JP2018513671A - Enterovirus 71 animal model - Google Patents

Abstract

The present invention relates to enterovirus 71 (EV71), development of animal models, and screening of candidate anti-EV71 compounds. More specifically, the invention relates to an enterovirus 71 (EV71) strain that is adapted for rodent cell line infection, or a cloning-derived virus that contains a mutation in VP1 is an immunocompetent rodent. Relates to the discovery that it is possible to cause disease. [Selection] Figure 23

Description

Cross reference to related applications
[0001] This application is related 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, They claim priority. Each application is incorporated herein in its entirety.

Submission of sequence listing
[0002] This application has been filed with an electronic sequence listing. The sequence listing is 2577243 PCTS sequence Listing. entitled txt, created on December 29, 2015, and the size is 32 kb. The information in electronic form of the sequence listing is incorporated herein in its entirety.

[0003] The present invention relates to enterovirus 71 (EV71), development of animal models, and screening of candidate anti-EV71 compounds.
[0004] Publications and other materials used herein to elucidate the background of the invention, as well as examples providing more details regarding implementation, in particular, are incorporated herein and for convenience, the following: Are listed by author name and date, and listed in alphabetical order by author in the accompanying reference list.

  [0005] Enterovirus 71 (EV71) is a small non-enveloped virus approximately 30 nm in diameter. The viral capsid exhibits icosahedral symmetry and is composed of 60 identical units (protomers), each consisting of four viral structural proteins VP1-VP4. The capsid surrounds the core of a single-stranded positive-sense RNA genome with a length of 7,450 nucleotides (nt). The genome encodes a 2193 amino acid (aa) polyprotein and includes a 745 nt long 5 'untranslated region (UTR), and a 3' variable length poly A tract containing a shorter 3 'UTR. 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 nonstructural proteins 2A-2C and 3A-3D [1-3]. By phylogenetic analysis of the major capsid protein (VP1) gene, EV71 is divided into six genotypes (referred to as AF) [66], and genotypes B and C are further subgenotypes B1-B5. And subdivided into C1-C5 [3].

  [0006] EV71 causes a number of clinical diseases, mainly in infants and young children, including hand and foot disease (HFMD), aseptic meningitis, encephalitis and poliomyopathy-like paralysis [4, 5]. The virus was first isolated from a child with acute encephalitis in 1969 in California, USA, and was subsequently characterized in 1974 as a new serotype of the Enterovirus genus [6]. A pandemic of HFMD with or without neurological complications and death has been reported in various parts of the world [7-20]. Since 1997, EV71 infection has become a major public health burden and is a certain epidemiological concern in the Asia-Pacific region. A very neurotoxic HFMD pandemic due to EV71 occurred in Malaysia, resulting in 48 deaths in 1997 [21, 22], followed by a larger pandemic in Taiwan in 1998. In particular, HFMD exceeded 129,000 cases, 405 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 to date, including 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 and cynomolgus monkeys can be experimentally infected [30-32]. ]. This is mainly due to the inability of EV71 to efficiently bind to its receptor in order to unwrap and enter the mouse cell, which recently became a scavenger. 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 thus show significant structural differences [49, 67] and thus underlie the natural resistance of non-human cells to EV71 infection. Cause receptor incompatibility; When the virus successfully binds to the SCARB2 receptor on the cell surface and unwraps and enters the cytoplasm, the viral RNA is translated, resulting in the expression of various viral nonstructural proteins. Viral RNA is subsequently replicated, packaged in capsids, and released freely into the environment to re-infect healthy cells.

  [0008] Since EV71 is naturally unable to infect small rodents, there is no suitable small animal model, which has hindered understanding its pathogenesis and 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 serial passage in young suckling mice [33-39]. Some models were able to reproduce the symptoms of clinical illness, but none have been reported to cause disease in immunocompetent mice 2 weeks of age or older. Furthermore, the clinical features of disease and pathology of EV71 infection in humans and experimental monkeys were unable to replicate in mice, with the exception of readily infectious 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 a slight minimal improvement in susceptibility to EV71 infection. Only show.

  [0009] RNA viruses are replicated as a group of related mutant sequences known as pseudospecies [43, 44] because of their error-prone replication and high mutation rates [40-42]. It consists of a master spectrum showing the best fit in a particular environment and a mutation spectrum consisting of a closely related mutant sequence collection with a specific probability distribution [44, 45]. These impart genomic plasticity to RNA viruses, which is reflected in their ability to quickly adapt 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 that rhesus and cynomolgus monkeys [114-120], experimental mice [112, 121- 129], and experimental animals including other mammals [130-133] have been attempted to reproduce the pathology of human infection. Unfortunately, none of these models show the full spectrum of neurological features observed in human cases, and in particular can contribute to acute brainstem encephalitis with fulminant neurogenic pulmonary edema (NPE) Features are not shown [21, 22, 134-137]. Indeed, even in experimental systems that replicate more accurately 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 replicated EV71-induced NPE critically. An important difference is that EV71 is confined 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-neural tissues including Efforts have been made 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]. While these models have been made, none of these models show NPE, and thus their usefulness for identifying new interventions for human patients is limited.

  [0011] There are no suitable animal models 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), development of animal models, and screening of candidate anti-EV71 compounds. More specifically, the present invention relates to an enterovirus 71 (EV71) strain adapted for infection with a rodent cell line, or a cloning-derived virus containing a mutation in VP1, wherein the immunocompetent rodent and It relates to the discovery that it is possible to cause disease in readily infectious rodents.

  [0013] Furthermore, the present invention relates to an EV71-induced neurological disease by infecting BALB / c mice with a modified strain adapted to infect NIH / 3T3 mouse fibroblasts (eg EV71: TLLmv). It relates to the development of a clinical authentic model. Using this approach, modified EV71 is used to induce acute encephalomyelitis associated with neurogenic pulmonary edema, characterized by lung swelling and organ weight increase in mice compared to sham-infected lungs. Despite the absence of lung or heart tissue inflammation, localized hemorrhage and proteinaceous fluid in the alveoli, high serum levels of catecholamines and thorough tissue damage in the brainstem, especially the medulla were 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 present invention relates to an animal model comprising a rodent infected with enterovirus 71, sometimes referred to herein as modified enterovirus 71, capable of infecting rodents. . 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, the mutation in VP1 allows CDV to infect rodent cells with a rodent SCARB2 protein. In one embodiment, the rodent is an immunocompetent rodent. In another embodiment, the rodent is an infectious rodent. Suitable animals for use as models 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 aspect, 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 a mutation in VP1 is CDV: BS _VP1 [K98E / E145A / L169F]. Animal models are useful for studying systemic transmission of viruses and the human disease spectrum 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 full spectrum of EV71-induced neurological infections, diseases and pathologies observed in humans. In some embodiments, the method comprises the step of infecting a rodent described herein with the modified enterovirus 71 described herein and breeding the infected rodent 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 other embodiments, the infected rodent is raised for about 1 week to about 4 weeks. In some embodiments, the rodent is a mouse as described herein. In other embodiments, the rodent is infected by inoculating the rodent with a modified enterovirus 71. In one embodiment, the inoculation is intraperitoneal (IP). In another embodiment, the inoculation is intramuscular (IM). In some embodiments, the viral dose inoculated in the rodent is a median cell culture infectious dose (CCID ₅₀ ) between about 10 ³ and about 10 ⁷ .

  [0016] In a further aspect, the present invention provides a method of screening for antiviral agents. In accordance with this aspect, the method includes the following steps: providing a test group of animals and a control group of animals, wherein each group of animals is an animal of the animal model described herein; 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 Selecting an antiviral drug candidate 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 an animal. In another embodiment, the antiviral agent is first screened in a test rodent cell line infected with a clone-derived virus (CDV) containing a mutation in VP1 prior to screening in the animal.

  [0017] In a further aspect, the present invention provides a method of screening for effective antiviral vaccines. In accordance with this aspect, the method includes the following steps: providing a test group of animals and a control group of animals, wherein each group of animals is an animal of the animal model described herein; Administer antiviral vaccine 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 Selecting an antiviral vaccine candidate to be caused. In one embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a rodent cell line matched 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-10 show the cytopathic effect (CPE) observed following 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) Primate cells infected with any of the viruses: RD cells (FIGS. A1 to 1C), HeLa cells (FIGS. 1D to 1F), HEp-2 cells (FIGS. 1G to 1l), Vero cells (FIGS. 1J to 1L), And COS-7 cells (FIGS. 1M-1O) were observed at 48 hpi for cytopathic effects or cell monolayer cell death. Images are representative of results in 3 independent experiments. [0019] FIGS. 2A-20 show viral antigen detection in cell lines infected with EV71: BS, EV71: TLLm and EV71: TLLmv. Overnight planted mammalian cell lines: HeLa (FIGS. 2A-2C), HEp-2 (FIGS. 2D-2F), CHO-K1 (FIGS. 2G-2I), NRK (FIGS. 2J-2L), and TCKM (FIG. 2M) ~ 20) were infected with each virus at 1 MOI. Cells were harvested at 48 hpi, coated on Teflon slides and fixed in cold acetone. Cells were probed with pan-enterovirus antibody and stained with FITC-conjugated anti-mouse IgG. Images are representative of two independent experiments. [0020] FIGS. 3A-3D show the growth kinetics of EV71: BS, EV71: TLLm and EV71: TLLmv determined in NIH / 3T3 and Vero cells. Supernatants from various mammalian cells infected with 1 MOI of each virus were collected at various time points and subjected to titration and counted using the Reed and Muench method. FIG. 3A: EV71: BS virus titer determined in Vero cells. FIG. 3B: EV71: TLLmv virus titer determined in NIH / 3T3 cells. Figures 3C and 3D: EV71: TLLm virus titer determined in NIH / 3T3 cells. Growth curves from cell lines that do not show productive infection are not shown. [0021] FIGS. 4A-4O show the cytopathic effect (CPE) observed after viral infection of various rodent cell lines. 1 MOI EV71: BS (FIGS. 4A, 4D, 4G, 4J and 4M), EV71: TLLm (4B, 4E, 4H, 4K and 4N), or EV71: TLLmv (FIGS. 4C, 4F, 4I, 4L and 4O) viruses 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 (FIG. 4J) ~ 4L), and NRK cells (Figures 4M-4O) were observed at 48 hpi for cytopathic effects or cell monolayer cell death. Images are representative of results from three independent experiments. [0022] FIGS. 5A-5D show the viral fitness assessment of EV71: BS, EV71: TLLm and EV71: TLLmv in NIH / 3T3 as determined by titer ratio. Viral fitness 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 force values shown in FIGS. 3A-3D, the virus fit of (FIG. 5A) EV71: BS, (FIG. 5B) EV71: TLLmv and (FIGS. 5C and 5D) EV71: TLLm was calculated. Virus-matched assays from cell lines that do not show productive infection are not shown. [0023] FIGS. 6A and 6B show that transfection of NIH / 3T3 with EV71: BS viral RNA induces a productive infection. NIH / 3T3 and Vero cells seeded overnight were inoculated with viral supernatants taken from NIH / 3T3 cells previously transfected with viral RNA extracted from EV71: BS, EV71: TLLm, and EV71: TLLmv. FIG. 6A: Cells were imaged at 24 hpi using an inverted light microscope to observe induced CPE. FIG. 6B: Cells were harvested at 7 dpi, coated on Teflon slides, probed with pan-enterovirus antibodies, and stained with anti-mouse FITC conjugated antibodies. [0024] FIGS. 7A and 7B show viral fitness evaluation of EV71: BS, EV71: TLLm, and EV71: TLLmv in NIH / 3T3 and Vero cells at 30 ° C. Overnight planted infected with EV71: BS (panels a, d, g), EV71: TLLm (panels b, e, h), or EV71: TLLmv (panels c, f, i) (FIG. 7A) NIH / 3T3 and (FIG. 7B) Vero cells were incubated at 30 ° C. and observed at 24 hpi (panels ac), 48 hpi (panels df), and 72 hpi (panels g-i) under a phase contrast optical microscope. . The images taken are representative of two independent experiments. [0025] FIGS. 8A and 8B show viral fitness evaluation 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 at 24 hpi (panels a-c), 48 hpi (panels df), and 72 hpi (panels g-i) under a phase contrast optical microscope. . The images taken are representative of two independent experiments. [0026] FIGS. 9A and 9B show viral fitness evaluation 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 contrast optical microscopy at 24 hpi (panels a-c), 48 hpi (panels df), and 72 hpi (panels g-i) . The images taken are representative of 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. Overnight planted NIH / 3T3, Neuro-2A, and TCKM cells were infected with 1000 CCID ₅₀ EV71: BS virus (FIGS. 10A, 10C, and 10E) or transfected with an equal amount of viral RNA (FIG. 10B, 10D, and 10F) and at 48 hpi for viral antigen detection. Virus in the supernatant was harvested at 7 dpi and passaged on fresh Vero (FIGS. 10G, 10I, and 10K) and NIH / 3T3 cells (FIGS. 10H, 10J, and 10L). Cells were harvested at 48 hpi and stained for viral antigen. [0028] FIGS. 11A-11D show the position in VP1 and VP2 of matched mutations in the EV71: TLLm and EV71: TLLmv genomes. Using DeepView / SwissPDBviewer v3.7 and the 3D structure of EV71 capsid P1 region (PDB ID 4AED), VP1 of EV71: TLLm (FIGS. 11A and 11C) and EV71: TLLmv (FIGS. 11B and 11D) (FIGS. 11A and 11B) And matched mutations observed in the VP2 (FIGS. 11C and 11D) regions. Mutations were observed to be most localized in the surface exposure loop of the proteins shown. [0029] FIG. 12 shows the potency in NIH / 3T3 cells compared to the titer in Vero cells of viral supernatants collected from cells either transfected with EV71: BS viral RNA or infected with live virus. The valence ratio is shown. Supernatants from 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 Reed and Muench method. Figure 3 shows the ratio of log (titer) determined in NIH / 3T3 cells compared to the titer determined in Vero cells. 3T3-TRANS: RNA transfected NIH / 3T3 cells; 3T3-INF: virus infected NIH / 3T3 cells. Asterisk indicates Student's t-test with p-value <0.05. [0030] FIGS. 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: Body weight changes were plotted to determine the overall health of the animals. [0031] FIGS. 14A-14D show the symptoms and pathology of infected animals. Most of the infected animals showed disease symptoms. FIG. 14A: hind limb paralysis (arrow). FIG. 14B: Gross anatomical observation of the expanded lung after necrosis (arrow). Tissue sections were also stained with hematoxylin and eosin staining (10 × FIG. 14C and 20 × FIG. 14D). The black arrows point to mucous substances that infiltrate the alveolar space. [0032] FIGS. 15A-15E show that transfection of viral genomic RNA into both primate and rodent cells yields live virus. FIG. 15A: 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 collected and seeded on Vero or NIH / 3T3 cells (P1) and assessed for viability of virus progeny. Infection of P0 cells was assessed 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] FIGS. 16A-16F show that the capsid coding region of mouse cell adapted EV71: TLLm drives proliferative infection of mouse cells with EV71: BS. Figure 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 [M-P1]. FIG. 16B: Cells were infected with clone-derived virus (CDV) from either EV71: BS or EV71: BS [M-P1] and lytic cytopathic effect (CPE) (FIG. 16C) and viral antigen expression ( Infection was assessed by induction of FIG. 16D). The supernatant was replated on fresh cells and from passage (P1 and P2) obtained from infected Vero (FIG. 16E), and passage P1 of NIH / 3T3 (3T3) and Neuro-2A (N2a) cells (FIG. 16F), virus titers were measured. Error bars indicate SD. ^* P <0.05. [0034] FIGS. 17A-17G show that the VP1-L169F amino acid substitution in the capsid is sufficient to allow EV71: BS entry into murine cells. FIG. 17A: Amino acid substitutions in VP1: K98E, E145A, and L169F; and amino acid substitutions in VP2: S144T and K149I were incorporated into the full-length EV71: BS genome to generate diverse mutant cDNA clones. Mutations corresponding to amino acid substitutions are listed in parentheses. The infection of various cell lines with clone-derived virus (CDV) was monitored by assessing induction of lytic 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 progeny. Error bars indicate SD. [0035] FIGS. 18A-18E show that EV71: BS virus containing a combined VP1 amino acid substitution in the capsid shows improved mouse cell infection. FIG. 18A: Diverse mutant cDNA clones were generated by incorporating amino acid substitution combinations at VP1 and VP2 within the full-length EV71: BS genome. Mutations corresponding to amino acid substitutions are listed in parentheses. Evaluate cytopathic effect (FIG. 18B), viral antigen expression (FIG. 18C), and virus yield 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). Other clones not producing virus are not 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 neuronal cell line Neuro-2a. Clone-derived virus was passaged twice in Neuro-2a and NIH / 3T3 cells. At each passage, infection was monitored (FIG. 19B) by evaluation of viral antigen expression (FIG. 19A) and virus titer measured in Vero cells (FIG. 19B). Error bars indicate SD. ^* P <0.05; ^** p <0.005; ^*** p <0.0005. FIG. 19C: Genomic sequence of representative CDV: BS _VP1 [K98E / E145A / L169F] was determined to evaluate evidence for amino acid substitutions K98E (A2734G), E145A (A2876C), and L169F (C2947T). 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 EV71: TLLmv Inhibits binding. The fluorescence intensity on the membrane was measured using Imaris imaging software (n = 100). NSP (non-specific rabbit serum). Prior to inoculation on NIH / 3T3 cells, EV71: TLLmv of either mSCARB2 (FIG. 20C) or human SCARB2 (hSCARB2) (FIG. 20D) is preincubated with recombinant soluble protein, as assessed by an immunofluorescence assay As such, the severity of viral infection is reduced. Preincubation of either live cell NIH / 3T3 cells with either hSCARB2 (FIG. 20E) or mSCARB2 (FIG. 20F) prior to infection with EV71: TLLmv reduces the viral titer in the culture supernatant. ^* P <0.05; ^** p <0.005; ^*** p <0.0005. [0038] FIGS. 21A-21D show that incubation of murine SCARB2 rabbit antiserum with Neuro-2A cells reduced the severity of infection with CDV mutants. 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 [M-P1] (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. Figure 22a: Mice infected intraperitoneally 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 titers in sera collected from IP). Figures 22b and 22c: I.D. P. 10 ⁶ CCID ₅₀ (median cell culture infectious dose) of EV71: BS, EV71: TLLm, or EV71: TLLmv through either the route (FIG. 22b) or the intramuscular (IM) route (FIG. 22c) Kaplan-Meier survival curve of mice inoculated with. Statistical significance was determined using t-test with Welch correction for unequal variation (Figure 22a) or Mantel-Cox log rank test (Figures 22b and 22c). ^* P <0.05; ^** p <0.005; ^*** p <0.0005. [0040] FIGS. 23a-23h show that EV71: TLLmv infection in mice is characterized by acute severe disease resembling the human disease spectrum. Figures 23a and 23b: Dose-dependent lethality of EV71: TLLmv infection in 1 week old mice. Figure 23a: Various doses of EV71: TLLmv P. Kaplan-Meier survival curve of 6 day old pups injected. FIG. 23b: Median humane endpoint (HD50) was equivalent to a viral dose of 3.98 × 10 ³ CCID ₅₀ . FIG. P. Or I. M.M. Kaplan-Meier survival curve of 1 week old mice inoculated with EV71: TLLmv through the route. FIG. 23d: Age and route-dependent lethality induced by EV71: TLLmv infection in mice inoculated with a viral dose of 10 ⁶ CCID ₅₀ . Figures 23e and 23f: Clinical signs observed in end-stage mice, some of which showed hind limb (grey arrows) and / or forelimb paralysis. Others also showed small hairless lesions on the trunk (black arrows). Figures 23g and 23h: I.D. P. Pathway (FIG. 23g) or I.V. M.M. Disease classification of 1 week old mice inoculated with EV71: TLLmv through the route (FIG. 23h). [0041] FIGS. 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. Figures 24a and 24b: groups of 8-10 mice P. Pathway (FIG. 24a) or I.D. M.M. By route (Figure 24b), 10 ⁶ CCID _{50 of the} virus was inoculated and Kaplan-Meier survival curves were determined for animals of different age groups. Figures 24c and 24d: I.D. P. Pathway (FIG. 24c) or I.I. M.M. Neutralizing antibody titers in sera collected from mice of various ages inoculated through any of the routes (FIG. 24d) at the end of the observation period; 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) (IP) Neutralizing antibody titers in serum collected from mice. Statistical significance was determined by Mantel-Cox log rank test (FIGS. 24a and 24b) or t test with Welch correction for unequal variances (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. FIGS. 25a-25d: of lungs obtained from mock-infected mice (FIG. 25a) or 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 collapse of the lung that appears in FIG. 25b (white arrow). FIG. 25e: Harvested from mock-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 images of lung tissue sections (5 μm) stained with hematoxylin & eosin (H & E). Shown are mock-infected mice (FIG. 25f) or disease class IA (FIG. 25g) Low and high magnification images of lungs from EV71: TLLmv infected mice showing signs of class IB (FIG. 25h) or class II (FIG. 25i) Pink proteinic fluid in alveolar space (FIG. 25g, high magnification asterisk), some of which are also filled with red blood cells (FIG. 25g, high magnification gray arrows)). Figures 25j and 25k: in mock 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 Mann-Whitney test (FIG. 25e) or t-test with Welch correction for unequal variance (FIGS. 25j and 25k). ^* P <0.05; ^** p <0.005. [0043] FIGS. 26a and 26b show the lack of viral replication or inflammation in lung and heart tissue of class IA mice. FIG. 26a: EV71: from a diverse group of mice infected with TLLmv and stained with hematoxylin & eosin (H & E) for histopathology or with rabbit serum against EV71 antigen for viral antigen localization Representative images of labeled (EV71 IHC), lung tissue sections (5 μm). FIG. 26b: Representative images of heart tissue sections (5 μm) processed for H & E and EV71 IHC. [0044] FIGS. 27a-d 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) (Figures 27a, 27b and 27c); hypothalamus (HY) (Figures 27a and 27b); hippocampus (HP) (Figures 27b and 27c); thalamus (TH) (Figure 27b); midbrain (MB) ) And pons (P) (FIG. 27c); and cerebellum (CBX) and medulla (MY) (FIG. 27d). The areas where viral antigens and lesions are detected are detected and indicated accordingly. Larger dots indicate stronger signal / lesion size. The template image is brainstars. downloaded from org1 and licensed under Creative Commons Japan. A brain tissue coronal section map was obtained from the Interactive Mouse Brain Atlas (http: //mouse.brain-map/org/static/atlas) [113]. [0045] FIGS. 28a-28n show that EV71: TLLmv infection in mice is associated with neural tissue destruction and massive viral replication. Figures 28a-281: Representative images of brain tissue sections (5 [mu] m) 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). Lesions in the brain include edema (dotted box), infiltrating cells (diagonal area in the upper left quadrant and diagonal area in the lower right quadrant of FIG. 28k), nerve phagocytosis (in the left panel of FIGS. 28a-28c). , Neurodegeneration (black asterisks), and Purkinje cell degeneration (grey asterisks). Figures 28m and 28n: Representative maps of spinal coronal sections from mice in disease class IA (Figure 28m) or class IB (Figure 28n). Highlight areas where viral antigens and lesions were detected. A larger dot size indicates a larger signal / lesion. V, dorsal; D, ventral. [0046] FIGS. 29a-29c show EV71 antigen and virus-induced lesions in other regions of the hindbrain of class IA mice. FIG. 29a: Representative images of dental nuclei stained with either hematoxylin and eosin (H & E) for histopathological examination, or rabbit serum against EV71 antigen (EV71 IHC) for viral antigen localization. . The enclosed area is shown enlarged in the inset. FIG. 29b: Representative image of caudal brainstem from class I mice. The image shows cerebellar cortex (CBX) and medulla oblongata (MY). The last field (AP: asterisk) and the solitary nucleus (NTS; dotted circle) are also shown for reference. Regions where viral antigens and lesions are detected are indicated. Larger dots indicate stronger signal / lesion size. The template image is brainstars. downloaded from org1 and licensed under Creative Commons Japan. A brain tissue coronal section map was obtained from the mouse brain atlas (http: //mouse.brain-map/org/static/atlas) [113]. FIG. 29c: Representative image of medulla from class IA mice showing H & E and EV71 IHC staining patterns in AP and NTS. [0047] FIGS. 30a-30f show histological sections of neural tissue from mock-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 localization of viral antigen. image. Brain slices show 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); Purkinje cell layer in the cerebellar cortex (FIG. 30e) is shown. Note the normal Purkinje cell morphology (black asterisks in the left panel of FIG. 30e); and the reticulated neurons in the medulla (FIG. 30f). [0048] FIGS. 31a-31e show EV71: TLLmv-induced pathology and viral antigen distribution in other neural tissues. Representative images of mouse tissue sections (5 μm) stained either with hematoxylin & eosin (H & E) for histopathology or with rabbit serum (EV71 IHC) against EV71 antigen for immunohistochemical analysis. Brain sections were obtained from EV71: TLLmv infected or mock infected mice. The coronal cross-sections are the cortical pyramidal neurons of the motor (FIG. 31a); the pontine gray gray neurons (FIG. 31b); Show. Note the cellular infiltrates (lower right quadrant of the left panel of FIG. 31a) and neuronal necrosis (black asterisk of the second panel of FIG. 31) found in the motor cortex. 31c to 31e are shown enlarged in the respective insets.

  [0049] The present invention relates to enterovirus 71 (EV71), development of animal models, and screening of candidate anti-EV71 compounds. More specifically, the present invention relates to an enterovirus 71 (EV71) strain adapted to infect a rodent cell line or a clone-derived virus containing a mutation in VP1, wherein the immunocompetent rodent And relates to the discovery that it is possible to cause disease in infectious rodents. These EV71 strains are sometimes referred to herein as modified enterovirus 71.

  [0050] Furthermore, the present invention relates to an EV71 induced neurological disease by infecting BALB / c mice with a modified strain adapted to infect NIH / 3T3 mouse fibroblasts (eg EV71: TLLmv). It relates to the development of a clinical authentic model. Using this approach, modified EV71 is used to induce acute encephalomyelitis associated with neurogenic pulmonary edema, characterized by lung swelling and organ weight increase in mice compared to sham-infected lungs. Despite the absence of lung or heart tissue inflammation, localized hemorrhage and proteinaceous fluid in the alveoli, high serum levels of catecholamines and thorough tissue damage in the brainstem, especially the medulla were observed. These data demonstrate that the model accurately reproduces the signs and symptoms of human EV71-induced neurogenic pulmonary edema.

[0051] Accordingly, in one aspect, the present invention relates to an animal model comprising a rodent infected with enterovirus 71, sometimes referred to herein as modified enterovirus 71, capable of infecting rodents. . In one embodiment, such modified enterovirus 71 is a 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, the mutation in VP1 allows CDV to infect rodent cells using the rodent SCARB2 protein. In one embodiment, the rodent is an immunocompetent rodent. In another embodiment, the rodent is an infectious rodent. Suitable animals for use as models 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 aspect, 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 a mutation in VP1 is CDV: BS _VP1 [K98E / E145A / L169F]. Animal models are useful for studying systemic transmission of viruses and the human disease spectrum in animal models. Animal models are also useful for screening antiviral drugs and vaccines.

  [0052] Animal models are prepared as needed. A large standardized stock of rodent cell line matched EV71 strain is prepared, titered and maintained in a deep freezer (minus 80 ° C.). Several "normalized" (based on statistical calculations) rodents, such as BALB / c mice or NSG mice, are infected with a standard titer of virus strain to produce an animal model. The animal model of the present invention develops neurological symptoms upon infection (similar to those that can develop in humans). As shown herein, modified enteroviruses 71, eg, rodent cell line matched EV71 virus strains, affect a variety of neurological diseases 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 serial passage of the human EV71 BS strain in the NIH / 3T3 mouse cell line for a minimum of 60 cycles. In one embodiment, EV71: TLLm was deposited on 12 January 2015, under the terms of the Budapest Treaty, at the Type Culture Collection China Center located in Wuhan University, Wuhan 430072, People's Republic of China, and the deposit number Assigned CCTCC V2014437. In another embodiment, when viral RNA is synthesized using a viral RNA sequence (GenBank accession number KF514879; SEQ ID NO: 1), EV71: TLLm can be recovered using advanced reverse genetics. Advanced reverse genetics techniques are 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 the NIH / 3T3 mouse cell line for an additional 40 cycles. In one aspect, EV71: TLLmv was deposited on 12 January 2015, under the terms of the Budapest Treaty, at the Type Culture Collection China Center located in Wuhan University, Wuhan 430072, People's Republic of China, and the deposit number Assigned CCTCC V201438. In another embodiment, when viral RNA is synthesized using a viral RNA sequence (GenBank accession number KF514880; SEQ ID NO: 2), EV71: TLLmv can be recovered using advanced reverse genetics. Advanced reverse genetics techniques are 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, which allows the modified enterovirus 71 to use the rodent SCARB2 protein to infect rodent cells. It is a derived virus (CDV). A modified enterovirus 71 having a mutation in VP1 is generated using techniques known to those skilled 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-directed mutagenesis or CRISPR techniques (see, eg, PCT Publication No. WO2014 / 127287). Live virus (clone-derived virus (CDV)) is prepared from cDNA clones using techniques known to those skilled in the art or as described herein. CDVs with different mutations or collections of mutations are then tested for the ability to infect rodent cells. Alternatively, CDVs with different mutations or collections of mutations are then tested for their ability to bind to rodent SCARB2 protein as an initial screen. Any suitable enterovirus 71 strain can be used to develop a CDV with a mutation in VP1. The number of mutations and the specific mutations can vary for each strain in order to produce sufficient CDV in the target rodent cells to produce full-fledged infection. Thus, it is possible to produce a 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 CDV with a mutation in VP1 is the enterovirus 71 BS strain. In one embodiment, the modified enterovirus 71 is CDV: BS _VP1 [K98E / E145A / L169F].

[0056] In another aspect, the present invention provides a method for preparing an animal model that includes the full spectrum of E71-induced neurological infections, diseases and pathologies observed in humans. In some embodiments, the method comprises the step of infecting a rodent described herein with the modified enterovirus 71 described herein and breeding the infected rodent 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, the infected rodent is raised from about 1 week to about 4 weeks. In other embodiments, the infected rodent is raised from about 1 week to about 3 weeks. In other embodiments, the infected rodent is raised from about 1 week to about 2 weeks. In one embodiment, the infected rodent is raised for about 1 week. In another embodiment, the infected rodent is raised for about 2 weeks. In a further embodiment, the infected rodent is raised for about 3 weeks. In a further embodiment, the infected rodent is raised for about 4 weeks. In some embodiments, the rodent is an infectious rodent. In some embodiments, the rodent is a mouse as described herein. In one embodiment, the susceptible mouse is a BALB / c mouse. In other embodiments, the rodent is infected by inoculating the rodent with a modified enterovirus 71. In one embodiment, the inoculation is intraperitoneal (IP). In another embodiment, the inoculation is intramuscular (IM). In some embodiments, the viral dose inoculated in the rodent is a median cell culture infectious dose (CCID ₅₀ ) between about 10 ³ and about 10 ⁷ . In other embodiments, the viral dose inoculated in the rodent is a CCID ₅₀ between about 10 ³ and about 10 ⁶ . In one embodiment, the viral dose inoculated in the rodent is a CCID ₅₀ between about 4 × 10 ³ and about 10 ⁶ . In another embodiment, the viral dose inoculated in the rodent is a CCID ₅₀ between about 10 ⁴ and about 10 ⁶ . In a further embodiment, the viral dose inoculated in the rodent is a CCID ₅₀ between about 10 ⁵ and about 10 ⁶ . In one embodiment, the viral dose inoculated in the rodent is a CCID ₅₀ of about 10 ⁶ .

  [0057] Animal models prepared in this manner show superficial validity, ie these animals are observed over the entire spectrum of neurological diseases induced by EV71 infection in human patients, including NPE. A true mouse model of EV71 nerve infection showing the full range of possible clinical signs. This animal model also shows construct validity with respect to the gross and histopathological features of the disease, which are very similar to those reported in fatal human cases. This new in vivo model identifies important events in EV71 neuropathogenesis, dissects the mechanism of EV71-induced NPE, develops new therapeutic modalities and potential antiviral therapies, and preclinical evaluation of new vaccines Equivalent to a powerful tool for doing.

  [0058] In a further aspect, the present invention provides a method of screening for antiviral agents. In accordance with this aspect, the method includes the following steps: providing a test group of animals and a control group of animals, wherein each group of animals is an animal of the animal model described herein; 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 Selecting an antiviral drug candidate 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 an animal. In another embodiment, the antiviral agent is first screened in a test rodent cell line infected with a clone-derived virus (CDV) containing a mutation in VP1 prior to screening in the animal.

  [0059] In a further aspect, the present invention provides a method of screening for effective antiviral vaccines. In accordance with this aspect, the method includes the following steps: providing a test group of animals and a control group of animals, wherein each group of animals is an animal of the animal model described herein; Administer antiviral vaccine 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 Selecting an antiviral vaccine candidate to be caused. In one embodiment, antiviral vaccine candidates are first screened in a test rodent cell line infected with a rodent cell line matched 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] According to the method of the present invention, a large standardized stock of rodent cell line matched EV71 strain is prepared, titered and maintained in a deep freezer (minus 80 ° C.). Alternatively, a large standardized stock of clone-derived virus (CDV) containing a mutation in the VP1 strain is prepared, titered, and maintained in a deep freezer (−80 ° C.). Several animals that are “normalized” (based on statistical calculations), such as Balb / c mice or NSG mice, are infected with a virus strain of standardized titer. A variety of standardized dosages of candidate antiviral drugs or antiviral vaccines to infected rodents, prior to the onset of disease (for assaying prophylactic effects) or at the onset of disease (for assaying therapeutic effects of drugs) To administer. In one aspect, 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. Perform high-throughput in vitro screening for anti-EV71 compounds. In another aspect, 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. Used to perform 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, serial passage of human EV71 isolates (EV71: BS) produced virus strains that gained the ability to infect in vitro cultured rodent cell lines. The mouse adherent 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 represents the early stage of the adaptation process (passage number 60), and EV71: TLLmv (Passage number 100). Based on the appearance of virus-induced CPE, measurement of high potency value, and positive detection of viral antigens through immunostaining, we have identified viral-induced infection in cells as either proliferative or non-proliferative. Classified. Productive infections show positive viral antigen detection, as well as high viral titers, regardless of CPE observation. On the other hand, non-proliferative infections are characterized by an unmeasurable viral titer at the cutoff assay limit, despite viral antigen detection and / or observation of CPE.

  [0062] Clinical isolate EV71: BS only infects primate cell lines, whereas EV71: TLLm proliferates infects both primate and rodent cell lines. It is important to note that the EV71: TLLm virus has successfully achieved the ability to infect rodent cells, but the degree of adaptation to NIH / 3T3 cells is less pronounced. Viral titers determined using Vero cells are much higher than those determined in NIH / 3T3 cells, as shown by the negative values in the relative replication rate (RRR) assay (FIGS. 5C and 5D). Furthermore, EV71: TLLm successfully infects Vero cells leading to complete CPE at various incubation temperatures, whereas in infected NIH / 3T3, complete CPE can only be achieved at 37 ° C. (Table 1). Further adaptation in mouse cells that produced EV71: TLLmv virus showed a higher degree of adaptation to mouse cells (FIG. 5B), but in exchange resulted in a virus strain with a narrow permissive host cell spectrum. EV71: TLLmv does not infect primate cell lines as efficiently as mouse cells, but shows successful infection, leading to complete CPE of NIH / 3T3 cells incubated at a wider range of temperatures (Table 1). However, compared to ancestral EV71: TLLm, EV71: TLLmv appears to have lost the ability to enter and replicate in monkey kidney COS-7 cells, as well as human HeLa and Hep-2 cells. (FIGS. 3B-3C; FIGS. 2B-2C, 2E-2F, and 2H-2I). The ability to replicate efficiently in 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 matching in mouse cells in exchange for losing infectivity in cell lines of other origins.

  [0063] Although it is impossible to point out which amino acid substitutions are compatible with mouse NIH / 3T3 host cells, viral whole-genome sequencing may shed light on potential fitness mechanisms . Most of the amino acid substitutions identified are in the P1 (capsid) and RNA polymerase (3D region) proteins (Tables 2, 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. The capsid protein forms the structural background that the virus initiates interaction with permissive 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 exocytosis receptor for EV71 [48], and it is also utilized by several members of the human enterovirus A (HEV-A) species Yes. The human SCRB2 protein shares approximately 99% sequence identity with that of other primates. On the other hand, the mouse SCARB2 protein showed 15% sequence dissimilarity compared to the primate protein [49], showing a significant structural deviation from the primate SCARB2, and this is probably a rodent against native EV71 infection. Contributes to cell refractory. Compatibility mutations in the viral capsid make the virus eligible for binding to mouse cell receptors and result in successful entry and new host infection.

  [0064] The mapping of capsid protein mutations indicates that the majority of the amino acid substitutions identified in the viral P1 region are largely exposed to exposed regions of the protein (FIGS. 11A-11D), particularly BC on the VP1 surface. , 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 a major VP1 neutralizing epitope at amino acids 163-177 [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 radius from Gln-172. It has previously been reported that the VP1 S241L mutation resulted from mouse passage of CHO cell line compatible EV71 in association with K244E [50]. This mutation was found to be associated with an avirulent phenotype in 5 day old mouse pups in combination with VP2 K149I. However, a fit in NOD / SCID mouse brain tissue has been reported to result in a reverse mutation of VP1 241 from Leu to Ser [51], and it has been found that the mutation is associated with a mouse pathogenic phenotype. It was issued. The VP1 E145A mutation, separate from the SCARB2 docking site and located in the DE loop, is another candidate that confers the ability to infect murine cells. The VP1 145 mutation has been previously reported [34, 37] and a single E145A mutation leads to virulence in NOD / SCID mice [51]. Another mutation in VP1 of C4 genotype EV71, Q145E, is associated with virulence in 5 day old mice [52]. Mouse cytocompatibility mutations in VP2, particularly those within the neutralization epitope at residues 136-150 [53], may also contribute to the ability of the virus to infect rodent cells. Two substitutions in the VP2 EF loop were observed in EV71: TLLm (FIG. 11C), while there were three substitutions in EV71: TLLmv (FIG. 11D). None of these mutations have been previously described, but the near locus of VP2 149 in the EF loop has been referred to in the literature [34, 50, 54] and can be found in PSGL-1 overexpressing cells. It has been described as a generation compatible mutation [55].

  [0065] To examine 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. Direct introduction of EV71: BS RNA into the mouse cytoplasm as indicated by observation of virus-induced CPE and measurable virus titer in the culture supernatant when assayed in Vero cells is NIH / 3T3 cells. Results in a productive infection (Figure 12). However, reinoculation of virus supernatant on fresh NIH / 3T3 cells failed to induce productive 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 (Figures 10C and 10D) and measurable virus titer (Figure 12) in Neuro-2A cells, but directly virus Infection did not cause 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 show that avoiding the need for receptor binding for entry into the host cell led to successful infection and production of viral progeny in NIH3T3 and Neuro-2A cells. These data also confirm that human EV71: BS cannot successfully enter mouse NIH / 3T3 and Neuro-2A cells through the murine cell receptor. Furthermore, these data indicate that mutations within the P1 region of the EV71: TLLm and EV71: TLLmv genomes give the virus the ability for efficient receptor binding and thus allow host cell entry. Suggest to do.

  [0066] Several amino acid mutations are also observed in the P2 and P3 regions, which are critical for viral replication and hijacking of host cell protein translation machinery [56]. These matched mutations can function in optimizing EV71 genomic replication and translation in mouse cells. The viral 3D protein alone accumulates 8 amino acid substitutions in EV71: TLLmv and 4 substitutions in EV71: TLLm, and both strains each have one mutation in 3B and two mutations in 3C each. Mutation was shown. Direct inoculation of viral RNA into TCKM cells provides insight into the possible role of matched mutations in viral nonstructural proteins. TCHK cells have been shown to be permissive for EV71: TLLm and EV71: TLLmv infection (FIGS. 3B and 3D; FIGS. 4Q and 4R), but EV71: BS viral RNA into TCKM cells. Transfection did not result in successful infection. Viral antigen signal was not detected in infected and transfected cells (FIGS. 10E-10F), and passage of viral supernatant on fresh NIH / 3T3 and Vero cells did not result in positive viral antigen detection. (FIGS. 10K and 10L). Furthermore, there were no assayable virus 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 necessary for successful infection of TCKM cells. Suggest.

  [0067] This was originally derived from a human clinical sample that, after serial passage in a mouse cell line, has successfully acquired the ability to proliferatively infect several rodent cell lines. This is considered to be the first report of the EV71 strain. The relatively high mutation rate during RNA replication yields a mutant genome that serves as a genetic reservoir of phenotypic attributes for future relevance and the resulting replication as a pseudospecies distribution [57, 58] Guides the dynamic plasticity of RNA viral genomes and provides adaptability to changing environments [59, 60]. EV71 infected mammals [34, 37, 38, 51] and other rodents (eg, gerbils) [39] have been previously reported, but have been reported to proliferate infect in vitro cultured rodent cells. There was nothing. This may be due to high genetic barriers associated with large changes in phenotype and host range [58]. Interestingly, this is the first report of a number of matched mutations within the EV71 consensus whole genome sequence. Previously documented mouse-matched EV71 reported less than 10 amino acid substitutions in the genome [34, 38, 51], but we were in EV71: 21 in TLLm and 36 in EV71: TLLmv. Which was still greater than the maximum number of matched mutations previously identified [37]. These may not be sufficient for several passages of virus in mouse tissue [34, 36, 37] to break genetic barriers and successfully adapt the virus to infect cultured mouse cells. Suggest that there is sex. Instead, as observed in this study, hundreds of consecutive passages may be necessary.

  [0068] The host cell restriction of EV71 has recently been confirmed that EV71 is a scavenger receptor class B member 2 (SCARB2) as a functional receptor for host cell entry [47] and viral unwrapping in endosomes [72]. Has been proven and explained. Human and murine SCARB2 proteins show only 84% amino acid sequence identity [67], suggesting structural differences between the two proteins. This scenario is that EV71 clinical isolates are generally unable to infect mouse-derived cells, while mice and other rodents are generally resistant to EV71 experimental infections. Explaining this, this makes it difficult to develop small animal models of EV71 infection. However, despite this virus-receptor incompatibility, we have found that when the initial stage of infection, ie receptor-mediated cell entry, is bypassed through viral RNA transfection into the cell, Several murine cells have been shown to support EV71 infection. EV71: BS does not infect Neuro-2a and NIH / 3T3 cells [71], but transfection of viral genomic RNA induces expression of EV71 protein, induction of lytic cytopathic effect (CPE), and production of live virus progeny Produce. Similarly, live virus was previously produced after transfection of poliovirus RNA into mammalian cells [73-75], but the cells used (HeLa) were permissive for poliovirus infection. It was known that there was. In our experiments, non-permissive mouse neurons Neuro-2a and fibroblast NIH / 3T3 cells support viral replication and generate live virus progeny upon transfection of EV71: BS RNA into the cytoplasm The internal environment of the murine cells contains host factors necessary for EV71 infection and supports the completion of the viral infection cycle, and suggests that the EV71 protein is functional in the murine cytosol It 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 uncoating. This is mainly the function of the capsid protein. These results are similar to our previous observations (above or [71]), and we have shown that certain amino acid substitutions in the EV71: BS capsid protein allow binding to the receptor. And therefore leads to the hypothesis that virus entry and unwrapping can be led and subsequently infecting mouse cells. Two unique tools, mouse cell line (NIH / 3T3) adapted EV71 strain (EV71: TLLm and EV71: TLLmv) and two mouse cell lines (Neuro-2a and NIH / 3T3) provide an opportunity to examine this hypothesis did.

[0069] The infection phenotype of the chimeric clone-derived virus (CDV: BS [M-P1]) expressing the EV71: TLLm capsid protein but expressing the EV71: BS nonstructural protein is "mouse cell entry phenotype" Was confirmed to be provided by the capsid protein from the mouse cell line matched EV71 strain. These chimeric CDVs behaved similarly to the mouse cell-adapted EV71 strain and induced proliferative infection in Vero and NIH / 3T3 and Neuro-2a cells, which resulted in the completion of the viral infection cycle, ie generation of viable viral progeny Accompanied by direct evidence. Furthermore, mutagenesis introducing 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 the CDV (CDV: BS _VP1 [K98E / E145A / L169F]) with the EV71: TLLm P1 region substitution (CDV: BS [M-P1]) and the combined amino acid substitution VP1-K98E / E145A / L169F, Successfully passaged in Neuro-2a and NIH / 3T3 cells, it was possible to produce viable virus progeny in the resulting culture supernatant. The remainder of the CDV containing other amino acid substitutions, either individually or in various combinations, is limited entry into murine cells as evidenced by observation of CPE and detection of viral antigens in the inoculated cells. Only one cycle of replication was shown. Live virus progeny were not present in the infected cell culture supernatant because reinoculation on healthy mouse cells failed to induce infection. Although the reason for the failure of production of viable virus progeny in the culture supernatant of cells infected with these CDVs is not clear, one possibility is that the capsid that occurs after mutation is structurally unstable. That is. We speculate that the introduced amino acid is incompatible with the other amino acids that make up the protein, thus affecting the entire capsid protein fold and possibly altering the capsid structure. This assumption emphasizes the complexity of capsid protein assembly, where the four viral proteins (VP1-4) interact in concert to produce a functional capsid. Thus, amino acid substitutions that can allow a virus to bind to a novel receptor may also have devastating consequences, especially because it is incompatible with other amino acid residues in the protein This may be the case if the complex is destabilized undesirably.

[0070] The results shown in Examples 10-17 show that three VP1 substitutions: K98E, E145A, and L169F are necessary and sufficient for binding to receptors on mouse cells and allow EV71 entry Make it clear. Although VP1-169 residues have not been previously mentioned in the literature, VP1-98 and VP1-145 residues have previously been shown as markers for binding to the human PSGL-1 receptor protein on leukocytes. [76]. Analysis of 1,702 VP1 sequences of EV71 clinical isolates published in Genbank confirmed that the mutant combination, VP1 98E / 145A, was rare but viable, these were only 5 In one isolate (0.3% of the database). On the other hand, the VP1-169F mutant was not observed in the survey database of the EV71 VP1 sequence, indicating that it was extremely rare. CDV: BS _VP1 [K98E / E145A / L169F] was stably passaged on Neuro-2a cells and live virus progeny while retaining the introduced mutation in the viral genome for three passages. This suggested that the virus is viable and stable at least in Neuro-2a cells. Thus, introduction of these three residues: VP1 98E, 145A, and 169F into other EV71 clinical isolates may allow the isolate to infect murine cells, but this still Must prove.

  [0071] Similar to previous findings that EV71 utilizes the scavenger receptor class B member 2 (SCARB2) protein as a receptor for host cell entry and cytosolic uncoating [47, 72, 77 ] Our data also demonstrate that mouse cell-compatible EV71: TLLmv utilizes mouse SCARB2 (mSCARB2) to infect murine cells. Virus bound directly to recombinant soluble mSCARB2 in vitro and preincubation of 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 to mSCARB2 reduced virus binding on fixed cells and also inhibited live cell infection. The results also show EV71: TLLmv binding to the human SCARB2 (hSCARB2) protein that the virus uses to infect primates. Binding and infection of primate cells with EV71: TLLmv was also blocked by antibodies to hSCARB2. The data presented here shows only EV71: TLLmv, but the results can also be extended to EV71: TLLm. EV71: TLLmv was obtained from further passage of EV71: TLLm in NIH / 3T3c cells and only a few 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 [M-P1] 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 virus titer compared to the control. Recent data indicate that SCRAB2 binding to the EV71 canyon is a lipid in the capsid canyon, stabilizes mature virions, and induces the release of “pocket factor” predicted to be sphingosine in EV71 [ 78], a series of events in viral unwrapping: capsid expansion (forming 135-S or A particles), VP1 N-terminus and VP4 protrusion from the 5-fold capsid junction into the endosomal membrane, and in the host cytoplasm It has been shown to cause release of viral genomic RNA into [78-81]. Recent human SCARB2 crystal structure data also revealed a lipid tunnel across the entire protein [67], which is not relevant in the context of SCARB2 function to deliver β-glucocerebrosidase to lysosomes, In contrast, Dang et al. [72] proposed that it served as a conduit for removal and transport of sphingosine from the capsid canyon during SCARB2 binding. SCARB2 amino acid residues 140-151 vary greatly in sequence between human and murine proteins and are the main binding site 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 during viral unwrapping. The VP1-169 residues are in the capsid canyon and probably have a direct function in SCARB2 binding. A dramatic change from leucine to phenylalanine at this position alters the canyon structure, resulting in a better fit with the murine SCARB2 protein. VP1 98 and 145 residues, on the other hand, are on the fringe surrounding the Capsid Canyon and may have a function separate from SCARB2 binding. These are just hypotheses, and in order to elucidate the exact mechanism by which three VP1 amino acid substitutions can confer a “mouse cell entry phenotype”, the structure of the murine SCARB2 protein was analyzed, and the viral capsid You will need to map the interaction.

  [0073] This is believed to be the first scientific exploration of EV71 capsid site-specific mutations that induce productive infection of murine cells that are naturally non-permissive. Previous studies have generated mutants with improved viral infection profiles in mice through selective viral adaptation, as in the case of passage in surviving animals [34, 37, 38]. 82], or have been attempted to modify cells by ectopic expression of human SCARB2 protein [47, 69, 70, 83]. The incompatibility of EV71 to murine SCARB2 is generally due to the resistance of mice to EV71 infection, thus hindering the development of mouse models of EV71 infection. This also probably explains why the majority of mouse models of EV71 infection using either wild type or mouse compatible virus strains fail to reproduce the spectrum of disease observed in severe human infections [ 34, 37, 38]. On the other hand, transgenic mice expressing human SCARB2 showed a broader range of EV71 infection characteristics without the need for viral mouse adaptation, but the full spectrum of human disease was clearly not reproducible [69, 70]. Since it has been demonstrated herein that specific amino acid substitutions in EV71 enable EV71 to infect murine cells, the mouse cell line matched EV71: TLLm and EV71: TLLmv efficiently infect cultured rodent cells. An initial step to understand the molecular mechanism that can be made is provided. Furthermore, this may provide another approach to generate a mouse model of EV71 infection, study detailed pathogenesis, and reproduce the full spectrum of neurological diseases seen in human infection.

  [0074] In Examples 22-25, young BALB / c mice inoculated with mouse cytocompatibility enterovirus 71 (EV71) are similar to the pathology observed in infected human patients with neurogenic pulmonary edema (NPE). It is demonstrated to show acute encephalomyelitis associated with. Animals exposed to the matched virus strain EV71: TLLmv are presented as paralysis, ataxia and tremor and are consistent with CNS localization pathology identified in fatal cases of EV71 infection Various levels of virus-induced tissue damage were demonstrated in both regions [91-95]. In addition, some mice showed dyspnea compatible with autonomic dysfunction. Based on disease presentation at the time of euthanasia, the animals could easily be divided into four groups: class IA, class IB, class II and survivors. Survivors present no signs of disease, class II mice show persistent flaccid paralysis and severe weight loss, while class IA and class IB mice further suffer from acute neurological disease, which It was universally lethal within 3-7 DPI. Class IA mice also showed obvious 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, particularly the medulla, which is accompanied by high serum levels of catecholamines, and the respiratory signs observed in these mice are neurogenic pulmonary edema ( NPE) was strongly suggested. Gross pathological comparison of lungs from class IA mice with those from other groups revealed incomplete lung collapse and a significant increase in lung wet weight upon necropsy, which was probably due to bleeding and alveoli. This was due to liquid leakage into the space. These features were very similar to those observed in experimental animal models of lethal human cases with non-virus-induced and fulminant NPEs [96, 97]. Indeed, histopathological analysis of class IA animals further revealed a localized region of 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 an extravascular increase 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 obvious signs of disease, we are able to eliminate cardiogenic PE and in the lung parenchyma, virus replication or Since there was no inflammation, direct virus-induced lung injury was eliminated. Instead, we detect high serum levels of catecholamines in class IA mice, which has been demonstrated previously as a result of catecholamine storms induced by severe sympathetic discharges. Strongly demonstrated to exhibit neurogenic PE (NPE) [96, 101]. In this scenario, autonomic nervous system 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 by direct lung endothelial injury [101-104]. . Recently, several experimental animal models of non-viral inducible NPE have been developed (see Sedy [103] and Davison [104] for review), but our model is EV71 infection. Is the first to successfully induce classical signs of pulmonary edema. Thus, our new model represents a significant advance in pursuing antiviral therapy and therapeutic measures that can limit EV71 infection and prevent the onset of fulminant NPE in human patients. To do. Although pulmonary edema may also be inducible by host cytokine storms in response to EV71 infection [105-107], the data show that the primary mechanism of EV71-induced NPE is associated with a large inflammatory response And instead 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 hypothalamic periventricular and dorsal medial nuclei [101, 103], and ventrolateral and dorsal medulla, NTS And the AP region [96, 104, 108-110]. EV71-induced NPE has previously been shown to contribute to extensive damage to brainstem tissue [21, 22, 93, 111] and in our novel murine model, we In the brain stem and spinal cord, both viral antigens and extensive damage were detected. Class IA and class IB mice show similar distribution of lesions and viral antigens in the brainstem and spinal cord, along with comparable upregulation of serum catecholamines, while the absence of NPE in class IB animals is NTS, medullary network Can be explained by reduced number and severity of brain lesions and / or lower viral antigen intensity in the nuclei-like nucleus (MdRN) and the AP region of the medulla, the dentate nucleus of the cerebellum, and the dorsal medial nucleus of the hypothalamic cortex . We have therefore found that, in EV71 infected hosts, acute and severe destruction of brainstem tissue, particularly those related to vasomotor areas, lead to catecholamine storms and well-known trigger zones are sufficiently damaged. If so, we suggest that this can proceed to the NPE.

  [0077] In summary, the present invention shows superficial validity [112], ie, these animals are observed across the entire spectrum of neurological diseases induced by EV71 infection in human patients, including NPE. A true mouse model of EV71 nerve infection showing the full range of possible clinical signs. Observations of prominent features in EV71: TLLmv infected mice presenting class IA signs of disease were made by a video containing two video clips of two different class IA mice. Both animals were incapable of self-right and were in a coma. Severe dyspnea presented as tachypnea with subcostal retraction was evident in the first mice. Panting, subcostal retraction, and foamy fluid exiting the nostril were seen in the second mouse. Observations of prominent features in EV71: TLLmv infected mice presenting class IB signs of disease were made by video of one class IB mouse. He was unable to hold his posture and was stupid. Ipsilateral paralysis of the right limb and persistent tremor of the left hind limb were also observed. This model also shows construct validity with respect to gross and histopathological features of the disease [112], which are very similar to those reported in fatal human cases. This new in vivo model identifies important events in EV71 neuropathogenesis, dissects the mechanism of EV71-induced NPE, develops new therapeutic modalities and potential antiviral therapies, and preclinical evaluation of new vaccines Equivalent to a powerful tool for doing.

  [0078] The practice of the present invention is within the skill of the art, unless otherwise indicated, is within the skill of the art, chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and Conventional techniques of transgenic biology are used. For example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, New York (Cold Spring Harbor Co .; Cold Spring Harbor, New York) Russell, 2001, Molecular Cloning, 3rd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Green and Sambrook, 2012, Molecular Cl oning, 4th edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992, Current Protocols in Molecular Biology (including John Wiley & Sons, L Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); ues 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.H. J. et al. Transgition And Translation (BD D Hames and S. J. Higgins 1984); Culture Of Animal Cells (R. I. Fredney, C. R. I. Fredney, 1994); (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); Papers, Methods In Enzymology (Academic Press, Inc., NY); Methods In Enzymology. 154 and 155 (supervised by Wu et al.), Immunochemical Methods In Cell And Molecular Biology (supervised by Mayer and Walker, Academic Press, London, 1987); Handbook Of. Ex. Supervised by Blackwell, 1986); Riot, Essential Immunology, 6th edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al., RNA Interference Technology: Fc. rug 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), Human Press, Totowa, NJ, 2004; Sohail , Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.

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

Example 1
Materials and methods for Examples 2-8.
[0080] Cell lines and virus lines: All cell lines used in this study were purchased from the American Tissue Type Culture Collection (ATCC, USA). Various mammalian cell lines; human adenocarcinoma cell lines, HeLa (CCL-2) and HEp-2 (CCL-23), and rhabdomyosarcoma RD (CCL-136); African green monkey kidney Vero (CCL-81), and Vervet monkey kidney fibroblast COS-7 (CRL-1651); mouse neuroblastoma Neuro2A (CCL-131), embryonic fibroblast NIH / 3T3 (CRL-1658) , And kidney epithelium TCHK (CCL-139); hamster ovary epithelium-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 brain stem of a dead patient infected with EV71. The virus was passaged in Vero cells for 4 cycles before storage at -80 ° C until further use. The mouse cell (NIH / 3T3) matched EV71: TLLm strain was obtained from the EV71: BS strain through serial passage (> 60 cycles) in mouse NIH / 3T3 cells. The EV71: TLLm strain was further passaged in NIH / 3T3 cells (40 cycles) to generate a mouse cytocompatible pathogenic strain (EV71: TLLmv).

[0082] infection with maintenance and virus cell lines: 10% (v / v) fetal bovine serum (FBS, i-DNA Singapore) and 0.22% (w / v) sodium bicarbonate _(NaHCO 3, Sigma Aldrich All cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with USA, 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), And it incubated at 30 degreeC, 37 degreeC, or 39 degreeC for 2 hours. Cells were washed twice in sterile phosphate buffered saline (PBS, pH 7.4) solution 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 virus growth kinetic 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. Plates were subjected to 3 cycles of freeze-thaw and lysates were collected and clarified by exhaustive vortexing followed by centrifugation at 1500 × g for 10 minutes. Clarified supernatants were stored at −80 ° C. in frozen vials (Nunc, Fisher Scientific) until further use.

  [0085] For temperature compatible 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 at −80 ° C. in frozen vials until further use.

  [0086] Various mammalian cell lines: 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 parental EV71: BS or the resulting NIH / 3T3-matched EV71 strain at a MOI of 1 (multiplicity of infection) and incubated at 37 ° C. for 10 days. Cultures were observed daily for the appearance of CPE.

[0087] Determination of virus titer and relative replication rate (RRR): Virus supernatants were subjected to endpoint titration and assayed in both NIH / 3T3 and Vero cells. Viral titers were counted using the Reed and Muench method [61] and the Reed and Muench calculator [62]. Briefly, NIH / 3T3 (1 × 10 ⁴ cells per well) and Vero cells ( ⁴ × 10 ³ cells per well) were seeded overnight in 96 well plates. Frozen virus thawed to room temperature was diluted (10 ⁻¹ ) in sterile 1% aqueous sodium deoxycholate (Sigma Aldrich, USA) and mixed vigorously for 15 minutes to disaggregate the virus. The disaggregated virus was subjected to 10-fold serial dilution in maintenance medium, and 100 μl diluted virus from 10 ⁻³ dilution was added onto each well of cells. Plates were incubated at 37 ° C. and observed daily for the appearance of obvious CPE under an inverted light microscope. Viral titers were reported as 50% cell culture infectious dose per volume (CCID ₅₀ / ml).

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

  [0089] Viral antigen detection by immunofluorescence assay: For infected cells that did not show significant CPE, immunofluorescence (IF) staining was performed to verify infection. Cells were trypsinized to 72 hpi, washed twice in sterile PBS and coated on Teflon slides (Erie, USA). Slides were air-dried in a biosafety cabinet and UV treated for 15 minutes to inactivate live virus and then fixed in cold acetone at 4 ° C. for 10 minutes. Slides were probed 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] Transfection of cells with EV71 viral RNA: Vero and NIH / 3T3 cells (3 × 10 ⁴ cells per well) seeded overnight in 24-well plates were subjected to Lipofectamine 2000 (Life technologies, USA) according to the manufacturer's protocol. ) Was used to transfect viral RNA extracted from 4 × 10 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. At 7 dpi, supernatants were collected from infected cells and passaged on freshly seeded 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.

  [0091] Full-length genome sequencing and gene mapping of EV71 strain: Viral RNA of EV71: BS, EV71: TLLm, and EV71: TLLmv strains were 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). The amplicon was purified and cloned into the pZero2 vector (Lifetech, USA) using PCR cleanup kit (Geneid Biotech, Taiwan). Clones were selected from kanamycin plates, 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, the plasmid was sequenced by the BigDye terminator method (Applied Biosystems, USA) using the same primers. Using BioEdit v7.0.9.0 [63], the resulting 500 bp fragment sequence was aligned against the entire genome sequence of EV71 Singapore isolate 3799-SIN-98 (GenBank accession number DQ3412354.1). 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 server [64, 65].

Example 2
Primate cell lines are permissive for infection by EV71: BS, but rodent cell lines are not permissive
[0092] All primate cell lines used in this study were permissive for infection with EV71: BS virus. Human RD cells, as well as monkey Vero and COS-7 cells, show fully lytic cytopathic effect (CPE) within 48 hours (hpi) after infection (FIGS. 1A, 1J, and 1M) and viral antigens fixed. Detected in infected cells (Figures 2A-2L; data not shown for RD, COS-7 and Vero cells). Virus growth kinetic curves taken from supernatants of various infected cell lines confirmed proliferative infection in RD, Vero, and COS-7 cells (FIG. 3A). Virus harvested from RD and Vero cells reached an endpoint titer of 3 × 10 ⁹ CCID ₅₀ / ml, while the virus titer from COS-7 was 10 ⁶ CCID ₅₀ / ml (FIG. 3A). EV71: BS did not induce complete CPE in HeLa and Hep-2 cells (FIGS. 1D and 1G) and the resulting virus titer was not measurable within the assay cutoff limit. However, viral antigens were detected by indirect immunofluorescence staining in both HeLa cells (Fig. 2a) and Hep-2 cells (Fig. 2D), and the virus was successful in entering the cells, but insufficiently Or replication deficiency, which may have resulted in no measurable virus titer.

  [0093] All tested rodent cell lines were determined to be non-permissive for EV71: BS infection. Cell lytic CPE is not present after infection (FIGS. 4A, 4D, 4G, 4J and 4M), viral titers from the supernatant are not measurable, and viral antigens are not detectable in the inoculated cells (FIGS. 2G, 2J and 2M; FIGS. 10A, 10C and 10E).

Example 3
Mouse cell (NIH / 3T3) matched EV71: TLLm virus proliferatingly 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. All primate and rodent cells tested were permissive for proliferative infection 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 NIH / 3T3 and Neuro-2A cells (FIG. 4B, E). High supernatants can be measured in supernatants collected from all cell lines infected except NRK (Figures 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 virus titer are not observed in NRK cells (FIG. 4N; FIG. 3D), but viral antigen is detectable (FIG. 2K), and viral entry into NRK cells by EV71: TLLm is successful However, it has been shown that inefficient virus replication may have resulted in an unmeasurable virus titer.

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

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

Example 5
EV71: TLLmv virus showed a higher degree of adaptation to 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 potency assayed in NIH / 3T3 to the potency assayed in Vero was used as a surrogate measure of the degree of virus adaptation to NIH / 3T3 cells. The parental EV71: BS virus shows high negative RRR values for RD, Vero, and COS-7 (FIG. 5A), and the virus titer assayed in Vero cells is higher than the titer assayed in NIH / 3T3 cells. It was shown that it was far beyond. Relative growth ratio values for other cell lines were indeterminate because virus titer was not measurable. On the other hand, EV71: TLLmv virus showed a positive RRR value with the exception of viruses grown in Vero cells (FIG. 5B). Positive RRR values were an indication of more efficient replication in NIH / 3T3 cells and thus higher potency values compared to Vero cells. Negative RRR values determined for EV71: TLLmv harvested from Vero cells were consistent with the observed slow growth kinetics (FIG. 3B) and lower virus titers. EV71: TLLm showed negative RRR values (FIGS. 5C and 5D), but to a lesser degree than the RRR values for EV71: BS. This suggested that EV71: TLLm could infect some rodent cell lines proliferatively, but still be more compatible with replication in Vero than NIH / 3T3 cells.

Example 6
EV71: TLLm showed better adaptability to changing temperature than EV71: TLLmv
[0098] Vero and NIH / 3T3 cells infected with parental EV71: BS and the resulting NIH / 3T3-compatible EV71: TLLm and EV71: TLLmv strains were incubated at various temperatures, 30 ° C., 37 ° C., 39 ° C. Virus suitability to temperature fluctuations or changes was determined. EV71: BS showed the most limited suitability and complete CPE was observed only in Vero cells incubated at 37 ° C. (FIG. 8B; Table 1). EV71: TLLmv is 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. (FIG. 8A, FIG. 9A; Table 1). The degree of suitability was shown. EV71: TLLm, on the other hand, showed the best suitability, where it induced full CPE in Vero cells for all incubation temperatures (FIGS. 7B, 8B, 9B), whereas in NIH / 3T3 cells, Only at 37 ° C. (FIG. 8A; Table 1).

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

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

^Three complete CPE observations are shown.
⁴ indicates lack of complete CPE, the numbers in parentheses indicate the maximum degree of CPE observed in the cells.

Example 7
The EV71: TLLm and EV71: TLLmv viral genomes accumulated numerous missense mutations as a result of adaptation to NIH / 3T3 cells.
[0099] EV71: BS, EV71: TLLm, EV71: TLLmv viral RNA was subjected to Sanger sequencing to determine consensus genomic sequence and to identify possible matching mutations resulting from the matching process in NIH / 3T3 cells . Genomic consensus sequences representing a dominant population of pseudospecies have been deposited with GenBank, NCBI (National Center for Biotechnology Information). The alignment of the entire genome sequence of EV71: TLLm (GenBank accession number KF514879; SEQ ID NO: 1) relative to EV71: BS (GenBank accession number KF514878; SEQ ID NO: 3) reveals 60 nucleotide mutations, of which 21 are amino acids Substitution occurred (Table 2). On the other hand, 83 mutations with 36 amino acid substitutions were found between EV71: TLLmv (Genbank accession number KF514880; SEQ ID NO: 2) and EV71: BS genome. The majority of missense mutations were located in the P1 (capsid protein gene) region (Table 2), particularly in 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 the palm and thumb domains of the enzyme (Table 4). On the other hand, EV71: TLLmv accumulated 8 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 base changes, EV71: TLLm showed a 1 base insertion, while EV71: TLLmv 5'UTR showed a 4 bp insertion and a 20 bp deletion (Tables 2 and 3). On the other hand, no amino acid substitution was observed in the VP3 and 3A regions of EV71: TLLm, and neither was observed in the 3′UTR of both EV71: TLLm and EV71: TLLmv.

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

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

Numbering of amino acids in the uncleaved polyprotein before ^one maturation.
Numbering of amino acids in ^two mature proteins
³ Internal ribosome entry site.
Mutations are based on alignment with the reference EV71: BS genome.

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

Numbering of amino acids in the uncleaved polyprotein before ^one maturation.
Numbering of amino acids in ^two mature proteins
³ Mutation located in the ring finger domain of RNA-dependent RNA polymerase.

⁴ Mutation 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 the reference EV71: BS genome.

Example 8
Transfection of EV71: BS viral RNA into murine cells resulted in a productive infection, but the virus progeny failed to reinfect the same mouse cell.
[0100] Vero and NIH / 3T3 cells transfected with viral RNA showed complete CPE 7 days after transfection (dpt) (data not shown). Viral antigens were detected in NIH / 3T3 cells transfected with EV71: BS viral RNA (FIG. 10B) but not in NIH / 3T3 cells subjected to infection with the virus (FIG. 10A). . Viral supernatants replated on fresh Vero and NIH / 3T3 cells produced productive infection (100% CPE) only in Vero cells, but not in NIH / 3T3 cells (FIG. 6A) and virus 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: Plasmid (pMD18-mSCARB2) (Genbank accession number NP_031670.1) encoding murine SCARB2 cDNA was obtained from Sino Biological, Inc. (China / Beijing) 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 Life Sciences Laboratory, Singapore). A low copy number plasmid pACYC177 (New England Biolabs, Singapore) was used to generate a plasmid encoding EV71 full length cDNA. The plasmid construct (pCMV-T7pol) expressing T7 polymerase was a generous gift from Dr. Peter McMinn, University of Sydney, New South Wales. Plasmid pZero-2, used for sequencing the clone-derived virus fragment, was purchased from Invitrogen (Life Technologies, USA).

  [0102] The clinical isolates used in this study EV71: BS (Genbank accession number KF514878), EV71: TLLm (Genbank accession number KF51489.1), and EV71: TLLmv (Genbank accession number KF514880.1) Or as previously described [71].

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

  [0104] The E. coli cell line BL21 (New England Biolabs, Singapore) is used for high level protein expression, the TOP10 strain (Life Technologies, USA) is 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.

  [0105] Construction of EV71: BS full-length genomic cDNA clone, capsid chimeric clone, and VP1 / VP2 mutant clone: The EV71: BS cDNA clone was 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]. A genomic proximal fragment encoding the 5′UTR and P1 region was amplified using the primer pair: EV71_BamHI-PfF and EV71_Pf-AatIIR (Table 5), these primers were used for cloning into plasmid pACYC177 for BamHI and Contains an AatII restriction site. Distal fragments encoding P2, P3, and 3'UTR were amplified using primer pairs EV71_HindIII-DF and EV71_D-BamHIR, which 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, a MluI restriction site was engineered within the boundary between 5'UTR and P1 (primer pair SDM_MluIF and SDM_MluI-R). EV71: The P1 cDNA sequence of TLLm was amplified (primer pairs MluI-TLLm-P1F and EagI-TLLm-P1R), digested with MluI and EagI, and cloned into the construct harboring the proximal fragment. This modified proximal fragment was subsequently ligated to the distal fragment as described.

  [0107] To generate clones containing amino acid substitutions in the VP2 and VP1 proteins, site-directed mutagenesis was performed on the proximal fragment before ligation to the distal fragment. A VP2 S144T (nt G1385C) amino substitution was introduced into the proximal fragment containing the specific primer pair VP2_G1385C-F and VP2_G1385C-R. VP2 S144T (nt G1385C), VP1 K98E (nt A2734G), E145A (nt A2876C), and E167E (nt C2947T) were also generated in a similar manner using different primer pairs (Table 5).

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

  [0109] To assess the infection phenotype, the progress 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 cells pelleted from the culture supernatant were combined, washed twice in sterile phosphate buffered saline (PBS) and fixed on a Teflon slide. Fixed cells were incubated with a pan-enterovirus monoclonal antibody (Merck Millipore, USA) and detected with a standard FITC-conjugated anti-mouse IgG antibody. Infected cell culture supernatants were also collected, cleared, and subjected to serial dilution for virus titer determination using the Reed and Muench method [61]. Once the titer was known, the supernatant was passaged at 1 MOI on freshly seeded NIH / 3T3 and Neuro-2a cells and the infection 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) was paired with primer pairs pQE-mSCARB2F and pQE-mSCARB2R And introduced BamHI and HindIII restriction sites to facilitate cloning into the pQE30 protein expression vector. Clones were transformed into BL21 E. coli cells and protein expression was induced overnight with 1 mM IPTG. Harvested cells were digested with lysozyme (1 mg / ml) and the crude extract was purified using a Ni-NTA column (Qiagen®, Germany). The cleared lysate was incubated in 1 ml of 50% Ni-NTA slurry overnight at 4 ° C. 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 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 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 . A final bleed 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. Clarified serum was collected and stored at −20 ° C. for further use. The production of the SCRAB2 rabbit antiserum was approved by the Temasek Lifesciences Laboratory Institutional Animal Care and Use Committee (TLL-IACUC) [Authorization Number 047/17].

  [0112] Viral competition assay with murine SCARB2 protein: An 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 seeded overnight on sterile Teflon-coated slides (Erie, USA). Prior to virus inoculation, 100 MOI EV71: TLLmv was applied to 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) for 2 hours at 37 ° C. in a shaking platform. Infected cells were observed daily for signs of CPE and fixed in absolute acetone (4 ° C., 10 minutes) 48 hours after infection. Fixed cells were assayed immunofluorescently with a pan-enterovirus antibody (Merck Millipore®, USA). Slides were imaged with an upright fluorescent microscope (Nikon, Japan).

  [0113] Virus-SCARB2 binding assay: An antibody-mediated SCARB2 blocking assay was performed on fixed cells to assess whether masking cell surface SCARB2 protein would affect 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. Slides were incubated for 1 hour at 37 ° C. in polyclonal rabbit serum (1: 100) raised against mSCARB2. For negative controls, cells were incubated with polyclonal rabbit sera raised against Safford virus L protein. Slides were washed in PBS, then incubated with live EV71: TLLmv (1000 MOI) for 1 hour at 37 ° C., probed with pan-enterovirus antibody, and detected with FITC-conjugated antibody. Slides were imaged with a Zeiss LSM 510 Meta inverted confocal laser microscope (Zeiss, Germany) and fluorescence intensity was measured using Imaris (BitPlane Scientific Software, Germany) imaging software. Statistical analysis of fluorescence intensity differences was performed using Prism GraphPad version 6.01 (GraphPad Software, Inc., USA).

[0114] Cell protection assay from viral infection using rabbit anti-SCARB2 polyclonal serum: To assess the effect on cell infection, antibody-mediated SCARB2 blocking assays were also performed on viable cells. Overnight-planted NIH / 3T3 cells (1 × 10 ⁴ cells per well in a 96-well plate) were serially diluted (1:20 to 1: 2) in rabbit polyclonal sera made against either mouse or human SCARB2 protein. 640) at 37 ° C. for 1 hour. Cells were subsequently inoculated with 100 MOI of EV71: TLLmv or clone-derived virus mutants CDV: BS [M-P1] and CDV: BS _VP1 [K98E / E145A / L169F] at 37 ° C. for 1 hour. 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 post infection (dpi). The supernatant was subjected to virus titration after the virus disaggregation process by vortexing vigorously for 15 minutes at room temperature in [6, 71], 1% sodium deoxycholate as before. Viral titers were counted with the Reed and Muench method [61] and reported as CCID ₅₀ / ml on an infection calculator [62].

Example 10
Transfection of EV71: BS genomic RNA into mouse neuronal Neuro-2a and fibroblast NIH / 3T3 cells yields live virus progeny
[0110] Murine fibroblasts NIH / 3T3 and neuroblastoma Neuro-2a cells were previously demonstrated to be non-permissive for EV71: BS infection, whereas Vero cells were permissive ( Or [71]). Two strains, both EV71: BS 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 nonpermissive cells, genomic RNA from EV71: BS was extracted and transferred into Vero, NIH / 3T3, and Neuro-2a cells. Erected. Similarly, genomic RNA from EV71: TLLm and EV71: TLLmv was transfected into these three cell lines for comparison. To assess the viability of the resulting virus progeny, the transfection supernatant was subsequently replated on fresh cells (FIG. 15A).

  [0111] Genomic RNA transfection of all three virus strains into Vero cells produced a lytic cytopathic effect (CPE) in the transfected cell monolayer (Figure 15B). Viral antigen expression was also observed in dead cells (FIG. 15C), indicating successful viral replication. Similar results were observed from transfection of NIH / 3T3 and Neuro-2a cell monolayers with either EV71: TLLm or EV71: TLLmv viral RNA (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 showing viral antigen expression, but did not result in complete CPE of the cell monolayer. Further passage of EV71: BS transfection supernatant from both NIH / 3T3 and Neuro-2a cells to fresh Vero cells induced induction of complete lysis of the cell monolayer (FIG. 15D) and viral antigen in dead cells Detection (Figure 15E). However, passage of the transfection supernatant on fresh NIH / 3T3 cells did not result in either CPE (FIG. 15D) or viral antigen expression (FIG. 15E).

Example 11
The 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
[0112] Previous analysis suggests that the 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 [M-P1], which represents the capsid protein of EV71: TLLm and expresses the nonstructural protein of EV71: BS. These CDVs were re-inoculated into various cell lines to assess the infection phenotype (Figure 16B).

  [0113] EV71: BS clone-derived virus (CDV: BS) induced CPE in Vero cells but not in NIH / 3T3 and Neuro-2a cells, whereas CDV: BS [M-P1] 48 hours post infection (hpi), CPE was induced in all three cell lines (FIG. 16C). Murine cells infected with CDV: BS [M-P1] produced detection of viral antigen in dead cells but not with CDV: BS (FIG. 16D). To assess the production and release of viable virus progeny at the first passage (P1), the clarified culture supernatant from infected cells was replated onto a fresh monolayer of the same cell line and at 72 hpi Virus yield was measured. Passage of both CDV: BS and CDV: BS [M-P1] on Vero cells showed high virus titers and a significant increase in titer was observed after further passages (P2) (FIG. 16E). On the other hand, passage of CDV: BS [M-P1] (P1) resulted in high virus yield in both NIH / 3T3 and Neuro-2a cells, but not CDV: BS (FIG. 16F).

Example 12
EV71: VP1-L169F amino acid substitution in BS capsid allows virus to enter murine cells and induce limited infection
[0114] The mouse cytocompatibility EV71: TLLm capsid protein allows entry of EV71: BS into murine cells, and we are interested in the identity of specific residues that confer this novel phenotype. I got it. Previous data regarding the comparison of polyprotein sequence alignments of EV71: BS and mouse cell-compatible EV71 strains showed numerous amino acid substitutions in VP1 and VP2 proteins that may be involved in viral receptor binding on host cells ( Or [71]). These residues include VP1 K98E, E145A, and L169F, and VP2 S144T and K149I. In order to determine which of these amino acid substitutions are required for mouse cell entry, individual mutations that produce these amino acid substitutions are transferred into the EV71: BS full-length cDNA clone through standard site-directed mutagenesis. It was introduced (FIG. 17A). These modified cDNA clones were independently transfected into Vero cells to generate clone-derived virus (CDV) and harvested supernatants were placed on freshly seeded Vero, NIH / 3T3, and Neuro-2a cells. Inoculated and evaluated for infection phenotype.

[0115] Vero cells infected with all mutant clone-derived virus (CDV) showed 100% CPE but CDV carrying the VP1 amino acid substitution, CDV: BS _VP1 [K98E], CDV: BS _VP1 [E145A ], And only CDV: BS _VP1 [L169F] 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: _BSVP1 ) and VP2 (CDV: _BSVP2 ) but infected with CDV: BS _VP2 . Only NIH / 3T3 cells showed viral antigen expression (FIGS. 17D and 17E). Moreover, Vero cells infected with all mutant CDVs produced measurable virus titers (FIG. 17F), suggesting virus viability, but only CDV: BS _VP1 [L169F] was assayed in Vero cells. When produced, it produced measurable virus titers in the 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 productive infection in murine cells requires a combined amino acid substitution at VP1
[0116] To assess whether combinations of amino acid substitutions in VP1 and VP2 are also able to allow EV71: BS to enter mouse cells, EV71: BS full-length genomic cDNA containing various combinations of amino acid substitutions Clones (BS _VP2 [S144T / K149I], BS _VP [K98E / E145A], BS _VP1 [K98E / E145A / L169F], and BS [VP1 / VP2]) were generated (FIG. 18A). Plasmid clones were independently transfected onto Vero cells and the resulting supernatant was used to inoculate Vero, NIH / 3T3, and Neuro-2a cells to assess the infection phenotype.

[0117] CDV assayed all 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 NIH / 3T3 cells when comparing murine cell lines (FIG. 18C). High virus 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 virus titers in the culture supernatants of infected NIH / 3T3 and Neuro-2a cells when assayed in Vero cells (FIG. 18E).

Example 14
EV71: BS virus containing a combined amino acid substitution of VP1 K98E, E145A, and L169F was successfully passaged in mouse Neuro-2a cells
[0118] Four of the clone-derived virus isolates: 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, the virus supernatant is passaged twice in the same cell line, ie from Neuro-2a to fresh Neuro-2a cells. did. Infection was monitored by assessment of CPE induction and viral antigen expression, and production of live virus progeny.

[0119] CDV: BS _VP1 [K98E / E145A / L169F] and CDV: BS [M-P1] only, as evidenced by detection of viral antigen (Figure 19A) and measurable viral titer (Figure 19B) Was able to succeed in succession 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 as compared to the first passage. On the other hand, CDV: BS [M-P1] was able to induce viral antigen expression in NIH / 3T3 cells (FIG. 19A), but no viable virus progeny 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 mutation (FIG. 19C).

Example 15
Mouse cell line compatible strain EV71: TLLmv binds to 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]. To confirm whether the mouse cell line compatible EV71 strain also utilizes SCARB2 during mouse cell infection, a competitive virus binding assay was performed. 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 grown in vitro. EV71: conjugated with TLLmv. A pan-enterovirus monoclonal antibody was used to detect the fluorescence of bound cells, and the fluorescence intensity was quantified. Preincubation of NIH / 3T3 cells with anti-mSCARB2 serum resulted in a significant decrease in EV71: TLLmv binding (FIG. 20A), which was not observed when cells were preincubated with non-specific 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 the planted NIH / 3T3 cells and infected by fluorescent tagging of bound panenterovirus monoclonal antibody. Evaluated. Preincubation of soluble mSCARB2 with virus reduced the severity of cell infection in a dose-dependent manner (FIG. 20C). Similar results were obtained when human SCARB2 (hSCARB2) protein was used in the preincubation (FIG. 20D).

Example 16
Preincubation of mouse cells with sera raised against SCARB2 protein blocks cell infection with EV71: TLLmv
[0122] To assess whether cell infection is reduced by blocking the interaction of EV71: TLLmv and SCARB2, after inoculating the implanted NIH / 3T3 cells with various dilutions of SCARB2 protein antiserum, Infection was assessed by inoculating live EV71: TLLmv and measuring the titer of live virus progeny. Low incubation of hSCARB2 antiserum (1: 20-1: 80) and cell preincubation resulted in a significant dose-dependent decrease in virus titer compared to controls (FIG. 20E). Similar results were obtained when cells were preincubated with mSCARB2 antiserum (FIG. 20F).

Example 17
CDV: BS [M-P1] and _{CDV: BS VP1 [K98E / E145A} / L169F] utilizes murine SCARB2 protein as a functional receptor for entry into murine cells
[0123] Both CDV: BS [M-P1] and CDV: BS _VP1 [K98E / E145A / L169F] stably infect mouse Neuro-2a cells. These CDVs, as well as the mouse cell-matched EV71: TLLmv strain, were also incubated with Neuro-2a cells with mSCARB2 antiserum to determine whether to utilize mSCARB2 for viral entry and decoating, followed by CDV Mutants were infected. 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 [M-P1] (FIG. 21B). . The titer determination of the culture supernatant 7 days after infection (dpi) showed significant differences in CDV: BS _VP1 [K98E / E145A / L169F] virus titers in cells preincubated with SCARB2 antiserum compared to controls. Does not reveal. On the other hand, significant virus titer reduction was detected in CDV: BS [M-P1] infected cells preincubated with serum compared to controls (FIG. 21D).

Example 18
Example 19 Materials and Methods
[0124] Animal model: To determine the animal infection phenotype of mouse cell compatible strains (EV71: TLLm and EV71: TLLmv), 5-6 day old Balb / c mice were infected with 10 ⁶ CCID ₅₀ virus, They were then observed for symptoms of disease and neurological complications. Animals were followed for up to 28 days, after which the animals were sacrificed and serum collected for detection of EV71 specific antibodies.

Example 19
Neuropathogenicity studies of mouse cell line matched EV71 (EV71: TLLm and EV71: TLLmv) in Balb / c mice
[0125] Of immunocompetent animals infected with EV71: TLLm (n = 7), 2 (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. On the other hand, animals infected with EV71: TLLmv showed more severe clinical signs of the disease. Nine out of 10 infected animals (90%) succumbed to disease within 8 days of infection (FIG. 13A) and 6 out of 9 (66.7%) died within 4 days after 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%). There appears to be no difference in the body weight of mice infected with EV71: TLLm compared to sham-infected animals, but mice infected with EV71: TLLmv showed dramatic weight loss within the first 10 days of infection. A decrease was shown (FIG. 13B). More interestingly, we observed a new symptom in EV71 infected mice, whereby paralyzed animals (FIG. 14A, arrows) presented tachypnea with significant subcostal retraction. In addition, 6 of 9 (66.7%) dead animals presented this symptom prior to euthanasia. Upon necropsy, we also observed that the lung was uncollapsible upon opening the chest cavity, which is the usual procedure for necropsy when collecting lung and heart tissue (FIG. 14B, arrow) It was suggested that edema was present. Histological examination of the tissue reveals pulmonary edema and bleeding characteristics in the alveolar space of the lung (FIG. 14C) and higher magnification images indicate the presence of hemorrhagic proteinaceous material (FIG. 14D, Arrow).

[0126] This example was also performed using susceptible mice, such as NSG mice. Similar results were obtained with the exception of higher disease severity and higher mortality.
Example 20
Screening for candidate anti-EV71 compounds
[0127] High-throughput in vitro screening of candidate anti-EV71 compounds is performed using the mouse NIH / 3T3 cell line that has been shown to be susceptible to cytolytic infection by EV71: TLLm or EV71: TLLmv virus strains. Promising compounds selected from in vitro screening are then tested in vivo in animal models. To achieve in vivo screening, a standardized (based on statistical calculations) number of BALB / c mice were prepared, titered, and maintained in a deep freezer (−80 ° C.) From a standardized stock of matched EV71 strains (EV71: TLLm and EV71: TLLmv), infect with the standardized titer of the virus strain. Candidate anti-EV71 compounds can be varied either to assay the potential prophylactic effect of the candidate compound, either before the appearance of the disease, or after the onset of disease to assay the potential therapeutic effect of the candidate compound. Infected mice are administered at a standardized dose.

Example 21
Materials and Methods of Examples 22-25
[0128] Mice and virus strains: Adult BALB / c mice were purchased from InVivos (Singapore) and bred to yield pups. EV71 strains used for inoculation include EV71: BS, EV71: TLLm, and EV71: TLLmv, the details and characteristics of which have been described herein.

  [0129] Ethical Description: Animal treatment was approved by the Temasem Lifesciences Laboratory Institutional Animal Care and Use Committee (IACUC) (approval number TLL-14-023). Infected animals that are moribund are: P. Euthanized by injection of 90 mg / kg pentobarbitone through the route. Neurological examinations were performed according to guidelines and standard procedures established by the Institutional Animal Care and Use Committee at the University of California, San Francisco (IACUC). Criteria for euthanasia included previously established guidelines [34]: (1)> 20% loss of maximum recorded body weight, (2)> 48 hours of paralysis, (3) lack of eating or Altered consciousness presented as either inability to eat, (4) inability to maintain posture, and (5) either stupor or coma. The pups were observed for a total of 28 days, and animals that survived this period were treated with Pentobarbitone I.V. P. Euthanized by injection.

[0130] Animal handling and infection: Groups of 8 mice of various ages (6, 14, 21, or 28 days of age) received EV71: TLLmv (dose 10 ⁶ CCID ₅₀ ) I.V. P. Or I. M.M. Inoculated by either injection. EV71: To determine the optimal dose of TLLmv, groups of 6 day old pups (8 per group) were transferred to various doses of virus (10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , or 10 ² CCID ₅₀ ). , I. P. Exposure through the route. In the first week after infection, infected animals were observed twice daily for disease presentation. Both moribund 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] Necropsy, gross pathological observations, and tissue collection: Using standard protocols, euthanized animals were necropsied and organs were harvested. Gross pathological examination was also performed and photographs were taken with IACUC approval. The lung surface was flushed twice with sterile PBS and blotted dry with filter paper before measuring wet weight. Organs collected for histological studies were stored in 10% neutral buffered formalin (NBF) for 1 week at 4 ° C.

[0132] Determination of tissue viral load: Frozen tissue was softened using a Teflon pestle and reconstituted in 1 ml DMEM (1% FBS). The sample was then mixed for 1 hour and centrifuged twice (20 g, 30 minutes, 4 ° C.) to remove tissue debris and obtain a clear virus. Viral samples were disaggregated in 1% sodium deoxycholate [88], then serially diluted 10-fold 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]. Viral titers were counted and reported as CCID ₅₀ per g tissue.

  [0133] Tissue processing for histological analysis: Fixed tissues were dehydrated in increasing concentrations of 70%, 95% and 100% ethanol. Tissues were incubated in 2 changes of alcohol and 3 changes of Histoclear II (Electron Microscience Sciences, USA) and finally infiltrated with 4 changes of molten paraffin wax. All incubations were at room temperature for 1 hour with gentle shaking at 100 rpm. Paraffin infiltration was performed in an oven set at 65 ° C. Using a microtome, paraffin-embedded tissue blocks were sectioned (5 μm), loaded onto polylysine-coated glass slides, dried at 42 ° C. overnight, and then stored at room temperature until further use.

  [0134] Staining of tissue sections: tissue sections were dewaxed by incubation in two exchanges of Histoclear II and then slowly dehydrated in decreasing alcohol concentrations of 100%, 95%, 70%, and 50% did. 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 incubating for 15 minutes at room temperature (RT). The slide was then rinsed in water, destained with 1% acid alcohol (95% ethanol, 1% HCl), soaked in 0.2% NH 4 OH, and rinsed in water for 10 minutes before contrasting in eosin solution. Stained. The slides were then destained in 95% ethanol and dehydrated by 3 changes of absolute alcohol and 2 changes of Histoclear II. Finally, the tissue was set in DPX mounting solution (Sigma Aldrich, USA).

[0135] Immunohistochemistry: After dewaxing and rehydration, slides were subjected to heat-induced antigen recovery by incubation at 96 ° C for 30 minutes in histological grade microwave oven and citrate buffer (pH 6.0). did. Slides were allowed to cool to RT for 3 hours and subsequently 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. Then, slides Tris-buffered saline (pH 7.4), washed five times in 0.05% Tween-20 (TBS- T), and rinsed twice with TBS, a _3% H 2 _{O 2} Endogenous peroxidase was quenched by addition for 1 hour at RT. 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, the slides were incubated in diaminobenzidine (DAB) substrate and counterstained with hematoxylin.

  [0136] Mapping of viral antigens and virus-induced lesions in tissue sections: Template images of representative coronal sections of mouse brain, 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. Coronal fragment mouse brain atlas (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. The area where the presence of viral antigen and virus-induced lesion was shown was then marked on the template image.

  [0137] Measurement of serum catecholamine levels: During autopsy, blood samples were collected by cardiac puncture of moribund animals. Serum was obtained by centrifuging the clotted sample at 3000 g, 4 ° C. for 30 minutes, and then using a 2-CAT (AN) ELISA kit (Labor Diagnostics Nova, Germany) according to the manufacturer's protocol. Stored at −20 ° C. until catecholamine levels were determined.

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

  [0139] Comparison of infection phenotypes induced by virus strains EV71: TLLm and EV71: TLLmv: 106 CCID 50 doses of EV71: BS, EV71: TLLm, or EV71: TLLmv in 6 day old BALB / c mouse pups Were inoculated by intraperitoneal (IP) or intramuscular (IM) route (n = 10 mice per group). During the first week after inoculation, animals were observed twice daily for signs of disease and euthanized when critical signs were detected as described above.

[0140] Measurement of neutralizing antibody levels in serum from infected mice: Blood samples were collected by cardiac puncture at necropsy and centrifuged at 3000 g, 4 ° C for 20 minutes before clotting at room temperature, and serum was collected. Got. Samples were stored at −20 ° C. until further analysis. Random samples of frozen stock were assayed for neutralizing antibody titers. Two-fold serial dilutions of serum (1: 20-1: 1280) were prepared in 96 well plates and mixed with 100 CCID ₅₀ virus. After the mixture was incubated at 37 ° C. for 1 hour, NIH / 3T3 cells (6,000 cells / well) were added. Plates were incubated at 37 ° C for several days and observed for CPE for 4-10 days. Reed and Muench methods were used to determine neutralizing antibody titers (reported as units per ml serum).

Example 22
Infection kinetics of modified EV71 strain in murine hosts
[0141] Current animal models of enterovirus 71 (EV71) -induced neurological disease and pathology only partially replicate human disease. We therefore use the murine cell (NIH / 3T3) compatible virus strains EV71: TLLm and EV71: TLLmv to proliferatively infect both rodent and primate cell lines, and clinical disease pathology. The objective was to develop a model of authentic authenticity [88]. First, the relative pathogenicity of EV71: TLLm and EV71: TLLmv compared to the parental strain EV71: BS is shown in the pathology of disease in 1 week old BALB / c mice infected with the virus via the intraperitoneal (IP) route. Evaluated by evaluating. Although seroconversion was observed in all inoculated animals, only mice infected with either EV71: TLLm or EV71: TLLmv progressed to lethal disease (FIGS. 22a and 22b). Similarly, when matched strains were administered through the alternative intramuscular (IM) route, they were again associated with reduced host survival compared to the parental strain EV71: BS (57% survival; FIG. 22c). I. P. And I. M.M. When considering the route of infection together, the median survival time after inoculation was 4 days post infection (DPI) for EV71: TLLmv infection and 7 DPI for EV71: TLLm infection (χ ² = 6.840; p = 0.089). Taken together, these data indicate that the murine cytocompatible virus strain EV71: TLLmv is associated with the maximum level of lethality and was therefore used in all subsequent experiments.

[0142] The optimal viral dose, inoculation route, and mouse age used for further model development were then evaluated. First, disease severity in EV71: TLLmv-infected mice is virus dose dependent and the lowest survival rate is observed in animals inoculated with a median cell culture infectious dose of 10 ⁶ (CCID ₅₀ ) (FIG. 23a). ), And the median humane endpoint (HD ₅₀ ) was confirmed to be equivalent to 3.98 × 10 ³ CCID ₅₀ (FIG. 23b). The effects of animal age and infection route on host survival were then assessed; the survival curves of 1 week old mice injected with EV71: TLLmv were not significantly affected by the inoculation route (FIG. 23c), Regardless of the site of virus injection, young animals showed consistently poorer survival than older animals (FIG. 23d). Only in older animals it is possible to identify any effect of the route of infection on disease severity; P. Although completely resistant to infection, some animals were M.M. Viral injection through the route did not survive (FIGS. 24a and 24b). Seroconversion 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 1 to 3 weeks of age. Of the conditions tested herein, disease severity was greater in 1 week old mice injected with a viral dose of 10 ⁶ CCID ₅₀ , either intraperitoneally or intramuscularly.

Example 23
EV71: Disease progression in TLLmv infected mice
[0143] The majority of 1 week old mice inoculated with EV71: TLLmv succumbed to disease and showed numerous clinical signs of neurological disease. Infected animals showed either transient or persistent ataxia, local or systemic tremor, unstable gait, and limb paralysis and paralysis by the time of euthanasia. Based on clinical presentation, sick animals could be easily classified into 4 groups (Table 6). Survivors included mice that did not appear moribund at any time during the 28 day observation period. Class I animals showed severe signs, including inability to maintain posture and either stupor or coma after only 3-7 DPI. All mice in this group showed spastic paraparesis and / or paralysis (fore limbs, hind limbs, or both), but some animals lacked respiratory symptoms (Class 1B), others were frequent It was further characterized by symptoms of dyspnea (class IA), including breathing, hiccups, panting, and subcostal retraction. The observation of prominent features in EV71: TLLmv infected mice presenting class IA signs of disease was made by a 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. Panting, subcostal retraction, and foamy fluid exiting the nostril were seen in the second mouse. Observation of prominent features in EV71: TLLmv infected mice presenting class IB signs of disease was made by video of one class IB mouse. The animal was unable to hold posture and was stupid. 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% maximum body weight), as well as persistent flaccid limbs (> 48 hours duration) (FIG. 23e). . In all disease classes, some of the infected animals showed hairless lesions or back hair loss spots that persisted for several days (FIG. 23f). EV71: TLLmv P. The majority of inoculated pups are classified as class I (FIG. 23g); class IA animals constitute 19.3% (n = 11; obvious respiratory signs), whereas class IB animals are 43.9. % (N = 25; unclear respiratory signs). Class II4 animals represented only 12.3% of infected pups (n = 7) and survivors made up 24.5% (n = 14) of all infected mice. A similar pattern of distribution among disease categories is M.M. It was also observed in offspring inoculated through the route (FIG. 23h). Taken together, these data indicate that mice infected with EV71: TLLmv show diverse incidences and severity of both neurological and respiratory symptoms that reflect the full spectrum of disease observed in human patients. Indicated.

Table 6
Clinical characteristics 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 Animals were unable to maintain posture but responded to physical toe stimulation.
^c Animals were unable to maintain posture and did not respond to physical toe stimulation.

^d Tachycardia was observed in 35% of class IB mice Example 24
Neurogenic pulmonary edema in mice presenting class IA symptoms
[0144] It was further investigated whether dyspnea observed in class IA mice was a sign of virus-induced pulmonary edema (PE). Comparison of macroscopic pulmonary pathology between class IA, class IB, and class II mice resulted in swollen class IA lungs, incomplete crushing at necropsy (FIGS. 25a-25d), and lungs from other groups In comparison, it was found to show increased wet weight (FIG. 22E). Comparison of sham inoculation (FIG. 25f) and lung tissue sections from class IA lungs reveals that there is a localized area of bleeding and proteinaceous and red blood cell filling fluids only in the alveolar space of infected animals. (FIG. 25g). These pathological features were not present in the lungs of class IB and class II mice (FIGS. 25h and 25i). Furthermore, we found no evidence of inflammatory infiltrates or viral antigens in lungs collected from any group of mice (FIG. 26a). Similarly, myocardial histochemical analysis 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 cannot be attributed to either pneumonia or congestive heart failure. We therefore attempted to determine if the PE observed in this group is neurogenic, so we next measured serum levels of catecholamines to It was determined whether the transmitter concentration was 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 more significant in class IA mice than those detected in either class II or mock-infected animals. Were observed to be higher (FIGS. 25j and 25k). These data strongly indicated that class IA mice exhibited EV71-induced neurogenic pulmonary edema (NPE) before death.

Example 25
EV71: TLLmv virus neurotropic 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 only, the distribution of EV71: TLLmv and virus-induced lesions in the brain and spinal cord of animals from each disease group was then evaluated. The majority of Class IA and Class IB animals showed ubiquitous staining of viral antigens (> 10 positive neurons detected) and mild lesions (> 5 lesions) in all CNS regions evaluated. ) (Table 7). In contrast, only 1 out of 5 mice with class II disease showed viral antigens and / or lesions in the sensory cortex, hippocampus, diencephalon, midbrain, medulla, 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 route; data not shown).

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

^a Density of viral antigens detected per slide; +, <10 positive neurons; ++ 10-20 positive neurons; +++,> 20 positive neurons
^b Proportion of animals showing viral antigens in the specified brain area (n = 5)
^c Distribution of lesions in neural tissue: +, <5 lesions; ++ 5-10 lesions; +++,> 10 lesions
^d Proportion of animals showing lesions in specified brain area (n = 5)
[0146] When comparing CNS pathology between class IA and class IB mice, both tissue lesions and viral antigens are located 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 Figures 27a-27d). Indeed, when compared to class IB mice, class IA animals showed more extensive neurodegeneration, phagocytosis, and necrosis in hippocampal CA3 neurons (FIGS. 28a and 28b). Class IA mice also showed strong viral antigen staining in the hypothalamus, which was accompanied by significant 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 are also capable of ventro-postoral complex of the theramus (Figures 28e and 28f and Figure 27b), periaqueductal gray matter (PAG), mesencephalic reticular region, and motor Features of more severe virus-induced pathology in mesencephalic related tissues including associated superior hills (FIGS. 28g and 28h and FIG. 27c), and Purkinje cells and dentate nuclei of cerebellum (FIGS. 28i and 28j, FIGS. 27d and 29a) And the virus antigen strength was presented.

  [0147] In both disease groups (Class IA and Class IB), the most extensive distribution of viral antigens and lesions with neuronal damage and tissue inflammation is the medulla oblongata (Figs. 28k and 28l), particularly the intermediate reticulated nucleus (IRN), It was detected in the motion-related areas of the small cell reticular nucleus (PARN) and the spinal tract nucleus of the trigeminal nerve (sptV) (FIGS. 27d and 29b). However, only class IA mice showed viral antigens and tissue lesions in the ventral and dorsal regions of the medullary reticular nucleus (MdRN), the solitary nucleus (NTS) and the terminal area (AP) (FIGS. 29b and 29c). . For comparison, representative images of hippocampus, hypothalamus, thalamus, midbrain, cerebellum, and medulla from mock-infected mice are also shown (FIGS. 30a-30f). In contrast, Class IA and Class IB mice do not differ in terms of the distribution, localization or degree of tissue lesions or viral antigen staining within the anterior horn of motor cortex, somatosensory cortex, pons or spinal cord gray (Figure 28m and 28n, FIGS. 27a-27c and FIGS. 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 across all tissues 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" and similar reference controls are indicated elsewhere herein. Unless otherwise clearly contradicted by context, it shall be considered to include both the singular and plural. The terms “including”, “having”, “included” and “containing” are to be considered open-ended terms (ie, including but not limited to) unless otherwise indicated. ”). The recitation of value ranges herein is intended to serve as a simplification for individually referring to each distinct value within the range, unless otherwise indicated herein, and each distinct value is It is incorporated within this specification as if it were individually mentioned in the specification. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by background. The use of any examples or exemplary language (eg “for example”) provided herein is merely intended to facilitate an understanding of the invention and, unless otherwise claimed, the scope of the invention No restrictions are imposed on No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  [0149] Aspects of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these aspects will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors contemplate that those skilled in the art will properly use such variations and intend to practice the invention differently from those specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

  List of references

Claims (33)

  An animal model of enterovirus 71 (EV71) nerve infection, comprising a rodent infected with EV71 modified to infect rodents.   2. The animal model of claim 1, wherein the modified EV71 is a rodent cell line matched EV71.   The animal model of claim 1 or 2, wherein the modified EV71 is a rodent cell line matched EV71 which is EV71: TLLmv.   The animal model of claim 3, wherein EV71: TLLmv has been deposited with the Type Culture Collection China Center and has been assigned the deposit number CCTCC V201438.   The animal model of claim 1 or 2, wherein the modified EV71 is a rodent cell line matched EV71 which is EV71: TLLm.   The animal model of claim 5, wherein EV71: TLLm has been deposited with the Type Culture Collection China Center and has been assigned the deposit number CCTCC V2014437.   The animal model of claim 1, wherein the modified EV71 is an EV71 clone-derived virus (CDV) containing a mutation in VP1. The animal model according to claim 1 or 7, wherein the modified EV71 is EV71 CDV CDV: BS _VP1 [K98E / E145A / L169F].   The animal model according to any one of claims 1 to 6, wherein the rodent is immunocompetent.   The animal model of claim 9, wherein the rodent is a mouse.   11. The animal model of claim 10, wherein the rodent cell line is the mouse cell line NIH / 3T3.   9. The animal model of claim 7 or 8, wherein the rodent is immunocompetent.   The animal model of claim 12, wherein the rodent is a mouse.   14. The animal model of claim 13, wherein the rodent cell line is mouse cell line NIH / 3T3 or mouse cell line Neuro-2a.   3. The animal model of claim 1 or 2, wherein the rodent is immunocompetent.   16. The animal model of claim 15, wherein the rodent is a mouse.   17. The animal model of claim 16, wherein the mouse is a BALB / c mouse.   A method for screening antiviral agents comprising: a test group of animals and a control group of animals, wherein each group of animals comprises the animal model of any one of claims 1-17; Antiviral drug candidates are administered; disease progression is monitored in the test and control groups; disease progression in the test group is compared to disease progression in the control group; and disease progression in the test group compared to the control group Selecting said antiviral drug candidate to be reduced.   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 the animal.   A method for screening an antiviral vaccine comprising: a test group of animals and a control group of animals, wherein each group of animals comprises the animal model of any one of claims 1-17; Antiviral vaccine candidates are administered to the patient; disease progression is monitored in the test and control groups; disease progression in the test group is compared to disease progression in the control group; and disease progression in the test group compared to the control group Selecting said antiviral vaccine candidate to be reduced.   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 an animal. Infecting the rodent with EV71 modified to infect the rodent and breeding the infected rodent,
The method of preparing an animal model of claim 1, comprising the step of providing an animal model of EV71 nerve infection thereby.   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. 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.   26. The method of any one of claims 22-25, wherein the modified EV71 is a rodent cell line compatible EV71 that is EV71: TLLmv.   27. The method of claim 26, wherein EV71: TLLmv has been deposited with the Type Culture Collection China Center and has been assigned the deposit number CCTCC V201438.   26. The method of any one of claims 22-25, wherein the modified EV71 is a rodent cell line compatible EV71 that is EV71: TLLm.   30. The method of claim 28, wherein EV71: TLLm has been deposited with the Type Culture Collection China Center and has been assigned the deposit number CCTCC V201437.   26. 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. 31. The method of claim 30, wherein the modified EV71 is EV71 CDV CDV: BS _VP1 [K98E / E145A / L169F].   32. The method of any one of claims 22-31, wherein the rodent is a mouse.   35. The method of claim 32, wherein the mouse is a BALB / c mouse.