under an inverted light microscope for the appearance of apparent CPE. Viral titers were reported as 50% cell culture infection dose per volume (CCID ₅₀ / ml).

[0088] In order to assess the suitability of EV71: TLLm and EV71: TLLmv in NIH / 3T3 cells, virus supernatants collected from pre-infected primate and rodent cell lines were taken from NIH / 3T3 and Vero cells. Both were used for virus titration assay. Titer ratios used to measure relative replication (RRR) in NIH / 3T3 and Vero cells were calculated using the following formula:
RRR = log (A / B)
In the formula, A is the viral titer assayed in NIH / 3T3 cells and B is the viral titer assayed in Vero cells.

Viral antigen detection by immunofluorescence assay: Infected cells that did not show significant CPE were tested for infection by immunofluorescence (IF) staining. Cells were trypsinized to 72 hpi, washed twice in sterile PBS and coated on Teflon slides (Erie, USA). The slides were air dried in a biosafety cabinet and UV treated for 15 minutes to inactivate the live virus and then fixed in cold acetone at 4 ° C. for 10 minutes. Slides were explored with pan-enterovirus antibody (Merck Millipore, USA) and subsequently with FITC-conjugated mouse IgG (DAKO Cytomation, Denmark) mixed with 0.01% (w / v) Evan blue counterstain. Stained.

[0090] EV71 viral RNA in the cell Transfection: the 24-well plates overnight planting was Vero and NIH / 3T3 cells (3x10 ⁴ cells per well), following the manufacturer's protocol, Lipofectamine 2000 (Life technologies, USA ) Was used to transfect viral RNA extracted from 4x10 ⁶ CCID ₅₀ virus. RNA was extracted from EV71: BS, EV71: TLLm, and EV71: TLLmv using a viral RNA kit (Qiagen, Germany) and incubated on cells with Lipofectamine 2000 at 37 ° C. for 6 hours. Transfected cells were observed daily for the appearance of CPE. Supernatants were collected from infected cells at 7 dpi and passaged on freshly planted Vero and NIH / 3T3 cells. Cells were observed daily for the appearance of CPE, and at 7 dpi, cells were trypsinized and processed for immunofluorescent virus antigen detection.

Full-length genomic sequencing and gene mapping of EV71 strains: viral RNA of EV71: BS, EV71: TLLm, and EV71: TLLmv strains was extracted using a viral RNA kit (Qiagen, Germany) and Superscript II ( Reverse transcription was performed using SII-RT, Life Technologies, USA). The resulting cDNA was amplified with GoTaq Green (Promega, USA) and degenerate EV71 primers (primer sequences are available upon request). Amplicon was purified using a PCR cleanup kit (Geneaid Biotech, Taiwan) and cloned into a pZero2 vector (Lifetech, USA). Clones were selected from the kanamycin plate, inoculated into LB broth (50 μg / ml kanamycin), and grown overnight at 37 ° C. for plasmid extraction with the QiaSpin miniprep kit (Qiagen, Germany). Subsequently, plasmids were sequenced by the BigDye terminator method (Applied Biosystems, USA) using the same primers. Using BioEdit v7.0.9.0 [63], the obtained 500 bp fragment sequence was aligned with the entire genome sequence of EV71 Singapore Isolate 3799-SIN-98 (GenBank Deposit No. DQ341354.1). , EV71: BS, EV71: TLLm, and EV71: TLLmv whole genome sequences were reconstructed. Molecular modeling of the EV71: TLLm and EV71: TLLmv promoters was performed using Deepview / Swiss pdvviewer version 3.7 (http://expasy.org/spdbv/) and SWISS-MODEL servers [64, 65].

Example 2
Primate cell lines are tolerant of EV71: BS infection, but rodent cell lines are not.
[0092] All primate cell lines used in this study were tolerant of infection with EV71: BS virus. Human RD cells, as well as monkey Vero and COS-7 cells, showed a fully lytic cytopathic effect (CPE) within 48 hours (hpi) after infection (FIGS. 1A, 1J, and 1M), and viral antigens were fixed. Detected in infected cells (FIGS. 2A-2L; no data presented for RD, COS-7 and Vero cells). Viral proliferative curves taken from the supernatants of various infected cell lines confirmed proliferative infections in RD, Vero, and COS-7 cells (FIG. 3A). Virus collected from RD and Vero ^cells, reaching the endpoint titer of 3x10 _{9 CCID} 50 / ml, whereas, the virus titers from COS-7 was ¹⁰ 6 _CCID 50 / ml (Figure 3A). EV71: BS did not induce complete CPE in HeLa and Hep-2 cells (FIGS. 1D and 1G), and the resulting viral titer was unmeasurable within the assay cutoff limit. However, the virus antigen was detected in both HeLa cells (Fig. 2a) and Hep-2 cells (Fig. 2D) by indirect immunofluorescence staining, and the virus succeeded in entering the cells, but was insufficient. It was shown that it was or was replication deficient and may not have produced measurable viral titers.

[0093] All rodent cell lines tested were determined to be intolerant of EV71: BS infection. Soluble CPE in cells is absent after infection (FIGS. 4A, 4D, 4G, 4J and 4M), viral titers from supernatants are unmeasurable, and viral antigens are undetectable in inoculated cells. (FIGS. 2G, 2J and 2M; FIGS. 10A, 10C and 10E).

Example 3
Mouse cell (NIH / 3T3) compatible EV71: TLLm virus proliferatively infected both primate and rodent cell lines
[0094] EV71: TLLm was obtained after continuous passage of EV71: BS in NIH / 3T3 mouse cells for a minimum of 60 cycles. The primate and rodent cells tested were all tolerant of proliferative infections with EV71: TLLm, with the exception of NRK cells. Complete CPE was observed at 48 hpi in RD, Vero, and COS-7 (FIGS. 1B, 1K, and 1N), and in NIH / 3T3 and Neuro-2A cells (FIGS. 4B, E). High viral titers are measurable in supernatants taken from all infected cell lines except NRK (FIGS. 3C and 3D), and infected cells are indirectly positive for viral antigens by immunofluorescence assay. It was a test result (Fig. 2B, 2E, 2H, 2K and 2N). Complete CPE and measurable viral titers are not observed in NRK cells (FIG. 4N; FIG. 3D), but viral antigens are detectable (FIG. 2K) and EV71: TLLm successfully enters the virus into NRK cells However, it was suggested that inefficient virus replication may have resulted in unmeasurable virus titers.

Example 4
Mouse cell (NIH / 3T3) compatible EV71: TLLmv virus proliferatively infects rodent cell lines, but not all primate strains.
[0095] The EV71: TLLmv virus strain was obtained from an additional 40 cycles of EV71: TLLm passage in NIH / 3T3 cells. EV71: TLLmv causes soluble CPE in fewer cell lines, RD, Vero, NIH / 3T3, Neuro-2A, and TCKM cells (FIG. 3B), and complete CPE is RD, NIH / 3T3, And were only observed in Neuro-2A cells (Fig. 1C; 4C, F). It was noted that TCKM, CHO-K1 and NRK cells were also tolerant of infection without progressing to complete CPE, as indicated by positive viral antigen detection in infected cells (FIGS. 2L, 2O). , And 2R).

[0096] On the other hand, the primate cell lines HeLa, Hep-2, and COS-7 have the absence of CPE (FIGS. 1F, 1I, and 1O), unmeasurable viral titers (FIG. 3B), and negative viral antigens. It was observed to be intolerant to EV71: TLLmv infection, as indicated by detection (FIGS. 2C, 2F, and data not presented).

Example 5
The EV71: TLLmv virus showed a higher degree of compatibility with NIH / 3T3 cells, whereas EV71: TLLm was more compatible with replication in Vero cells.
[0097] The amount of viable virus in the supernatant collected from infected cells was determined at various time points by counting virus titers in both Vero and NIH / 3T3 cells. The relative growth ratio (RRR) calculated by taking the ratio of the viral power value assayed in NIH / 3T3 to the power value assayed in Vero was used as a surrogate measure of the degree of viral compatibility with NIH / 3T3 cells. Parent EV71: BS virus showed high negative RRR values for RD, Vero, and COS-7 (FIG. 5A), and the viral titers assayed in Vero cells were the titers assayed in NIH / 3T3 cells. It was shown to be far beyond. The value of the relative growth ratio to other cell lines was undecidable because the virus titer could not be measured. On the other hand, the EV71: TLLmv virus showed a positive RRR value, with the exception of the virus propagated in Vero cells (FIG. 5B). Positive RRR values were an indicator of more efficient replication and therefore higher power value in NIH / 3T3 cells compared to Vero cells. Negative RRR values determined for EV71: TLLmv taken from Vero cells were consistent with the observed slow growth kinetics (FIG. 3B) and lower viral titers. EV71: TLLm showed a negative RRR value (FIGS. 5C and 5D), but to a lesser extent than the RRR value for EV71: BS. This suggested that EV71: TLLm could proliferatively infect several rodent cell lines, but was still more suitable for replication in Vero than NIH / 3T3 cells.

Example 6
EV71: TLLm showed better adaptability to varying temperatures than EV71: TLLmv
[0098] Parent EV71: BS and the resulting NIH / 3T3 compatible EV71: TLLm and EV71: TLLmv strain-infected Vero and NIH / 3T3 cells were incubated at various temperatures, 30 ° C, 37 ° C, 39 ° C. , Viral compatibility with temperature fluctuations or changes was determined. EV71: BS showed the most limited compatibility and complete CPE was observed only in Vero cells incubated at 37 ° C (FIG. 8B; Table 1). EV71: TLLmv is medium based on the complete CPE induction observed in Vero cells at 37 ° C. (FIG. S2B) and in NIH / 3T3 cells at both 37 ° C. and 39 ° C. (FIGS. 8A, 9A; Table 1). The degree of suitability was shown. EV71: TLLm, on the other hand, showed the highest suitability, where complete CPE was induced in Vero cells for all incubation temperatures (FIGS. 7B, 8B, 9B), but in NIH / 3T3 cells. It was only at 37 ° C (Fig. 8A; Table 1).

Table 1
Assessment of virus compatibility of EV71: BS, EV71: TLLm and EV71: TLLmv grown in NIH / 3T3 and Vero cells at various incubation temperatures

¹ Infected cells were observed daily for signs of lytic cell death and time of appearance of complete CPE.
² Shows a lack of complete CPE in infected cells.

³ Observation of complete CPE is shown.
⁴ Indicates the lack of complete CPE, the numbers in parentheses indicate the maximum degree of CPE observed in the cell.

Example 7
The viral genomes of EV71: TLLm and EV71: TLLmv accumulated a large number of missense mutations as a result of adaptation to NIH / 3T3 cells.
[0099] EV71: BS, EV71: TLLm, EV71: TLLmv viral RNAs were subjected to Sanger sequencing to determine consensus genomic sequences and identified possible matching mutations resulting from matching processes in NIH / 3T3 cells. .. Genome consensus sequences representing the dominant population of pseudospecies have been deposited with GenBank, NCBI (National Center for Biotechnology Information). Alignment of the entire genome sequence of EV71: TLLm (GenBank deposit number KF514879; SEQ ID NO: 1) with respect to EV71: BS (GenBank deposit number KF514878; SEQ ID NO: 3) revealed 60 nucleotide mutations, 21 of which were amino acids. Substitution occurred (Table 2). On the other hand, 83 mutations with 36 amino acid substitutions were found between the genomes of EV71: TLLmv (Genbank Deposit No. KF514880; SEQ ID NO: 2) and EV71: BS. The majority of missense mutations were located in the P1 (capsid protein gene) region (Table 2), especially within the VP protein gene (Table 3).

[00100] Within the P2 and P3 regions, most notably, amino acid changes were also observed with RNA-dependent RNA polymerase (3D region). EV71: TLLm acquired four amino acid changes, most of which were in the palm and thumb domains of the enzyme (Table 4). On the other hand, EV71: TLLmv accumulated eight amino acid changes, which were also mostly in the palm and thumb domains of the polymerase. Nucleotide mutations were also observed in the 5'untranslated region (UTR) of the genome. Apart from the base change, 1 base insertion was observed in EV71: TLLm, while 4bp insertion and 20bp deletion were observed in EV71: TLLmv 5'UTR (Tables 2 and 3). On the other hand, no amino acid substitutions were observed in the VP3 and 3A regions of EV71: TLLm, and no 3'UTRs of both EV71: TLLm and EV71: TLLmv.

Table 2
Nucleotide and amino acid changes in the genome of EV71: TLLm and EV71: TLLmv compared to EV71: BS

Table 3
Compatible mutations observed in the 5'UTR and P1 regions of the EV71: TLLm and EV71: TLLmv viral genomes

¹ Amino acid numbering in uncut polyproteins before maturation.
² Amino acid numbering in mature proteins
³ Internal ribosome entry site.
Mutations are based on alignment with reference EV71: BS genome.

Table 4
Compatible mutations observed in the P2 and P3 regions of the EV71: TLLm and EV71: TLLmv viral genomes

¹ Amino acid numbering in uncut polyproteins before maturation.
² Amino acid numbering in mature proteins
³ Mutation located in the ring finger domain of RNA-dependent RNA polymerase.

⁴ Mutations located in the palm domain of RNA-dependent RNA polymerase.
⁵ Mutation located in the thumb domain of RNA-dependent RNA polymerase.
Mutations are based on alignment with reference EV71: BS genome.

Example 8
Transfection of EV71: BS viral RNA into murine cells resulted in proliferative infection, but viral progeny were unable to reinfect the same mouse cells.
[0100] Vero and NIH / 3T3 cells transfected with viral RNA showed complete CPE 7 days after transfection (dpt) (data not presented). Viral antigens were detected in NIH / 3T3 cells transfected with EV71: BS viral RNA (FIG. 10B), but not in NIH / 3T3 cells infected with the virus (FIG. 10A). .. Virus supernatants re-inoculated on fresh Vero and NIH / 3T3 cells produced proliferative infections (100% CPE) only in Vero cells, but not in NIH / 3T3 cells (Fig. 6A), and viruses Antigen detection confirmed infection in Vero cells, but not in NIH / 3T3 cells (FIG. 6B).

Example 9
Materials and Methods of Examples 10-17
[0101] plasmids, viruses, bacteria, and cell lines: A plasmid encoding the murine SCARB2 cDNA (pMD18-mSCARB2) (Genbank Deposit No. NP_031670.1) is available from Sino Biological, Inc. Purchased from (Beijing, China). The pQE30 vector (Qiagen, Germany) for recombinant expression of soluble mSCARB2 protein in E. coli cells was a generous gift from Dr. Kian Hong Ng (Temasek Lifesciences Laboratory, Singapore). A low copy number plasmid pACYC177 (New England Biolabs, Singapore) was used to generate a plasmid encoding the full-length cDNA of EV71. The plasmid construct expressing T7 polymerase (pCMV-T7pol) was a generous gift from Dr. Peter McMinn of the University of Sydney, New South Wales. The plasmid pZero-2 used for fragment sequencing of the clone-derived virus was purchased from Invitrogen (Life Technologies, USA).

The clinical isolates EV71: BS (Genbank deposit number KF514878), EV71: TLLm (Genbank deposit number KF5148799.1), and EV71: TLLmv (Genbank deposit number KF514880.1) used in this study are described above. Or previously described [71].

[0103] All cell lines used in this study, African green monkey kidney Vero (CCL-81); mouse neuroblastoma Neuro-2a (CCL-131), and fibroblast NIH / 3T3 (CRL-1658) cells. , Purchased from the American Tissue Culture Collection (ATCC®, USA). Cells were grown and maintained as described above or as previously described [71].

[0104] E. coli cell BL21 strain (New England Biolabs, Singapore) was used for high-level protein expression, TOP10 strain (Life Technologies, USA) was used for fragment sequencing of individual clones, and XL-10 Gold. A supercompetent strain (Stratagene, USA) was used for the generation of full-length genomic cDNA clones.

Construction of EV71: BS full-length genomic cDNA clones, capsid chimeric clones, and VP1 / VP2 mutant clones: EV71: BS cDNA clones were generated by two-step cloning. Viral RNA extraction (Qiagen viral RNA kit, Germany) and conversion to cDNA (Life Technologies Superscript-II RT, USA) have been described above or previously [71]. Genome proximal fragments encoding the 5'UTR and P1 regions were amplified using primer pairs: EV71_BamHI-PfF and EV71_Pf-AatIIR (Table 5), and these primers were used for cloning into the plasmid pACYC177. Contains AatII restriction sites. Distal fragments encoding P2, P3, and 3'UTR are amplified with primer pairs EV71_HindIII-DF and EV71_D-BamHIR, and these pairs also contain HindIII and BamHI restriction sites for cloning. The proximal fragment contains a T7 polymerase promoter region upstream of the 5'UTR to facilitate transcription. After digestion with EagI and AatII, the proximal fragment was ligated to the distal fragment and a full length EV71: BS clone was produced.

Table 5
Primer

[0106] To replace the P1 region of EV71: BS with P1 of EV71: TLLm, the MluI restriction site was manipulated within the boundary between 5'UTR and P1 (primer vs. SDM_MluIF and SDM_MluI-R). EV71: The P1 cDNA sequence of TLLm was amplified (primer vs. MluI-TLLm-P1F and EagI-TLLm-P1R), digested with MluI and EagI, and cloned into a construct containing a proximal fragment. This modified proximal fragment was subsequently ligated to the distal fragment as described.

[0107] In order to generate clones containing amino acid substitutions in VP2 and VP1 proteins, site-specific mutagenesis was performed in the proximal fragment prior to ligation to the distal fragment. A VP2 S144T (nt G1385C) amino substitution was introduced into the proximal fragment containing the specific primer pairs VP2_G1385C-F and VP2_G1385C-R. VP2 S144T (nt G1385C), VP1 K98E (nt A2734G), E145A (nt A2876C), and E167E (nt C2497T) were also produced using different primer pairs in a similar manner (Table 5).

[0108] Assessment of "mouse cell entry phenotype": cDNA clones into Vero cells using Lipofectamine 2000 (Life Technologies, USA) according to the protocol recommended by the manufacturer to generate live virus. It was co-transfected with another construct (pCMV-T7pol) expressing T7 RNA polymerase. Transfection supernatants were collected 7-10 days after transfection and inoculated with 1 MOI on cell lines inoculated overnight, as described above or as previously described [71]. Cells were incubated with virus supernatant for 1 hour at 37 ° C., washed twice with PBS and then replaced with fresh DMEM, medium containing 1% FBS.

[0109] To assess the infectious phenotype, progression of lytic cytopathic effect (CPE) induction was recorded and images were taken at various time points. Infected cells were also harvested and processed for immunofluorescence detection of viral proteins as described above or as previously described [71]. Briefly, adherent cells were trypsinized and pelleted cells from the culture supernatant were combined, washed twice in sterile phosphate buffered saline (PBS), and fixed on a Teflon slide. Fixatives were incubated with a pan-enterovirus monoclonal antibody (Merck Millipore, USA) and detected with standard FITC-conjugated anti-mouse IgG antibody. Infected cell culture supernatants were also collected, clarified and subjected to serial dilution for viral titering using the Red and Muench method [61]. Once the titers were known, the supernatant was passaged on freshly planted NIH / 3T3 and Neuro-2a cells at 1 MOI, and the infectious phenotype was reassessed using the methods described herein.

[0110] Recombinant protein expression of soluble SCARB2 protein and production of SCARB2 rabbit antiserum: plasmid pMD18-mSCARB2 encoding the extracellular domain of mouse SCARB2 (aa Arg27-Thr432), primer vs. pQE-mSCARB2F and pQE-mSCARB2R Amplified with, BamHI and HindIII restriction sites were introduced to facilitate cloning into the pQE30 protein expression vector. Clones were transformed into BL21 E. coli cells and 1 mM IPTG overnight induced protein expression. The harvested cells were digested with lysozyme (1 mg / ml) and the unpurified extract was purified using a Ni-NTA column (Qiagen®, Germany). The clarified lysate was incubated overnight at 4 ° C. in 1 ml of 50% Ni-NTA slurry with gentle shaking. The protein was washed 5 times in wash buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0) and in elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0). Eluted with.

[0111] Two healthy male rabbits were immunized on day 0 with 1.4 μg of purified mouse SCARB2 protein mixed with Freund's complete adjuvant (Sigma-Aldrich®). Boosters containing 0.8 μg antigen mixed with Freund's incomplete adjuvant (Sigma-Aldrich®) were injected on days 21, 42, 63, 84, and 105. .. Final bleeding by cardiac puncture was performed on day 117, and the collected blood was incubated overnight at 4 ° C. and then centrifuged at 3,000 rpm for 30 minutes. Cleared serum was collected and stored at −20 ° C. for further use. The production of SCRAB2 rabbit antiserum was approved by the Temasek Lifescients Laboratory Institutional Animal Care and Use Committee (TLL-IACUC) [Authorization No. 047/17].

[0112] Viral competition assay with murine SCARB2 protein: In vitro binding assay was performed to confirm the interaction of mouse and human SCARB2 protein with EV71: TLLmv. NIH / 3T3 cells (6000 per well) were planted overnight on sterile Teflon-coated slides (Erie, USA). Prior to virus inoculation, 100 MOI EV71: TLLmv in various concentrations of recombinant mouse SCARB2 (mSCARB2) or human SCARB2 (hSCARB2) protein (4.0 μg, 2.0 μg, 1.0 μg, 0.5 μg, 0.25 μg, 0) .125 μg, and 0 μg) and incubated at 37 ° C. for 2 hours in a shaking platform. Infected cells were observed daily for signs of CPE and fixed in anhydrous acetone 48 hours after infection (4 ° C., 10 minutes). Immobilized cells were immunofluorescently assayed with pan-enterovirus antibody (Merck Millipore®, USA). The slides were imaged with an upright fluorescence microscope (Nikon, Japan).

[0113] Virus-SCARB2 Binding Assay: An antibody-mediated SCARB2 blocking assay was performed on fixed cells to assess whether masking the cell surface SCARB2 protein affected virus binding. NIH / 3T3 and Vero cells cultured on Teflon slides were fixed (4% PFA, 25 minutes, room temperature) and blocked with 5% BSA in PBS for 1 hour at 37 ° C. The slides were incubated in polyclonal rabbit serum (1: 100) prepared for mSCARB2 at 37 ° C. for 1 hour. Cells were incubated with polyclonal rabbit sera prepared against Safford virus L protein for negative control. After washing the slides in PBS, they were incubated with raw EV71: TLLmv (1000MOI) at 37 ° C. for 1 hour and explored with pan-enterovirus antibody and detected with FITC conjugated antibody. The slides were imaged with a Zeiss LSM 510 Meta inverted confocal laser scanning microscope (Zeiss, Germany) and fluorescence intensity was measured using Imalis (BitPlane Scientific Software, Germany) imaging software. A statistical analysis of the difference in fluorescence intensity was performed using Prism GraphPad version 6.01 (GraphPad Software, Inc., USA).

Cell defense assay from viral infections with rabbit anti-SCARB2 polyclonal sera: An antibody-mediated SCARB2 blocking assay was also performed on living cells to assess their effect on cell infections. (In 96-well plates, 1x10 ⁴ cells per well) NIH / 3T3 cells were seeded overnight, two-fold serial dilutions of the rabbit polyclonal sera generated against either mouse or human SCARB2 protein (1: 20 to 1: 640) and 37 ° C. for 1 hour incubation. Cells were subsequently inoculated with 100 MOI EV71: TLLmv or clone-derived virus mutant CDV: BS [MP1] and CDV: BS _VP1 [K98E / E145A / L169F] at 37 ° C. for 1 hour. The cells were washed twice with PBS and then replaced with fresh DMEM (1% FBS). Cells were observed daily for signs of CPE, and infected cell culture supernatants were harvested 3 days after infection (dpi). The supernatant was subjected to virus titer determination after a virus deagglomeration process by vigorous vortexing at room temperature for 15 minutes in [6,71], 1% sodium deoxycholate as previously. Virus titers were counted by the Red and Muench method [61] and reported on the infection calculator as CCID ₅₀ / ml [62].

Example 10
Transfection of EV71: BS genomic RNA into mouse neuronal Neuro-2a and fibroblast NIH / 3T3 cells yields viable viral progeny
[0110] Mouse fibroblasts NIH / 3T3 and neuroblastoma Neuro-2a cells were previously demonstrated to be intolerant to EV71: BS infection, while Vero cells were tolerated (Vero cells). Above or [71]). Two strains, both derived from EV71: BS, EV71: TLLm and EV71: TLLmv, successfully entered and replicated in these murine cells. To determine if the EV71: BS genome is replicable in these non-tolerant cells, EV71: BS-derived genomic RNA is extracted and transfected into Vero, NIH / 3T3, and Neuro-2a cells. Ection. Similarly, genomic RNAs derived from EV71: TLLm and EV71: TLLmv were transfected into these three cell lines for comparison. Transfection supernatants were subsequently re-inoculated onto fresh cells to assess the viability of the resulting virus offspring (FIG. 15A).

[0111] Genomic RNA transfection of all three viral strains into Vero cells produced a lytic cytopathic effect (CPE) in the transfected cell monolayer (FIG. 15B). Viral antigen expression was also observed in dead cells (Fig. 15C), indicating successful viral replication. Similar results were observed from transfection of either EV71: TLLm or EV71: TLLmv viral RNA into NIH / 3T3 and Neuro-2a cell monolayers (FIGS. 15B and 15C). On the other hand, transfection of EV71: BS RNA into NIH / 3T3 and Neuro-2a cells led to the death of some cells exhibiting viral antigen expression, but did not result in complete CPE of the cell monolayer. Further passage of EV71: BS transfection supernatants from both NIH / 3T3 and Neuro-2a cells into fresh Vero cells induces complete lysis of the cell monolayer (Fig. 15D) and viral antigens in dead cells. (Fig. 15E) was derived. However, passage of transfection supernatants on fresh NIH / 3T3 cells did not result in either CPE (FIG. 15D) or viral antigen expression (FIG. 15E).

Example 11
Capsid-coded P1 region of mouse cell line-matched EV71: TLLm is involved in successful viral entry into murine NIH / 3T3 and Neuro-2a cells
Previous analysis suggests that EV71: TLLm and EV71: TLLmv capsid proteins may be involved in successful entry into NIH / 3T3 and Neuro-2a cells (above or [71]). To confirm this, a full-length cDNA clone of the EV71: BS genome was generated by standard reverse genetics. The P1 (capsid) region of the EV71: BS full-length cDNA clone was subsequently replaced with the gene sequence of EV71: TLLm P1 to generate a chimeric plasmid clone (FIG. 16A). EV71: BS cDNA clones were transfected into Vero cells to generate clone-derived virus (CDV: BS). Similarly, chimeric clones were transfected to produce CDV: BS [MP1], which represents a capsid protein of EV71: TLLm and expresses a non-structural protein of EV71: BS. These CDVs were re-inoculated into various cell lines and evaluated for infectious phenotype (Fig. 16B).

[0113] EV71: BS clone-derived virus (CDV: BS) induced CPE in Vero cells but not in NIH / 3T3 and Neuro-2a cells, while CDV: BS [MP1] 48 hours after infection (hpi), CPE was induced in all three cell lines (Fig. 16C). Mice cells infected with CDV: BS [MP1] produced detection of viral antigens in dead cells, but not in CDV: BS (FIG. 16D). To assess the production and release of viable virus progeny in the first passage (P1), clear culture supernatants from infected cells were re-inoculated onto a fresh monolayer of the same cell line and at 72 hpi. The virus yield was measured. Passage on Vero cells of both CDV: BS and CDV: BS [MP1] showed high viral titers, and a significant increase in titers was observed after further passage (P2) (Figure). 16E). On the other hand, passage (P1) of CDV: BS [M-P1] produced high virus yields in both NIH / 3T3 and Neuro-2a cells, whereas CDV: BS did not (FIG. 16F).

Example 12
EV71: VP1-L169F amino acid substitution in BS capsid allows the virus to enter murine cells and induce limited infection
[0114] The capsid protein of mouse cell-matched EV71: TLLm allows the entry of EV71: BS into murine cells, and we are interested in the identity of specific residues that confer this novel phenotype. I had it. EV71: Previous data on comparison of polyprotein sequence alignment of BS and mouse cell-matched EV71 strains showed numerous amino acid substitutions in VP1 and VP2 proteins that may be involved in viral receptor binding on host cells ( Above or [71]). These residues include VP1 K98E, E145A, and L169F, as well as VP2 S144T and K149I. To determine which of these amino acid substitutions are required for mouse cell entry, the individual mutations that result in these amino acid substitutions are placed in the EV71: BS full length cDNA clone through standard site-specific mutagenesis. Introduced (Fig. 17A). These modified cDNA clones were independently transfected into Vero cells to generate clone-derived virus (CDV), and the collected supernatants were placed on freshly planted Vero, NIH / 3T3, and Neuro-2a cells. Inoculated and evaluated for infectious phenotype.

[0115] Vero cells infected with all mutant clone-derived viruses (CDV) showed 100% CPE, but CDV, CDV: BS _VP1 [K98E], CDV: BS _VP1 [E145A] harboring a VP1 amino acid substitution. ], And CDV: BS _VP1 [L169F] alone produced 100% CPE in Neuro-2a cells (FIGS. 17B and 17C). Viral antigen expression was detected in Vero and Neuro-2a cells infected with CDV containing amino acid substitutions in VP1 (CDV: BS _VP1 ) and VP2 (CDV: BS _VP2 ), but infected with CDV: BS _VP2 . Only NIH / 3T3 cells showed viral antigen expression (FIGS. 17D and 17E). In addition, all mutant CDV-infected Vero cells produced measurable viral titers (Fig. 17F), suggesting viral viability, but only CDV: BS _VP1 [L169F] was assayed in Vero cells. Produced measurable viral titers in culture supernatants of infected NIH / 3T3 and Neuro-2a cells (FIG. 17G). However, further passage of culture supernatants from NIH / 3T3 and Neuro-2a cells infected with CDV: BS _VP1 [L169F] on healthy murine cells failed to induce infection.

Example 13
Efficient proliferative infection in murine cells requires combinatorial amino acid substitutions at VP1
[0116] Full-length genomic cDNA of EV71: BS containing various combinations of amino acid substitutions to assess whether the combination of amino acid substitutions in VP1 and VP2 is also capable of allowing EV71: BS to enter mouse cells. Clone (BS _VP2 [S144T / K149I], BS _VP [K98E / E145A], BS _VP1 [K98E / E145A / L169F], and BS [VP1 / VP2]) were generated (FIG. 18A). The plasmid clones were independently transfected onto Vero cells and the resulting supernatant was inoculated into Vero, NIH / 3T3, and Neuro-2a cells to assess the infection phenotype.

[0117] All assayed CDVs induced CPE in Vero and Neuro-2a cells except CDV: BS _VP2 [S144T / K149I] (FIG. 18B). Viral antigens were also detected in all cells infected with various CDVs, but immunostaining was more pronounced in Neuro-2a than in NIH / 3T3 cells when comparing murine cell lines. (Fig. 18C). High viral titers were measurable in the culture supernatants of all CDV-infected Vero cells (Fig. 18D), but CDV: BS _VP1 [K98E / E145A] and CDV: BS _VP1 [K98E / E145A / L169F]. ] Produced measurable viral titers in culture supernatants of infected NIH / 3T3 and Neuro-2a cells when assayed in Vero cells (FIG. 18E).

Example 14
EV71: BS virus containing a combination of amino acid substitutions of VP1 K98E, E145A, and L169F was successfully passaged in mouse Neuro-2a cells.
[0118] Four of the clones derived from a virus _{isolate: CDV: BS [M-P1} ], CDV: BS VP1 [L169F], CDV: BS VP1 [K98E / E145A], and _CDV: BS VP1 [K98E / E145A / L169F] has so far allowed EV71: BS to enter and infect cultured mouse cell lines. To identify which of these CDVs can stably infect mouse cells for multiple cycles, passage the virus supernatant twice in the same cell line, ie from Neuro-2a to fresh Neuro-2a cells. did. Infection was monitored by CPE induction and viral antigen expression, as well as evaluation of viable viral offspring production.

[0119] CDV: BS _VP1 [K98E / E145A / L169F] and CDV: BS [MP1] only, as evidenced by the detection of viral antigens (FIG. 19A) and measurable viral titers (FIG. 19B). Was able to succeed in successive passages in Neuro-2a cells. Positive staining was observed at both the second and third passages, and an increase in virus titer was recorded at the second passage compared to the first passage. On the other hand, CDV: BS [MP1] was able to induce the expression of viral antigens in NIH / 3T3 cells (Fig. 19A), but no viable viral offspring were detected. The genomic sequence of CDV: BS _VP1 [K98E / E145A / L169F] obtained from the third passage in Neuro-2a cells showed changes in the introduced amino acid mutations (FIG. 19C).

Example 15
Mouse cell line compatible strain EV71: TLLmv binds to the SCARB2 protein both in vivo and in vitro
[0120] EV71 has recently been demonstrated to utilize the scavenger receptor class B member 2 (SCARB2) protein as its receptor for host cell entry [47]. A competitive virus binding assay was also performed to determine if the mouse cell line compatible EV71 strain utilizes SCARB2 during mouse cell infection. First, NIH / 3T3 and Vero cells grown overnight in Teflon-coated slides and gently fixed with 4% paraformaldehyde were incubated with rabbit serum against mouse SCARB2 protein (mSCARB2) and then live in vitro. EV71: Combined with TLLmv. Fluorescent detection of bound cells was performed using a pan-enterovirus monoclonal antibody and the fluorescence intensity was quantified. Preincubation of NIH / 3T3 cells with anti-mSCARB2 serum resulted in a significant reduction in EV71: TLLmv binding (FIG. 20A), which was not observed when the cells were preincubated with nonspecific serum (NSP). It was. Similar results were observed when using Vero cells (Fig. 20B).

[0121] In a second experiment, live EV71: TLLmv was incubated with recombinant soluble SCARB2 protein, then inoculated onto planted NIH / 3T3 cells and infected by fluorescent tagging of bound pan-enterovirus monoclonal antibody. Was evaluated. Preincubation of soluble mSCARB2 with the virus reduced the severity of cell infection in a dose-dependent manner (Fig. 20C). Similar results were obtained when the human SCARB2 (hSCARB2) protein was used in the preincubation (Fig. 20D).

Example 16
Preincubation of mouse cells with serum prepared for the SCARB2 protein blocks cell infection with EV71: TLLmv
[0122] EV71: Inoculated NIH / 3T3 cells with various dilutions of SCARB2 protein antisera to assess whether cell infection is reduced by blocking the interaction between TLLmv and SCARB2. Infection was assessed by inoculating live EV71: TLLmv and measuring the titer of live virus progeny. Pre-incubation of cells with a low dilution of hSCARB2 antiserum (1:20 to 1:80) resulted in a significant dose-dependent reduction in viral titers compared to controls (FIG. 20E). Similar results were obtained when cells were preincubated with mSCARB2 antiserum (Fig. 20F).

Example 17
CDV: BS [MP1] and CDV: BS _VP1 [K98E / E145A / L169F] utilize the murine SCARB2 protein as a functional receptor for entry into murine cells.
Both [0123] CDV: BS [MP1] and CDV: BS _VP1 [K98E / E145A / L169F] stably infect mouse Neuro-2a cells. These CDVs, like the mouse cell-matched EV71: TLLmv strain, are also CDVs after incubation of Neuro-2a cells with mSCARB2 antiserum to determine whether to utilize mSCARB2 for virus entry and decapitation. Infected with mutants. A dose-dependent decrease in soluble CPE was observed in cells infected with either CDV: BS _VP1 [K98E / E145A / L169F] (FIG. 21A) or CDV: BS [MP1] (FIG. 21B). .. The titer determination of the culture supernatant 7 days after infection (dpi) was a significant difference in CDV: BS _VP1 [K98E / E145A / L169F] virus titers in cells pre-incubated with SCARB2 antiserum compared to controls. Will not reveal. On the other hand, a significant decrease in virus titer was detected in CDV: BS [MP1] infected cells preincubated with serum as compared to controls (Fig. 21D).

Example 18
Materials and methods of Example 19
[0124] Animal models: mouse cell adapted strain (EV71: TLLm and EV71: TLLmv) for determining the infected animal phenotype, were infected with 5-6 day old Balb / c mice with the virus of ¹⁰ 6 CCID _50, Then we observed the symptoms of the disease and neurological complications. Animals were followed for up to 28 days, after which the animals were sacrificed and serum was collected for detection of EV71-specific antibodies.

Example 19
Neuropathogenicity studies of mouse cell line-matched EV71s (EV71: TLLm and EV71: TLLmv) in Balb / c mice
[0125] Of the immunoeligible animals infected with EV71: TLLm (n = 7), two (29%) died of severe and persistent (> 24 hours) paralysis 8 days after infection (FIG. 13A). Of the other surviving mice, 5 (5/7, 71.4%) showed tremor and ataxia, 5 showed paresis in one or both hind limbs, and 4 (4/7,) 57.1%) showed temporary paralysis in either one or both hind limbs. Animals infected with EV71: TLLmv, on the other hand, showed more severe clinical signs of the disease. Nine of the ten infected animals (90%) succumbed to the disease within eight days of infection (Fig. 13A), and six of the nine (66.7%) died within four days of infection. .. Other symptoms presented include tremor (5/10, 50%), paralysis of either one or both hind limbs (6/10, 60%), and paralysis of either one or both hind limbs (5). / 10, 50%) is included. There seems to be no difference in body weight of mice infected with EV71: TLLm compared to sham-infected animals, but mice infected with EV71: TLLmv have a dramatic weight within the first 10 days of infection. It showed a decrease (Fig. 13B). More interestingly, we observed novel symptoms in EV71-infected mice, whereby paralyzed animals (FIG. 14A, arrow) presented tachypnea with marked subcostal retraction. In addition, 6 of 9 (66.7%) dead animals presented this condition prior to euthanasia. Upon autopsy, we also observed that the lungs were incapable of crushing upon opening of the chest cavity, which is the usual procedure for autopsy when collecting lung and heart tissue (FIG. 14B, arrow). It was suggested that edema was present. Histological examination of the tissue reveals the characteristics of pulmonary edema and bleeding in the alveolar space of the lung (Fig. 14C), and higher magnification images indicate the presence of hemorrhagic proteinaceous material (Fig. 14D, Arrow).

[0126] The present embodiment was also carried out using immunocompromised mice, for example, NSG mice. Similar results were obtained, except that the severity of the disease was higher and the mortality rate was higher.
Example 20
Screening for candidate anti-EV71 compounds
High-throughput in vitro screening of candidate anti-EV71 compounds is performed using mouse NIH / 3T3 cell lines that have been shown to be susceptible to cytolytic infections by EV71: TLLm or EV71: TLLmv virus lines. Promising compounds selected from in vitro screening are then tested in vivo in animal models. Standardized (statistical) numbers of BALB / c mice were prepared, titrated, and maintained in a deep freezer (-80 ° C) to achieve in vivo screening. From a standardized stock of compatible EV71 strains (EV71: TLLm and EV71: TLLmv), the virus strain is infected with a standardized titer. Candidate anti-EV71 compounds vary, either before the onset of the disease, to assay the potential therapeutic effect of the candidate compound, or after the onset of the disease, to assay the potential therapeutic effect of the candidate compound. Administer to infected mice at standardized dosages.

Example 21
Materials and Methods of Examples 22-25
[0128] Mouse and Virus Strains: Adult BALB / c mice were purchased from InVivos (Singapore) and mated to give offspring. The EV71 strain used for inoculation includes EV71: BS, EV71: TLLm, and EV71: TLLmv, the details and characteristics of which have been described herein.

[0129] Ethical Statement: Animal treatment has been approved by the Temasek Lifesciences Laboratory Institutional Animal Care and Use Committee (IACUC) (Authorization No. TLL-14-023). The dying infected animals are I.I. P. Euthanasia was performed by injecting 90 mg / kg pentobarbital through the route. Neurological examination was performed according to the guidelines and standard procedures set by the University of California, San Francisco Facility Animal Care and Use Committee (IACUC). Euthanasia criteria included previously established guidelines [34]: (1) loss of> 20% of maximum recorded body weight, (2)> 48 hours of paralysis, (3) eating deficiency or Consciousness alterations presented as either inability to eat, (4) inability to maintain posture, and (5) stupor or coma. The pups were observed for a total of 28 days, and the animals that survived this period were described in Pent Barbiturates I. P. Euthanized by injection.

[0130] Animal handling and infection: various age groups of 8 mice (6 days, 14 days, 21 days, or 28 days of age), EV71: TLLmv (dose ¹⁰ 6 CCID ₅₀₎ I. P. Or I. M. Inoculated by one of the injections. EV71: To determine the optimum dose of TLLmv, the group of 6-day-old pups various doses of viruses (8 mice per group) ^{(10 6,} 10 ^5, 10 4, 10 ³ or ¹⁰ 2 CCID _50,) , I. P. Exposed through the route. Infected animals were observed twice daily for disease presentation during the first week after infection. Both dying animals and animals that survived the observation period were euthanized as described above. Terminal blood collection was performed through cardiac puncture using a 26G needle.

[0131] Autopsy, gross pathological observation, and tissue collection: Euthanized animals were necropsied and organs harvested using standard protocols. Gross pathological examinations were also performed and photographs were taken with IACUC approval. The lung surface was flushed twice with sterile PBS and blot-dried with filter paper before wet weight measurement. Organs collected for histological studies were stored in 10% neutral buffered formalin (NBF) at 4 ° C. for 1 week.

[0132] Tissue virus loading determination: Frozen tissue was softened using a Teflon pestle and reconstituted in 1 ml DMEM (1% FBS). The samples were then mixed for 1 hour and centrifuged twice (20 g, 30 minutes, 4 ° C.) to remove tissue debris and obtain a clear virus. Virus samples were disaggregated in 1% sodium deoxycholate [88], then diluted 10-fold serially and transferred onto NIH / 3T3 cells. Infected cells were observed daily for cytopathic effect (CPE) and stained with pan-enterovirus monoclonal antibody (Merck Millipore, USA) [88]. Virus titers were counted and reported as CCID ₅₀ per g tissue.

Tissue processing for histological analysis: Fixative was dehydrated in increasing concentrations of 70%, 95% and 100% ethanol. Tissues were incubated during two exchanges of alcohol and three exchanges of Histoclear II (Electron Microscopic Sciences, USA), and finally infiltrated with four exchanges of molten paraffin wax. All incubations were at room temperature for 1 hour with gentle vibrations at 100 rpm. Paraffin infiltration was performed in an oven set at 65 ° C. Paraffin-embedded tissue blocks were sectioned (5 μm) using a microtome, loaded onto polylysine-coated glass slides, dried overnight at 42 ° C., and then stored at room temperature for further use.

Staining of tissue sections: Tissue sections are dewaxed by incubation in two exchanges of Histoclear II and then slowly dehydrated in reduced alcohol concentrations of 100%, 95%, 70%, and 50%. did. The slides were incubated in PBS for 10 minutes before staining. Hematoxylin and eosin (H & E) staining was performed by first immersing the slides in Harris hematoxylin (Sigma Aldrich, USA) and then incubating at room temperature (RT) for 15 minutes. The slides are then rinsed in water, destained with 1% acid alcohol (95% ethanol, 1% HCl), soaked in 0.2% NH4OH, rinsed in water for 10 minutes and then compared in eosin solution. Stained. The slides were then destained in 95% ethanol and dehydrated by 3 exchanges of absolute alcohol and 2 exchanges of Histoclear II. Finally, the tissue was set in DPX mounting solution (Sigma Aldrich, USA).

Immunohistochemistry: After dewaxing and rehydration, slides are subjected to heat-induced antigen recovery by incubation in a histology-grade microwave oven and citrate buffer (pH 6.0) at 96 ° C. for 30 minutes. did. The slides were cooled to RT for 3 hours and then blocked with 5% normal porcine serum for 1 hour at RT. The slides were then incubated overnight at 4 ° C. in rabbit serum containing a polyclonal antibody against EV71 (NIID, a generous gift from Dr. Hiroyuki Shimizu, Japan) without further washing. The slides were then washed 5 times in Tris buffered saline (pH 7.4), 0.05% Tween-20 (TBS-T), and rinsed twice with TBS before 3% H ₂ O ₂ . Endogenous peroxidase was arrested by addition at RT for 1 hour. The slides were subsequently washed twice in TBS and then incubated with porcine anti-rabbit Ig-HRP (Dako Cytomation, Denmark) for 1 hour at RT. After washing, slides were incubated in a diaminobenzidine (DAB) substrate and counterstained with hematoxylin.

Mapping of viral antigens and virus-induced lesions in tissue sections: Template images of typical coronal sections of mouse brains are available at www. brainstars. Downloaded from org [89]. These images can be freely used and processed under license from Creative Commons Japan. Observed lesions and viral antigens were marked on the template image. A mouse brain atlas of coronary fragments (www.mouse.Brain-map.org/static/atlas) was used to identify affected brain regions [90]. Similarly, a representative coronal section of the thoracic spinal cord was used as a template for the spinal cord map. Regions showing the presence of viral antigens and virus-induced lesions were then marked on the template image.

Measurement of Serum Catecholamine Levels: Blood samples were collected by cardiac puncture of dying animals during autopsy. Serum is obtained by centrifuging 3000 g of blood clot sample at 4 ° C. for 30 minutes and then using a 2-CAT (AN) ELISA kit (Labor Diagnostika Nord, Germany) according to the manufacturer's protocol. , Stored at −20 ° C. until catecholamine levels were determined.

[0138] Statistical Analysis: Using GraphPad Prism (version 6.01) for Windows (GraphPad Software, USA, www.graphpad.com), all graphs were generated and statistical analysis was performed.

Comparison of infection phenotypes induced by viral strains EV71: TLLm and EV71: TLLmv: 106 CCID50 doses of EV71: BS, EV71: TLLm, or EV71: TLLmv in 6-day-old BALB / c mouse pups. Was inoculated via the intraperitoneal (IP) or intramuscular (IM) route (n = 10 mice per group). For the first week after vaccination, animals were observed twice daily for signs of disease and, as already described, euthanized when definitive signs were detected.

Measurement of Neutralizing Antibody Levels in Serum from Infected Mice: Serum by collecting blood samples by cardiac puncture at autopsy and centrifuging at 3000 g, 4 ° C. for 20 minutes before clotting at room temperature. Got Samples were stored at −20 ° C. until further analysis. Random samples of frozen stock were assayed for neutralizing antibody titers. 2-fold serial dilutions of serum (1:20 to 1: 1280) were prepared in 96-well plates and mixed with 100 CCID ₅₀ virus. After incubating the mixture at 37 ° C. for 1 hour, NIH / 3T3 cells (6,000 cells / well) were added. The plate was incubated at 37 ° C. for several days and observed for CPE for 4-10 days. Neutralizing antibody titers were determined using the Red and Muench methods (reported as units per ml serum).

Example 22
Infectious kinetics of modified EV71 strain in a murine host
[0141] Current animal models of enterovirus 71 (EV71) -induced neurological disease and pathology replicate only partially of human disease. We therefore clinically pathologically diseased using mouse cell (NIH / 3T3) compatible virus strains EV71: TLLm and EV71: TLLmv, which are proliferatively capable of infecting both rodent and primate cell lines. The aim was to develop an authenticity model [88]. First, the relative pathogenicity of EV71: TLLm and EV71: TLLmv compared to the parent strain EV71: BS was found in 1-week-old BALB / c mice infected with the virus through the intraperitoneal (IP) pathway. Evaluated by evaluating. Serum conversion was observed in all inoculated animals, but only mice infected with either EV71: TLLm or EV71: TLLmv progressed to lethal disease (FIGS. 22a and 22b). Similarly, when compatible strains were administered via the alternative intramuscular (IM) route, they were again associated with reduced host survival compared to parent strain EV71: BS (57% survival; FIG. 22c). I. P. And I. M. When the transmission routes were considered together, the median survival time after inoculation was 4 days after infection (DPI) for EV71: TLLmv infection and 7 DPI for EV71: TLLm infection (χ ² = 6.840; p = 0.089). Taken together, these data show that mouse cell-matched virus strain EV71: TLLmv is associated with the highest levels of lethality, and therefore used in all subsequent experiments.

[0142] Optimal virus doses, inoculation routes, and mouse ages used for further model development were then evaluated. First, EV71: TLLmv disease severity in infected mice are dependent on the virus dose, the lowest viability was observed in animals inoculated with 10 ⁶ median cell culture infective dose of (CCID ₅₀₎ (Fig. 23a ), and the median humane endpoint _{(HD 50)} is equivalent to 3.98x10 ³ CCID ₅₀ (Figure 23b) it was confirmed. The effects of animal age and transmission route on host survival were then evaluated; the survival curve of 1 week old mice injected with EV71: TLLmv was not significantly affected by the inoculation route (FIG. 23c). Regardless of the site of virus injection, younger animals showed consistently inferior survival than older animals (Fig. 23d). Only in older animals can it be possible to identify any effect of the transmission route on disease severity; 3-week-old mice have I. P. Although completely resistant to infection, some animals were described in I.I. M. Viral injections through the route did not survive (FIGS. 24a and 24b). Serum conversion was detected in all mice that survived the infection, including those that did not show any signs of disease (FIGS. 24c, 24d and 24e). These data demonstrated that EV71: TLLmv induces acute and severe infections that are lethal in mice aged 1-3 weeks. Of the conditions tested herein, be either the intraperitoneal or muscle tissue, in 1-week-old mice injected with virus doses of 10 ⁶ CCID _50, disease severity was greater.

Example 23
EV71: Disease progression in TLLmv-infected mice
[0143] EV71: The majority of 1-week-old mice inoculated with TLLmv succumbed to the disease and showed numerous clinical signs of neurological disease. By the time of euthanasia, infected animals exhibited either transient or persistent ataxia, local or general tremor, unstable gait, and limb paresis and paralysis. Based on clinical presentation, diseased animals could be easily classified into four groups (Table 6). Survivors included mice that did not appear moribund at any point during the 28-day observation period. Class I animals showed severe signs, including postural inability and either stupor or coma, after only 3-7 DPI. All mice in this group exhibited spastic limb insufficiency paralysis and / or paralysis (front limbs, hind limbs, or both), but some animals lacked respiratory symptoms (class 1B), others frequent. Further characterized by signs of dyspnea, including breathing, hiccups, gasping, and subcostal retraction (Class IA). Observation of prominent features in EV71: TLLmv-infected mice presenting with Class IA signs of disease was performed by video containing two video clips of two different Class IA mice. Both animals were unable to maintain posture and were in a coma. Severe dyspnea, presented as tachypnea with subcostal retraction, was evident in the first mice. Pant, sub-rib retraction, and foamy fluid coming out of the nostrils were seen in the second mouse. Observation of prominent features in EV71: TLLmv-infected mice presenting with Class IB signs of disease was performed by video of one Class IB mouse. The animal was unable to maintain its posture and was in a coma. Ipsilateral paralysis of the right limb and persistent tremor of the left hind limb were also observed. Finally, Class II mice showed signs of infection after 7 DPI, including severe weight loss (> 20% maximal body weight) and persistent flaccid paralysis of the extremities (> 48 hours period) (FIG. 23e). .. In all disease classes, some infected animals showed hairless lesions or back hair loss spots that persisted for several days (Fig. 23f). EV71: TLLmv I. P. The majority of inoculated pups are classified as Class I (Fig. 23 g); Class IA animals make up 19.3% (n = 11; obvious respiratory signs), while Class IB animals are 43.9. % (N = 25; unclear respiratory signs). Class II4 animals corresponded to only 12.3% (n = 7) of infected offspring, and survivors made up 24.5% (n = 14) of all infected mice. Similar patterns of distribution between disease categories are described in I.I. M. It was also observed in pups inoculated through the route (Fig. 23h). Taken together, these data show that EV71: TLLmv-infected mice show varying incidences and severity of both neurological and respiratory symptoms that reflect the full spectrum of disease observed in human patients. Indicated.

Table 6
Clinical features of EV71: TLLmv-infected BALB / c mice at the time of euthanasia

^a Animals were evaluated at the end of the observation period (28 DPI)
^{b The} animal was unable to maintain its posture but responded to physical stimuli on the toes.
^{c The} animal was unable to maintain its posture and did not respond to physical stimuli on the toes.

^d Tachycardia was observed in 35% of class IB mice Example 24
Neurogenic pulmonary edema in mice presenting with Class IA signs
[0144] We further investigated whether the dyspnea observed in class IA mice was a sign of virus-induced pulmonary edema (PE). Macroscopic lung pathology comparison between Class IA, Class IB, and Class II mice revealed that Class IA lungs were swollen, incompletely crushed at necropsy (FIGS. 25a-25d), and into lungs from other groups. In comparison, it was revealed to show increased wet weight (Fig. 22E). Comparison of sham inoculation (Fig. 25f) and lung tissue sections from class IA lungs reveals that only the alveolar space of infected animals has localized areas of bleeding and accumulation of proteinatic and erythrocyte-filled fluids. (Fig. 25 g). These pathological features were not present in the lungs of class IB and class II mice (FIGS. 25h and 25i). Moreover, we found no evidence of inflammatory infiltrates or viral antigens in lungs collected from any group of mice (Fig. 26a). Similarly, histochemical analysis of myocardium in each class of infected mice was also unable to find any evidence of inflammatory infiltrates, myocardial necrosis, or viral antigens (Fig. 26b). Taken together, these data demonstrated that apparent dyspnea in class IA mice could not result from either pneumonia or congestive heart failure. We therefore attempted to determine if the PE observed in this group was neurogenic, and therefore we then measured serum levels of catecholamines to determine the nerve. It was determined whether transmitter levels were regulated in EV71: TLLmv-infected mice (class IA) with respiratory signs. Using this approach, we found that blood levels of both adrenaline (epinephrine) and noradrenaline (norepinephrine) were significantly higher in class IA mice than were detected in either class II mice or sham-infected animals. Was observed to be higher (FIGS. 25j and 25k). These data strongly showed that Class IA mice exhibited EV71-induced neurogenic pulmonary edema (NPE) prior to death.

Example 25
EV71: TLLmv viral enterovirus in class IA, class IB, and class II mice
[0145] To identify factors that could contribute to the selective development of NPE in class IA mice alone, the distribution of EV71: TLLmv and virus-induced lesions in the brain and spinal cord of animals from each disease group was then evaluated. In the majority of Class IA and Class IB animals, ubiquitous staining of viral antigens (> 10 positive neurons were detected) and mild lesions (> 5 lesions were observed in all evaluated CNS regions. ) Was shown (Table 7). In contrast, only one of the five mice with Class II disease showed viral antigens and / or lesions in the sensory cortex, hippocampus, diencephalon, midbrain, medulla oblongata, or lumbar spinal cord. We were unable to detect viral antigens in any tissue tested outside the CNS (except for skeletal muscle after inoculation through the IM pathway; data not presented).

Table 7
CNS distribution of EV71 antigen and virus-induced lesions in end-stage infected BALB / c mice

Density of viral antigens detected per ^a- slide; +, <10 positive neurons; ++ 10-20 positive neurons; +++,> 20 positive neurons
^b Percentage of animals showing viral antigens in the specified brain region (n = 5)
^c Lesion distribution in nervous tissue: +, <5 lesions; ++ 5-10 lesions; +++,> 10 lesions
^d Percentage of animals showing lesions in the specified brain region (n = 5)
[0146] When comparing CNS pathology between class IA and class IB mice, both tissue lesions and viral antigens are localized in the same region within the hippocampus, diencephalon, midbrain, cerebellum, and medulla. However, the pathology was observed to be more severe in animals with class IA disease (Table 7 and FIGS. 27a-27d). In fact, when compared to class IB mice, class IA animals showed broader neurodegeneration, phagocytosis, and necrosis in hippocampal CA3 neurons (FIGS. 28a and 28b). Class IA mice also showed strong viral antigen staining in the hypothalamus, accompanied by marked tissue inflammation and neuronal necrosis, while these pathological features were limited in class IB animals (Fig. 28c and 28d and FIG. 27b). Similarly, class IA mice also have ventro-posterior complex of the thalamus (FIGS. 28e and 28f and 27b), periaqueductal gray (PAG), midbrain reticular region, and motor. Features of more severe virus-induced pathology in midbrain-related tissues (FIGS. 28g and 28h and FIG. 27c), including the associated superior colliculus, and in the purkinier cells and dentate nucleus of the cerebellum (FIGS. 28i and 28j, 27d and 29a). And the virus antigen intensity was presented.

[0147] In both disease groups (class IA and class IB), the widest distribution of viral antigens and lesions with neuronal damage and tissue inflammation is in the medulla oblongata (FIGS. 28k and 28l), especially the intermediate reticular nucleus (IRN). It was detected in the movement-related regions of the small cell reticular nucleus (PARN) and the spinal tract nucleus (sptV) of the trigeminal nerve (FIGS. 27d and 29b). However, only class IA mice showed viral antigens and tissue lesions in the ventral and dorsal regions of the medulla reticular nucleus (MdRN), the solitary tract nucleus (NTS) and the posterior area (AP) (FIGS. 29b and 29c). .. For comparison, representative images of the hippocampus, hypothalamus, thalamus, midbrain, cerebellum, and medulla from pseudoinfected mice are also shown (FIGS. 30a-30f). In contrast, Class IA and Class IB mice did not differ in distribution, localization or degree of tissue lesions or viral antigen staining within the anterior horn of the motor cortex, somatosensory cortex, pons or spinal gray matter (Fig. 28m and 28n, FIGS. 27a-27c and 31a-31e), consistent with the notion that NPE is caused by virus-induced damage to specific brain regions rather than a uniform increase in EV71-induced lesions throughout tissue. To do.

[0148] In the context of describing the present invention (particularly in the context of the following claims), the use of the terms "a" and "an" and "the" as well as similar reference controls is set forth herein. Or, unless clearly inconsistent by background, it shall be considered to include both the singular and the plural. The terms "include", "have", "include" and "include" shall be deemed to be open-end terms unless otherwise indicated (ie, include, but are not limited to). Means). The enumeration of the range of values herein is intended to serve as a simplification to individually refer to each distinct value within the range, unless otherwise indicated herein, and each distinct value is in the book. It is incorporated herein as if individually referred to in the specification. All methods described herein are feasible in any suitable order, unless otherwise indicated herein or are clearly inconsistent with background. The use of any example, or exemplary terminology (eg, "eg,") provided herein is merely intended to facilitate the understanding of the invention and, unless otherwise claimed, the scope of the invention. No restrictions are imposed on. None of the terms herein are considered to indicate that any non-claiming element is essential to the practice of the present invention.

[0149] Aspects of the invention are described herein, including optimal modes known to the inventors to carry out the invention. Modifications of these embodiments may be apparent to those skilled in the art by reading the above description. We anticipate that those skilled in the art will use these variants appropriately, and intend that the invention will be practiced differently than that specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter listed in the accompanying claims, as permitted by applicable law. In addition, any combination of the above elements in all possible variants is included in the invention unless otherwise indicated herein or as clearly contradicted by background.

List of references

Aspects of the Invention [Aspect 1] An animal model of enterovirus 71 (EV71) neural infection, comprising rodents infected with EV71 modified to infect rodents.
[Aspect 2] The animal model of claim 1, wherein the modified EV71 is a rodent cell line compatible EV71.
[Aspect 3] The animal model of claim 1 or 2, wherein the modified EV71 is a rodent cell line compatible EV71 of EV71: TLLmv.
[Aspect 4] EV71: The animal model of claim 3, wherein the TLLmv has been deposited at the Type Culture Collection China Center and has been assigned the deposit number CCTCC V201438.
[Aspect 5] The animal model of claim 1 or 2, wherein the modified EV71 is a rodent cell line compatible EV71 of EV71: TLLm.
[Aspect 6] The animal model of claim 5, wherein the EV71: TLLm has been deposited at the Type Culture Collection China Center and assigned the deposit number CCTCC V201437.
[Aspect 7] The animal model of claim 1, wherein the modified EV71 is an EV71 clone-derived virus (CDV) containing a mutation in VP1.
[Aspect 8] The animal model of claim 1 or 7, wherein the modified EV71 is EV71 CDV CDV: BS _VP1 [K98E / E145A / L169F].
[Aspect 9] An animal model according to any one of claims 1 to 6, wherein the rodent is immunoqualified.
[Aspect 10] The animal model of claim 9, wherein the rodent is a mouse.
[Aspect 11] The animal model of claim 10, wherein the rodent cell line is the mouse cell line NIH / 3T3.
[Aspect 12] The animal model of claim 7 or 8, wherein the rodent is immunoqualified.
[Aspect 13] The animal model of claim 12, wherein the rodent is a mouse.
[Aspect 14] The animal model of claim 13, wherein the rodental cell line is the mouse cell line NIH / 3T3 or the mouse cell line Neuro-2a.
[Aspect 15] The animal model of claim 1 or 2, wherein the rodent is immunoeligible.
[Aspect 16] The animal model of claim 15, wherein the rodent is a mouse.
[Aspect 17] The animal model of claim 16, wherein the mouse is a BALB / c mouse.
[Aspect 18] A method for screening an antiviral agent: a test group of animals and a control group of animals are provided, wherein the animals in each group are subjected to the animal model according to any one of claims 1 to 17. Includes; administer antiviral drug candidates to the test group; monitor disease progression in the test and control groups; compare disease progression in the test group to disease progression in the control group; and compare the test group to the control group The method comprising selecting an antiviral drug candidate that reduces disease progression in.
[Aspect 19] The method of claim 18, wherein the antiviral agent is first screened in a test rodent cell line infected with modified enterovirus 71 prior to screening in animals.
[Aspect 20] A method for screening an antiviral vaccine: a test group of animals and a control group of animals are provided, wherein each group of animals uses the animal model according to any one of claims 1 to 17. Includes; administers antiviral vaccine candidates to the test group; monitors disease progression in the test and control groups; compares disease progression in the test group to disease progression in the control group; and compares the test group to the control group The method comprising selecting an antiviral vaccine candidate that reduces disease progression in.
[Aspect 21] The method of claim 20, wherein the antiviral vaccine is first screened in a test rodent cell line infected with modified enterovirus 71 prior to screening in animals.
[Aspect 22] Rodents are infected with EV71 modified to infect rodents, and infected rodents are bred.
The method of preparing an animal model of claim 1, comprising the step of thereby preparing an animal model of EV71 neural infection.
[Aspect 23] The method of claim 22, wherein the age of the rodent at the time of infection is between about 1 week and about 4 weeks.
24. The method of claim 22 or 23, wherein the infected rodent is bred for up to about 4 weeks.
[Aspect 25] The median cell culture infective dose of about 10 ³ to about 10 ^7, used to infect rodent method of any one of claims 22 to 24.
[Aspect 26] The method according to any one of claims 22 to 25, wherein the modified EV71 is a rodent cell line-compatible EV71 of EV71: TLLmv.
[Aspect 27] EV71: The method of claim 26, wherein the TLLmv has been deposited at the Type Culture Collection China Center and assigned the deposit number CCTCC V201438.
[Aspect 28] The method according to any one of claims 22 to 25, wherein the modified EV71 is a rodent cell line compatible EV71 of EV71: TLLm.
29. The method of claim 28, wherein the EV71: TLLm has been deposited at the Type Culture Collection China Center and assigned the deposit number CCTCC V201437.
[Aspect 30] The method according to any one of claims 22 to 25, wherein the modified EV71 is an EV71 clone-derived virus (CDV) containing a mutation in VP1.
[Aspect 31] The method of claim 30, wherein the modified EV71 is an EV71 CDV CDV: BS _VP1 [K98E / E145A / L169F].
32. The method of any one of claims 22-31, wherein the rodent is a mouse.
[Aspect 33] The method of claim 32, wherein the mouse is a BALB / c mouse.

Claims (15)

An animal model of enterovirus 71 (EV71) neural infection, comprising mice up to 4 weeks of age, in which EV71 modified to infect mice was infected between 1 and 2 weeks of age.
The ^mice were modified EV71 of 105 ^{to 106} median cell culture infective dose between (CCID ₅₀₎ were infected by intraperitoneal injection or in muscle tissue,
The modified EV71 is EV71: TLLmv.
(A) is deposited with Type Culture Collection China Center, and either be assigned the accession number CCTCC V201438, or (b) by using an advanced reverse genetics including the synthesis of viral RNA by using the sequence of SEQ ID NO: 2 It has been recovered,
Animal models, acute encephalomyelitis, focal hemorrhage and proteinaceous liquid, high serum levels of catecholamines in the alveoli, and shows a thorough tissue loss scratches in the brainstem, they signs of neurogenic pulmonary edema And the symptom , said animal model. CCID ₅₀ is 10 ^6, an animal model of claim 1. The animal model of claim 1, wherein the mouse is immunoeligible. The animal model of claim 3, wherein the mouse is a BALB / c mouse. The animal model of claim 1, wherein the mouse is susceptible to infection. The animal model of claim 5, wherein the mouse is an NSG mouse. A method of screening for antiviral agents: a test group of animals and a control group of animals are provided, wherein the animals in each group include the animal model of any one of claims 1-6; test group. Administer antiviral drug candidates to the drug; monitor disease progression in the test and control groups; compare disease progression in the test group to disease progression in the control group; and compare disease progression in the test group compared to the control group. The method comprising selecting antiviral drug candidates to be reduced. The method of claim 7, wherein the antiviral agent is first screened in a test mouse cell line infected with modified enterovirus 71 prior to screening in animals. A method of screening for an effective antiviral vaccine: a test group of animals and a control group of animals are provided, wherein the animals in each group include the animal model of any one of claims 1-6; Antiviral vaccine candidates were administered to the test group; disease progression was monitored in the test and control groups; disease progression in the test group was compared to disease progression in the control group; and disease in the test group compared to the control group. The method comprising selecting an antiviral vaccine candidate that reduces progression. A method of preparing an animal model of enterovirus 71 (EV71) neural infection.
(I) Mice between 1 and 2 weeks of age were infected with EV71 modified to infect the mice, and (ii) infected mice were bred to 4 weeks of age.
Thereby, including the step of preparing an animal model of EV71 neural infection,
The ^mice were modified EV71 of 105 ^{to 106} median cell culture infective dose between (CCID ₅₀₎ were infected by intraperitoneal injection or in muscle tissue,
The modified EV71 is EV71: TLLmv.
(A) is deposited with Type Culture Collection China Center, and either be assigned the accession number CCTCC V201438, or (b) by using an advanced reverse genetics including the synthesis of viral RNA by using the sequence of SEQ ID NO: 2 It has been recovered,
Animal models, acute encephalomyelitis, focal hemorrhage and proteinaceous liquid, high serum levels of catecholamines in the alveoli, and shows a thorough tissue loss scratches in the brainstem, they signs of neurogenic pulmonary edema And the symptom , said method. CCID ₅₀ is 10 ^6, The method of claim 10. The method of claim 10 or 11, wherein the mouse is immunoqualified. 12. The method of claim 12, wherein the mouse is a BALB / c mouse. The method of claim 10 or 11, wherein the mouse is susceptible to infection. 14. The method of claim 14, wherein the mouse is an NSG mouse.