A neutralizing antibody protects Kunming mice against AKAV lethal challenge

A neutralizing antibody protects Kunming mice against AKAV lethal challenge | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A neutralizing antibody protects Kunming mice against AKAV lethal challenge Jingjing Wang, Ruyang Yu, Fang Wei, Chunyan Feng, Xiangmei Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6963641/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Akabane virus (AKAV) infection is associated with arthrogryposis-hydranencephaly syndrome in ruminants. Current commercialized AKAV live attenuated vaccines have safety concerns and cannot fully protect against all genotypes or emerging strains. Thus, developing a new type of vaccine or treatment is urgently required. The neutralizing antibodies (NAbs) directed against the Gc protein can efficiently neutralize the corresponding Bunyavirales viruses. We previously generated three NAbs against the Gc protein of AKAV that collectively recognize a highly conserved epitope among diverse AKAV genotypes and established a mouse model of AKAV infection. Here, our objective was to evaluate the protective efficacy of one of the produced NAbs, 4F12, against AKAV infection using the mouse model. Methods Suckling Kunming mice were first intraperitoneally administered varying doses of the NAb 4F12, followed by intraperitoneal (IP) or intracerebral (IC) challenge with a lethal dose of AKAV. Clinical symptoms, body weight, and mortality were then monitored and recorded daily for 14 days. The AKAV RNA, viral particles, antigens distribution, and microscopic lesions in the brain tissues of the experimental mice were analyzed. Results All mice that did not receive 4F12 pretreatment died before the experimental endpoint, regardless of the AKAV challenge routes. While a dose-dependent survival increase (50% ~ 83.33%) was observed in 4F12-pretreated mice, with higher antibody concentrations conferring greater protection against both IC and IP AKAV challenges. Moreover, all mice that survived AKAV challenge due to 4F12 pretreatment showed complete absence of AKAV RNA, viral particles, and antigens in brain tissues, with no observable virus-associated brain lesions. Conclusions We proved that the NAb 4F12 could reduce the AKAV-induced mortality in mice. 4F12 is a promising candidate suitable for clinical development as an AKAV therapeutic. The highly conserved epitope recognized by 4F12 provides critical insights for the design of new broadly protective AKAV vaccines. Akabane virus neutralizing antibody mouse model protective efficacy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Akabane virus (AKAV) is the etiological agent of Akabane disease in ruminants, including cattle, sheep, and goats. Akabane disease is characterized by abortion, stillbirth, premature birth, and congenital deformities in newborns, which have caused considerable economic losses to the cattle industry. Large outbreaks of Akabane disease occurred in Japan and Australia in the 1970s, causing abortion, stillbirth, and congenital arthrogryopsis and hydranencephaly (A-H syndrome) in more than 31,000 cases and more than 8000 cases, respectively [ 1 , 2 ]. Since then, epidemics of this disease have been reported frequently in Japan and South Korea [ 3 – 6 ]. Sporadic cases of Akabane disease were also found in other countries [ 7 – 12 ]. Transmitted primarily by the bites of midges of the genus Culicoides, including the species Culicoides brevitarisis, Culicoides oxystoma, and Cnebeculosus nebeculosus [ 13 ], AKAV is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa [ 14 ]. Although the use of live attenuated vaccines and inactivated vaccines has reduced the prevalence of Akabane disease, cases still occurred in areas where vaccines are administered [ 15 ]. Besides, inactivated vaccines often provide insufficient protection, while live attenuated vaccines carry the risk of virulence reversion and recombination with field AKAV strains. Therefore, it is important to develop effective vaccines and novel antiviral strategies to control this disease. AKAV is a Simbu serogroup virus belonging to the genus Orthobunyavirus within the family Bunyaviridae . AKAV harbors a tripartite, negative-strand RNA genome that contains the small (S), medium (M), and large (L) RNA segments. The S segment encodes a nucleocapsid (N) protein and a nonstructural protein NSs. The L segment encodes the RNA-dependent RNA polymerase (RdRp) in the ribonucleoprotein complex. The M segment encodes two viral envelope glycoproteins, Gn and Gc, derived from a single polyprotein precursor, and a nonstructural protein, NSm [ 16 ]. Glycoprotein Gc is a major AKAV-neutralizing antigen, which is important for the induction of the host’s immune response and is responsible for viral neutralization and attachment to cell receptors [ 17 , 18 ]. Antibodies directed against the Gc protein efficiently neutralize the corresponding Bunyavirales viruses [ 19 – 21 ] and may play a role in protection from the lethal infection [ 22 – 25 ]. Previously, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain, GenBank accession nos. MT761689, MT761688, and MT755621) and identified a broadly neutralizing epitope that is highly conserved across different genotypes of AKAV strains by using these mAbs [ 26 ]. We also established experimental mouse models infected with the TJ2016 strain [ 27 ]. In this study, we selected the NAb 4F12, with the highest neutralizing titers of the three mAbs, to evaluate its therapeutic protection against AKAV infection in a mouse model. 2 Materials and Methods Cells, antibodies, and virus Baby hamster kidney cells (BHK-21) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (SolarbioLife Sciences, Beijing, China). The hybridoma cells secreting the mAb 4F12 were prepared in our lab previously [ 26 ] and cultured in DMEM supplemented with 20% FBS and 1% penicillin-streptomycin. All the cells were stored in our laboratory (the Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection & Testing) and maintained at 37°C with 5% CO 2 . The mAb 2D3 specific to AKAV N protein and the rabbit polyclonal antibody (pAb) specific to AKAV were generated in our laboratory previously [ 28 ]. FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG were purchased from SolarbioLife Sciences (Beijing, China). The AKAV strain TJ2016 was isolated and maintained by our lab [ 28 ]. Animals Specific pathogen-free (SPF) female BALB/C mice aged 8 weeks and SPF Kunming mice aged 7 days were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Preparation and neutralizing identification of 4F12 To prepare enough NAb 4F12 for the animal trials, the hybridoma cells secreting the NAb 4F12 were cultured and injected into the abdominal cavity of the 8-week-old female BALB/C mice to generate mouse ascites as described previously [ 29 ]. The harvested ascites was mixed, aliquoted, and stored at -80℃. The neutralization of NAb 4F12 against the TJ2016 strain was assessed in BHK-21 cells. Briefly, the ascites 4F12 was serially diluted twofold (from 2 1 to 2 12 ) with three replicates using DMEM supplemented with 2% FBS and 1% penicillin-streptomycin. The dilutions were mixed with 200 TCID 50 of AKAV in equal volumes and incubated at 37°C for 1 h. Then the virus-mAb mixture was added to BHK-21 cells in 96-well plates and incubated at 37°C with 5% CO 2 . After 48 hours, the supernatant was discarded, and the BHK-21 cells were processed for indirect immunofluorescence assay (IFA). In detail, the cells were fixed with ice-cold absolute ethyl alcohol for 30 min and then incubated with primary antibody rabbit pAb (1:1000 dilution) against AKAV at 4°C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG (1:500 dilution) for 45 min. All cells were washed with PBS three times after each incubation step. The fluorescence was observed using the Invitrogen EVOS FL cell fluorescence imaging system (Thermo Fisher Scientific, USA), and the percentage of uninfected cells per well was recorded. 2.3 Animal trials In a pre-trial, 48 seven-day-old Kunming mice of both sexes were randomly divided into 12 groups of 4 animals each. The TJ2016 strain was 10-fold diluted in DMEM from 10 7 down to 10 0 TCID 50 /ml. Seven of the groups received intracerebral (IC) inoculation (10 µL/mouse) with viral titers of 10 − 2 ~10 4 TCID 50 per dose. Four of the groups received intraperitoneal (IP) inoculation (100 µL/mouse) with viral titers of 10 3 ~10 6 TCID 50 per dose. The remaining group was kept as the control, in which two mice received IC inoculation of 10 µL DMEM and the other two were administered 100 µL DMEM via IP injection. Detailed information was summarized in Table 1 . All mice were examined for clinical symptoms for 14 days. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial were euthanized. The minimum 100% lethal dose (LD100) of the TJ2016 strain in suckling Kunming mice by each inoculation route was recorded and used for the further experiments. Table 1 Mortality of the suckling Kunming mice inoculated with AKAV LgTCID 50 per mouse Inoculation route IC IP N/A 0/2 0/2 -2 0/4 N/A -1 0/4 N/A 0 4/4 N/A 1 4/4 N/A 2 4/4 N/A 3 4/4 0/4 4 4/4 0/4 5 N/A 2/4 6 N/A 4/4 N/A, not applicable (the mice were inoculated with DMEM by IC or IP routes, or no mice were present/analyzed in these groups); IP, intraperitoneal; IC, intracerebral. For the main trial, 30 mice were randomly assigned to 5 groups of 6 animals each. The mice were administered NAb 4F12 by IP route and inoculated with TJ2016 by IP or IC routes after 24 hours (Fig. 1 A and 1 B). For control groups, mice were inoculated as follows: four via IP route with TJ2016, four via IC route with TJ2016, two via IC route with DMEM, two via IP route with DMEM, and two via IP route with 4F12. Detailed information was summarized in Table 2 . All mice were weighed and examined daily. The clinical symptoms of each mouse were monitored and scored according to the scoring system established previously [ 27 ]. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial, 14 days post-inoculation (dpi), were euthanized. Brain tissues were collected immediately after premature death or euthanasia for further analysis. Table 2 Animal trail design and the survival rate Group Survival rate 4F12 AKAV / DMEM Route Volume (µL) per mouse Route TCID 50 per mouse IP-4F12-100 4/6 IP 100 IP 10 6 IP-4F12-200 5/6 IP 200 IP 10 6 IP-DMEM 2/2 N/A N/A IP N/A IP-AKAV 0/4 N/A N/A IP 10 6 IC-4F12-50 3/6 IP 50 IC 10 0 IC-4F12-100 4/6 IP 100 IC 10 0 IC-4F12-200 4/6 IP 200 IC 10 0 IC-DMEM 2/2 IP N/A IC N/A IC-AKAV 0/4 IP N/A IC N/A 4F12-Control 2/2 IP 200 N/A N/A N/A, not applicable (Mice in the corresponding groups were not inoculated with 4F12 or AKAV); IP, intraperitoneal; IC, intracerebral. 2.4 RNA extraction and RT-PCR amplification 2.4 RNA extraction and RT-PCR amplification Total RNA was extracted from brain tissues with the RNA Easy Fast Tissue/Cell Kit (TianGen Biotech, Beijing, China) following the manufacturer’s protocol. The extracted RNA was detected for the AKAV N gene by an RT-PCR assay using the HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China). The RT-PCR was conducted as previously described [ 27 ]. 2.5 Virus titration Approximately 100 mg of brain tissue was homogenized in 1 mL DMEM, followed by centrifugation at 12000 rpm for 15 min. The supernatant was collected for virus titration. All procedures were performed under low-temperature conditions. The homogenate supernatant was serially diluted 10-fold in DMEM supplemented with 2% FBS and 2% penicillin-streptomycin. The 10 − 2 to 10 − 7 dilutions were then added to confluent BHK-21 cell monolayers in 96-well plates at 100 µL per well. Four replicates were performed for each dilution, with three independent repetitions for each homogenate supernatant. The culture was continued at 37°C in a 5% CO 2 incubator for 48 hours. Then the supernatant was discarded, and the cells were fixed with ice-cold absolute ethyl alcohol for IFA analysis. Briefly, the mAb 2D3 (1:1000 dilution) was used as the primary antibody, and the FITC-conjugated goat anti-mouse IgG (1:500 dilution) was used as the secondary antibody. The fluorescence was observed, and the virus titers were calculated by using the Reed-Muench method [ 30 ]. 2.7 Histopathology and immunohistochemistry (IHC) Half of each collected brain was fixed immediately in 4% paraformaldehyde and sent to Bioss (Beijing, China) for histopathological analysis. The fixed brain tissues were embedded in paraffin wax and sectioned into 4 µm sections. Each sample was examined in two sections. One section was stained with hematoxylin and eosin (HE) to observe pathological changes. The other section was used to detect the AKAV antigen-positive cells, with the rabbit pAb (1:1000 dilutions) specific to AKAV as the primary antibody and the goat anti-rabbit IgG H&L (HRP polymer) (Bioss, Beijing, China) as the secondary antibody. The severity of microscopic brain lesions and AKAV antigen levels were evaluated and scored from 0 to 4, as previously described [ 27 ]. 2.8 Statistical analysis All statistical analyses were performed using GraphPad Prism version 8.0 software (GraphPad Software, USA). Multiple t test-one per row was used for assessing differences between two groups. For comparisons of the virus titers within the same group, a two-tailed unpaired t-test with Welch’s correction was used. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered extremely significant. 3 Results 3.1 NAb 4F12 inhibits the AKAV infection in vitro To confirm the neutralizing activity of our newly prepared mouse ascites 4F12, we evaluated the ability of 4F12 to inhibit AKAV infection in BHK-21 cells. As a result, neutralizing activity of 4F12 was observed, and the inhibition was dependent on the dose of 4F12 when the AKAV amount was fixed. The ascites 4F12 exhibited 100% inhibition against AKAV infection in BHK-21 cells at dilutions up to 1:8, while maintaining > 50% inhibitory activity at dilutions as high as 1:64 (Fig. 2 ). 3.2 Determination of LD100 for AKAV infection in suckling Kunming mice Previous study has shown that the AKAV strain TJ2016 can kill the suckling Kunming mice by IC or IP route [ 27 ]. In this study, we aim to study whether the NAb 4F12 can protect the Kunming mice against AKAV lethal challenge. Therefore, we did a pre-trial to determine the minimum LD100 of AKAV in 7-day-old Kunming mice by each inoculation route. As the results in Table 1 showed, the minimum LD100 of the TJ2016 strain in suckling Kunming mice was 10 0 TCID 50 /mouse by IC route or 10 6 TCID 50 /mouse by IP route, which was used for the subsequent animal experiment. 3.3 NAb 4F12 protects Kunming mice against AKAV lethal challenge In preliminary experiments, we found that administering 4F12 at 24 hours before AKAV infection provided stronger protection compared to administration at 24 hours post-infection (data not shown). Therefore, only prophylactic administration was employed in this experiment. Here, 4F12 showed great protection from clinical symptoms and mortality. In the IP infection groups, 66.67% (4/6) of mice in the IP-4F12-100 group and 83.33% (5/6) of mice in the IP-4F12-200 group survived until the end of the trial (Table 2 and Fig. 3 A). In the IC infection groups, survival rates of 50% (3/6), 66.67% (4/6), and 66.67% (4/6) were observed in AKAV-infected mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200, respectively (Table 2 and Fig. 3 B). The results demonstrate a dose-dependent increase in survival rates among AKAV-infected mice with escalating doses of 4F12. No clinical symptoms were observed in any mouse surviving until the end of the experiment in any group. While neurological symptoms such as tremors, ataxia, paddling movements, or paralysis were observed in all the mice that died or were euthanized before the end of the experiment in all the groups. After performing clinical scoring for all mice in each group, we found that the average clinical symptom scores in groups IP-4F12-100 and IP-4F12-200 were significantly lower than those in group IP-AKAV at 7 ~ 14 dpi; the scores in group IC-4F12-100 were significantly lower than those in group IC-AKAV at 7 ~ 14 dpi; and the scores in group IC-4F12-200 were significantly lower than those in group IC-AKAV at 3 ~ 14 dpi (Fig. 3 C and D). For average daily weight gain (ADWG), no significant differences were observed between the groups (Fig. 3 E and F). 3.4 Detection of AKAV in brains of the inoculated mice To determine whether AKAV was present in the brain tissues of the inoculated mice, we detected the AKAV-N gene in brain tissue samples using RT-PCR and quantified the viral load by a microtitration infectivity assay. RT-PCR results showed that positive bands with 649 bp were only observed in the brains of the mice that died or were euthanized before the end of the experiment in all the groups (Fig. 4 A and B). Consistent with the RNA detection results, viral titer determination demonstrated that replication-competent AKAV particles were detected exclusively in the brain tissues of the mice that died or were euthanized before the end of the experiment (Fig. 4 C and D). 3.5 NAb 4F12 protects against AKAV invasion into brain tissues and prevents virus-induced brain lesions in intraperitoneally infected mice Since no AKAV was detected in the surviving mice of groups IP-4F12-100 and IP-4F12-200, we hypothesized that the pre-administration of 4F12 prevented the invasion of intraperitoneally injected AKAV into brain tissues. To further validate this hypothesis, Brain tissues of these surviving mice were subjected to IHC analysis and HE staining. Brains of mice in groups IP-DMEM and 4F12-control were used as the negative control, and brains of mice in the IP-AKAV group were used as the positive control. As expected, neither AKAV antigens (Fig. 5 A and B) nor microscopic brain lesions (Fig. 5 C and D) were observed in the brain tissues of these surviving mice. While viral antigens in the neurons as well as the lesions, including perivascular cuffing (PVC) of lymphocytes and macrophages, glial nodules (GN) consisting of microglial cells, and tissue liquefaction with loose structure, were present in the brains of the mice in the IP-AKAV group (Fig. 5 ). 3.6 NAb 4F12 inhibits AKAV replication in brain tissues and prevents virus-induced brain lesions in intracranially infected mice We next investigated whether 4F12 pretreatment could suppress AKAV replication and prevent virus-induced brain lesions following IC inoculation. Histopathology examinations were performed on the brain tissues of the surviving mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200. Brains of mice in groups IC-DMEM and IC-AKAV served as the negative and positive controls, respectively. As a result, the AKAV antigen was only detected in brains from the IC-AKAV group (Fig. 6 A and B). Besides, brain lesions such as PVC and tissue liquefaction with loose structure were observed exclusively in the IC-AKAV group (Fig. 6 C and D). Histopathological analysis revealed no apparent abnormalities in the surviving mice from Groups IC-4F12-50, IC-4F12-100, and IC-4F12-200 relative to controls (Fig. 6 ). 4 Discussion Vaccination with the live attenuated vaccines has proven to be the most effective way to prevent Akabane disease. However, there are still some safety concerns regarding these vaccines. Additionally, existing live attenuated vaccines might not cover newly emerged AKAV strains arising from genetic mutations or recombination. Developing a safe and broadly applicable passive antibody treatment strategy could be a promising alternative. Studies on various viruses, including SARS-CoV-2, MERS-CoV, and EBV, have demonstrated that NAbs exhibit excellent prophylactic or therapeutic efficacy in animal models [ 31 – 35 ]. The Gc protein, which can mediate cell attachment and membrane fusion, as well as induce the production of NAbs to neutralize the infectivity of corresponding viruses, is very closely related to the pathogenicity and immunogenicity of bunyaviruses. Recent studies on bunyavirus, such as Rift Valley fever virus (RVFV), Schmallenberg virus (SBV), Crimean-Congo Hemorrhagic Fever Virus (CCHFV), Hantaviruses, and Severe fever with thrombocytopenia syndrome virus (SFTSV), have proved that the NAbs directed against part or the whole of the Gc protein hold great promise for the development of bunyavirus antiviral therapies [ 24 , 36 – 38 ]. In our previous study, three mAbs targeting the truncated Gc protein (aa991 ~ 1232) of AKAV were generated and were identified to possess in vitro neutralizing activity. All of the mAbs were mapped to the same linear epitope, 1134 SVQSFDGKL 1142 , which is highly conserved across different genotypes of AKAV strains [ 26 ]. In this present study, we selected one of the NAbs, 4F12, with the highest neutralizing titer, to analyze its ability to protect against AKAV infection in a mouse model. The NAb 4F12 was shown to reduce and prevent AKAV-induced morbidity and mortality (from 100–16.67% ~ 50%) via IC or IP inoculation in suckling Kunming mice upon prophylactic administration. The brain tissues of all the surviving mice showed no detectable AKAV RNA, viral particles, or antigens and exhibited no AKAV-induced pathological alterations. Given that 4F12 recognizes a highly conserved linear epitope, we speculate that this NAb may confer broad-spectrum protection against different AKAV genotypes, possibly even against emerging variants, and this epitope could serve as a target for the development of new broadly protective AKAV vaccines. However, since we do not have other AKAV strains in stock, it is currently impossible to conduct the relevant animal experiments to verify this. Although the mouse model is a classic and commonly used system for evaluating drug or vaccine efficacy, validation in large animal models (such as cattle or sheep) remains necessary before 4F12 can be applied clinically. Our recent study [ 27 ], as well as the positive controls in this study, showed that the AKAV strain TJ2016 can kill the suckling Kunming mice by both IC and IP routes, and AKAV particles can be detected in the brains of the dead mice. While in this study, we found that preemptive administration of 4F12 not only prevented intraperitoneally injected AKAV from invading the brain but also suppressed the replication of intracranially inoculated AKAV viral particles, ultimately achieving viral clearance. Whether 4F12 employs the same mechanism to counteract AKAV invading through different routes remains unclear. The mechanistic basis for these observations requires further investigation. Some of the antibodies can prevent viral entry into cells or induce lysis of infected cells through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) [ 39 ]. The ADCC and CDC effects of NAb 4F12 should be further explored. In addition to Gc-specific NAbs, Gn-derived NAbs represent an equally crucial component of the immune response. NAbs targeting SFTSV Gn protein can elicit a robust humoral response and inhibit the viral infection, which can serve as promising therapeutic drugs for treating SFTSV infection [ 25 , 38 , 40 ]. Future studies should further investigate (1) the therapeutic efficacy of AKAV Gn-specific NAbs, (2) the combinatorial effects of Gc- and Gn-targeting NAbs, and (3) the underlying protective mechanisms mediated by these antibodies. Our study revealed a positive correlation between 4F12 ascites administration and improved survival rates in AKAV-infected mice. However, the unpurified nature of the ascites fluid precluded precise antibody dosage determination, representing a key limitation. Future studies should employ purified antibodies with graded dose regimens to obtain more accurate results. Furthermore, although the increased survival rate strongly demonstrates 4F12's protective efficacy, we were unable to analyze antibody levels or viral load dynamics in blood due to the impracticality of collecting blood samples from 7-day-old Kunming suckling mice. Altogether, our study demonstrates that the NAb 4F12, targeting a highly conserved epitope, provides potent protection against lethal AKAV infection in a mouse model. These findings not only offer a novel therapeutic option for AKAV infection but also provide critical insights for the design of new broadly protective AKAV vaccines. Declarations Ethis statements Animal studies were carried out in strict accordance with the experimental animal care and use guidelines of Beijing Animal Control Committee. All experimental protocols were reviewed and approved by the Animal Welfare Ethics Committee of Beijing MDKN Biotechnology Co., LTD., with approval No. MDKN-2024-104. All efforts were made to minimize animal suffering. Consent for publication Not applicable. Availability of data and materials All data associated with this study are included in the paper. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2022YFD1800505), the Fundamental Research Funds of Chinese Academy of Quality and Inspection & Testing (2024JK002), and the Beijing Natural Science Foundation (6254044). Author Contributions JJ.W.: Conceptualization, methodology, formal analysis, funding acquisition, writing—original draft preparation. RY.Y. and F.W.: investigation, methodology. CY.F. and XM.L.: funding acquisition, formal analysis. DJ.C. and SQ.W.: supervision, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable. References Coverdale OR, Cybinski DH, St George TD. 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A broadly protective antibody targeting glycoprotein gn inhibits severe fever with thrombocytopenia syndrome virus infection. Nat Commun. 2024 2024 Aug 15;15(1):7009. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=39147753&query_hl=1 doi: 10.1038/s41467-024-51108-z Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6963641","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492368834,"identity":"d7a14454-5a44-478d-840e-b783400a7df7","order_by":0,"name":"Jingjing Wang","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Wang","suffix":""},{"id":492368835,"identity":"cd616cf6-2438-48ab-8309-f3f0287c87d1","order_by":1,"name":"Ruyang Yu","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Ruyang","middleName":"","lastName":"Yu","suffix":""},{"id":492368836,"identity":"a6775140-59cb-41ad-a558-c565d9949f16","order_by":2,"name":"Fang Wei","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Wei","suffix":""},{"id":492368837,"identity":"40e9abe6-d892-4bfe-91b4-64ca63d124ff","order_by":3,"name":"Chunyan Feng","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Feng","suffix":""},{"id":492368838,"identity":"a6045f49-0ea1-4b14-afbc-57a9eab9e754","order_by":4,"name":"Xiangmei Lin","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Xiangmei","middleName":"","lastName":"Lin","suffix":""},{"id":492368840,"identity":"2b395231-7446-4615-97c0-ce2cc4129a1b","order_by":5,"name":"Dongjie Chen","email":"","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":false,"prefix":"","firstName":"Dongjie","middleName":"","lastName":"Chen","suffix":""},{"id":492368843,"identity":"3046a7d2-dbdd-4c9f-aab4-92911f9380ee","order_by":6,"name":"Shaoqiang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAm0lEQVRIiWNgGAWjYLCCDwwSJOpgnEGyFmYekpTLt+eYSdv8scjjb2B++OgGMVoMzrwxk85tkyiWOMBmbJxDlBaJ3G3SuQ0SiQ0HeNikidIiPwOoxeKPROJ8orUw3ABqYWCTSNxAtBaDM+8/W/a2SSRuPEysX+Tb0xJv/PhTlzjvePPDx8Q5jCEBSjMTpxxZyygYBaNgFIwCXAAAgmwtfVT5l2gAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Quality and Inspection \u0026 Testing","correspondingAuthor":true,"prefix":"","firstName":"Shaoqiang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-06-24 08:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6963641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6963641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87948447,"identity":"8bb28b3c-820c-47fc-a96e-23490b0e652b","added_by":"auto","created_at":"2025-07-30 16:47:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":305081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental timeline for antibody delivery, virus challenge, and necropsy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The NAb 4F12 was administered by IP route at the doses of 100 μL or 200 μL per mouse. After 24 hours, the mice were challenged with AKAV (10\u003csup\u003e6 \u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mouse) by IP route. (B) The NAb 4F12 was administered by IP route at the doses of 50 μL, 100 μL, or 200 μL per mouse. After 24 hours, the mice were challenged with AKAV (10\u003csup\u003e0 \u003c/sup\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mouse) by the IC route. As the controls, mice were only injected with AKAV or DMEM by IC route or injected with AKAV, DMEM, or 4F12 by IP route. Mice were monitored daily and euthanized at 14 days post inoculation of AKAV. NAb, neutralizing antibody; IP, intraperitoneal; IC, intracerebral.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/2bc1e15ba4bc848f868726f8.jpg"},{"id":87949557,"identity":"be399186-a3bf-488c-945a-a6500f48375b","added_by":"auto","created_at":"2025-07-30 16:55:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":310030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe neutralization efficiency of NAb 4F12 against\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eAKAV \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e 100 μL of the AKAV strain TJ2016 and a series of two-fold serially diluted ascites 4F12, ranging from 1:2 to 1:4096, were mixed in equal volumes and incubated at 37℃ for 60 min. The ascites-virus mixture was used to infect BHK-21 cells (n = 3) and incubated at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 48 hours, the virus was then detected by IFA. Inhibition of infection was fitted to a dose-response curve.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/67666bb8844722b59f9c6dd3.jpg"},{"id":87949559,"identity":"85821296-ff7a-4fbe-ac97-c04a6799e6c0","added_by":"auto","created_at":"2025-07-30 16:55:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":747094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAb 4F12 protects the suckling Kunming mice when given before AKAV inoculation. \u003c/strong\u003e(A and B) Survival curve, (C and D) scores of clinical signs, and (E and F) ADWG of the inoculated mice. The data were shown as means ± SD (error bars). Statistical differences were labeled according to multiple t test-one per row. Asterisks (*) indicate a significant difference between IP-4F12-100 and IP-AKAV, or IP-4F12-200 and IC-AKAV, or IC-4F12-100 and IC-AKAV, or IC-4F12-200 and IC-AKAV groups (*, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01;***, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001). IC, intracerebral; IP, intraperitoneal; DPI, days post infection.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/2a6e438b4fdb06f4cd800a1f.jpg"},{"id":87949834,"identity":"af9ff846-474e-4af3-bdf8-b8364ffb0ef1","added_by":"auto","created_at":"2025-07-30 17:03:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":793733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection and determination of AKAV in brains of the inoculated mice.\u003c/strong\u003e (A) RT-PCR detection of the AKAV N gene in brains collected from the mice that died or were euthanized before the end of the experiment. (B) RT-PCR detection of the AKAV N gene in brains collected from the mice that survived until the end of the experiment (C and D) virus titers in brains of the mice in different groups. Virus titers in brain tissues were determined by microtitration infectivity assay in BHK-21 cells. A two-tailed unpaired t-test with Welch’s correction was used to compare the virus titers between two groups. No significant difference was observed between IP-4F12-100 and IP-AKAV, or IP-4F12-200 and IC-AKAV, or IC-4F12-50 and IC-AKAV, or IC-4F12-100 and IC-AKAV, or IC-4F12-200 and IC-AKAV groups. IC, intracerebral; IP, intraperitoneal; dead mice, the mice that died or were euthanized before the end of the experiment; survival mice, the mice that survived until the end of the experiment.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/0e1d5fce15e358d61c115714.jpg"},{"id":87949835,"identity":"34bfd2db-01b0-4931-880a-96b903cb5880","added_by":"auto","created_at":"2025-07-30 17:03:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7214183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAb 4F12 prevents AKAV invasion into brains and prevents the brain lesions in intraperitoneally infected mice.\u003c/strong\u003e (A and B) IHC examinations and average scores for AKAV antigen in the brains of the mice in the IP-AKAV group and the mice that survived until the end of the experiment in other groups. AKAV antigen is present in neurons with marked labeling intensity. Hollow triangles indicate positive signals. (C and D) Microscopic brain lesions stained with hematoxylin and eosin(HE) and average microscopic brain lesion scores from the dead mice in the IP-AKAV group and the surviving mice at the end of the experiment in other groups. Solid arrows indicate PVC of lymphocytes and macrophages. Hollow arrows indicate tissue liquefaction with loose structure.Solid triangles indicate GN consisting of microglial cells. IP, intraperitoneal.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/622c200959b93be5b7ae8baa.jpg"},{"id":87948457,"identity":"8f19ba6f-62ae-4036-ac51-e79538241ace","added_by":"auto","created_at":"2025-07-30 16:47:31","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":318230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNAb 4F12 inhibits AKAV replication in brain tissues and inhibits brain lesions in intracranially infected mice. \u003c/strong\u003e(A and B) IHC examinations and average scores for AKAV antigen in the brains of the mice in the IC-AKAV group and the mice that survived until the end of the experiment in other groups. AKAV antigen is present in neurons with marked labeling intensity. Hollow triangles indicate positive signals. (C and D) Microscopic brain lesions stained with HE and average microscopic brain lesion scores from the dead mice in the IC-AKAV group and the surviving mice at the end of the experiment in other groups. Solid arrows indicate PVC of lymphocytes and macrophages. Hollow arrows indicate tissue liquefaction with loose structure. IC, intracerebral.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/d9c0c0ddccecf551e443c9b3.jpg"},{"id":88713179,"identity":"11625a1d-fe7e-4bf5-80a0-12c16edc6e22","added_by":"auto","created_at":"2025-08-10 08:23:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10766502,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6963641/v1/7e925fff-0bb1-4a17-9864-448f42cd3549.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A neutralizing antibody protects Kunming mice against AKAV lethal challenge","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAkabane virus (AKAV) is the etiological agent of Akabane disease in ruminants, including cattle, sheep, and goats. Akabane disease is characterized by abortion, stillbirth, premature birth, and congenital deformities in newborns, which have caused considerable economic losses to the cattle industry. Large outbreaks of Akabane disease occurred in Japan and Australia in the 1970s, causing abortion, stillbirth, and congenital arthrogryopsis and hydranencephaly (A-H syndrome) in more than 31,000 cases and more than 8000 cases, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Since then, epidemics of this disease have been reported frequently in Japan and South Korea [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Sporadic cases of Akabane disease were also found in other countries [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Transmitted primarily by the bites of midges of the genus Culicoides, including the species Culicoides brevitarisis, Culicoides oxystoma, and Cnebeculosus nebeculosus [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], AKAV is widely distributed throughout Australia, Southeast Asia, East Asia, the Middle East, and Africa [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although the use of live attenuated vaccines and inactivated vaccines has reduced the prevalence of Akabane disease, cases still occurred in areas where vaccines are administered [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Besides, inactivated vaccines often provide insufficient protection, while live attenuated vaccines carry the risk of virulence reversion and recombination with field AKAV strains. Therefore, it is important to develop effective vaccines and novel antiviral strategies to control this disease.\u003c/p\u003e\u003cp\u003eAKAV is a Simbu serogroup virus belonging to the genus \u003cem\u003eOrthobunyavirus\u003c/em\u003e within the family \u003cem\u003eBunyaviridae\u003c/em\u003e. AKAV harbors a tripartite, negative-strand RNA genome that contains the small (S), medium (M), and large (L) RNA segments. The S segment encodes a nucleocapsid (N) protein and a nonstructural protein NSs. The L segment encodes the RNA-dependent RNA polymerase (RdRp) in the ribonucleoprotein complex. The M segment encodes two viral envelope glycoproteins, Gn and Gc, derived from a single polyprotein precursor, and a nonstructural protein, NSm [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Glycoprotein Gc is a major AKAV-neutralizing antigen, which is important for the induction of the host\u0026rsquo;s immune response and is responsible for viral neutralization and attachment to cell receptors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Antibodies directed against the Gc protein efficiently neutralize the corresponding \u003cem\u003eBunyavirales\u003c/em\u003e viruses [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and may play a role in protection from the lethal infection [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePreviously, we prepared and characterized three monoclonal antibodies (mAbs), 4D1, 4E6, and 4F12, against the Gc protein of AKAV (TJ2016 strain, GenBank accession nos. MT761689, MT761688, and MT755621) and identified a broadly neutralizing epitope that is highly conserved across different genotypes of AKAV strains by using these mAbs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We also established experimental mouse models infected with the TJ2016 strain [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, we selected the NAb 4F12, with the highest neutralizing titers of the three mAbs, to evaluate its therapeutic protection against AKAV infection in a mouse model.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCells, antibodies, and virus\u003c/h2\u003e\u003cp\u003eBaby hamster kidney cells (BHK-21) were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (SolarbioLife Sciences, Beijing, China). The hybridoma cells secreting the mAb 4F12 were prepared in our lab previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and cultured in DMEM supplemented with 20% FBS and 1% penicillin-streptomycin. All the cells were stored in our laboratory (the Institute of Animal Inspection and Quarantine, Chinese Academy of Quality and Inspection \u0026amp; Testing) and maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe mAb 2D3 specific to AKAV N protein and the rabbit polyclonal antibody (pAb) specific to AKAV were generated in our laboratory previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG were purchased from SolarbioLife Sciences (Beijing, China).\u003c/p\u003e\u003cp\u003eThe AKAV strain TJ2016 was isolated and maintained by our lab [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eSpecific pathogen-free (SPF) female BALB/C mice aged 8 weeks and SPF Kunming mice aged 7 days were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.\u003c/p\u003e\n\u003ch3\u003ePreparation and neutralizing identification of 4F12\u003c/h3\u003e\n\u003cp\u003eTo prepare enough NAb 4F12 for the animal trials, the hybridoma cells secreting the NAb 4F12 were cultured and injected into the abdominal cavity of the 8-week-old female BALB/C mice to generate mouse ascites as described previously [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The harvested ascites was mixed, aliquoted, and stored at -80℃.\u003c/p\u003e\u003cp\u003eThe neutralization of NAb 4F12 against the TJ2016 strain was assessed in BHK-21 cells. Briefly, the ascites 4F12 was serially diluted twofold (from 2\u003csup\u003e1\u003c/sup\u003e to 2\u003csup\u003e12\u003c/sup\u003e) with three replicates using DMEM supplemented with 2% FBS and 1% penicillin-streptomycin. The dilutions were mixed with 200 TCID\u003csub\u003e50\u003c/sub\u003e of AKAV in equal volumes and incubated at 37\u0026deg;C for 1 h. Then the virus-mAb mixture was added to BHK-21 cells in 96-well plates and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 48 hours, the supernatant was discarded, and the BHK-21 cells were processed for indirect immunofluorescence assay (IFA). In detail, the cells were fixed with ice-cold absolute ethyl alcohol for 30 min and then incubated with primary antibody rabbit pAb (1:1000 dilution) against AKAV at 4\u0026deg;C overnight, followed by incubation with FITC-conjugated goat anti-rabbit IgG (1:500 dilution) for 45 min. All cells were washed with PBS three times after each incubation step. The fluorescence was observed using the Invitrogen EVOS FL cell fluorescence imaging system (Thermo Fisher Scientific, USA), and the percentage of uninfected cells per well was recorded.\u003c/p\u003e\n\u003ch3\u003e2.3 Animal trials\u003c/h3\u003e\n\u003cp\u003eIn a pre-trial, 48 seven-day-old Kunming mice of both sexes were randomly divided into 12 groups of 4 animals each. The TJ2016 strain was 10-fold diluted in DMEM from 10\u003csup\u003e7\u003c/sup\u003e down to 10\u003csup\u003e0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/ml. Seven of the groups received intracerebral (IC) inoculation (10 \u0026micro;L/mouse) with viral titers of 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e~10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e per dose. Four of the groups received intraperitoneal (IP) inoculation (100 \u0026micro;L/mouse) with viral titers of 10\u003csup\u003e3\u003c/sup\u003e~10\u003csup\u003e6\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e per dose. The remaining group was kept as the control, in which two mice received IC inoculation of 10 \u0026micro;L DMEM and the other two were administered 100 \u0026micro;L DMEM via IP injection. Detailed information was summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All mice were examined for clinical symptoms for 14 days. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial were euthanized. The minimum 100% lethal dose (LD100) of the TJ2016 strain in suckling Kunming mice by each inoculation route was recorded and used for the further experiments.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eMortality of the suckling Kunming mice inoculated with AKAV\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLgTCID\u003csub\u003e50\u003c/sub\u003e per mouse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eInoculation route\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e4/4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2/4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e4/4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eN/A, not applicable (the mice were inoculated with DMEM by IC or IP routes, or no mice were present/analyzed in these groups); IP, intraperitoneal; IC, intracerebral.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor the main trial, 30 mice were randomly assigned to 5 groups of 6 animals each. The mice were administered NAb 4F12 by IP route and inoculated with TJ2016 by IP or IC routes after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For control groups, mice were inoculated as follows: four via IP route with TJ2016, four via IC route with TJ2016, two via IC route with DMEM, two via IP route with DMEM, and two via IP route with 4F12. Detailed information was summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All mice were weighed and examined daily. The clinical symptoms of each mouse were monitored and scored according to the scoring system established previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The mice showing severe paralysis were euthanized immediately and recorded as dead. The mice surviving to the end of the trial, 14 days post-inoculation (dpi), were euthanized. Brain tissues were collected immediately after premature death or euthanasia for further analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eAnimal trail design and the survival rate\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSurvival rate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e4F12\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003eAKAV / DMEM\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRoute\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eVolume (\u0026micro;L)\u003c/p\u003e\u003cp\u003eper mouse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eRoute\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTCID\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eper mouse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIP-4F12-100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIP-4F12-200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIP-DMEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIP-AKAV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIC-4F12-50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e0\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIC-4F12-100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e0\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIC-4F12-200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10\u003csup\u003e0\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIC-DMEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIC-AKAV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eIC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4F12-Control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2/2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c8\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eN/A, not applicable (Mice in the corresponding groups were not inoculated with 4F12 or AKAV); IP, intraperitoneal; IC, intracerebral.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003e2.4 RNA extraction and RT-PCR amplification\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e2.4 RNA extraction and RT-PCR amplification\u003c/div\u003e\u003cp\u003eTotal RNA was extracted from brain tissues with the RNA Easy Fast Tissue/Cell Kit (TianGen Biotech, Beijing, China) following the manufacturer\u0026rsquo;s protocol. The extracted RNA was detected for the AKAV N gene by an RT-PCR assay using the HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China). The RT-PCR was conducted as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Virus titration\u003c/h2\u003e\u003cp\u003eApproximately 100 mg of brain tissue was homogenized in 1 mL DMEM, followed by centrifugation at 12000 rpm for 15 min. The supernatant was collected for virus titration. All procedures were performed under low-temperature conditions. The homogenate supernatant was serially diluted 10-fold in DMEM supplemented with 2% FBS and 2% penicillin-streptomycin. The 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e dilutions were then added to confluent BHK-21 cell monolayers in 96-well plates at 100 \u0026micro;L per well. Four replicates were performed for each dilution, with three independent repetitions for each homogenate supernatant. The culture was continued at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 48 hours. Then the supernatant was discarded, and the cells were fixed with ice-cold absolute ethyl alcohol for IFA analysis. Briefly, the mAb 2D3 (1:1000 dilution) was used as the primary antibody, and the FITC-conjugated goat anti-mouse IgG (1:500 dilution) was used as the secondary antibody. The fluorescence was observed, and the virus titers were calculated by using the Reed-Muench method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2.7 Histopathology and immunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eHalf of each collected brain was fixed immediately in 4% paraformaldehyde and sent to Bioss (Beijing, China) for histopathological analysis. The fixed brain tissues were embedded in paraffin wax and sectioned into 4 \u0026micro;m sections. Each sample was examined in two sections. One section was stained with hematoxylin and eosin (HE) to observe pathological changes. The other section was used to detect the AKAV antigen-positive cells, with the rabbit pAb (1:1000 dilutions) specific to AKAV as the primary antibody and the goat anti-rabbit IgG H\u0026amp;L (HRP polymer) (Bioss, Beijing, China) as the secondary antibody. The severity of microscopic brain lesions and AKAV antigen levels were evaluated and scored from 0 to 4, as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e2.8 Statistical analysis\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism version 8.0 software (GraphPad Software, USA). Multiple t test-one per row was used for assessing differences between two groups. For comparisons of the virus titers within the same group, a two-tailed unpaired t-test with Welch’s correction was used. A \u003cem\u003eP\u003c/em\u003e value of less than 0.05 was considered statistically significant, and a \u003cem\u003eP\u003c/em\u003e value of less than 0.01 was considered extremely significant.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cb\u003e3.1 NAb 4F12 inhibits the AKAV infection\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm the neutralizing activity of our newly prepared mouse ascites 4F12, we evaluated the ability of 4F12 to inhibit AKAV infection in BHK-21 cells. As a result, neutralizing activity of 4F12 was observed, and the inhibition was dependent on the dose of 4F12 when the AKAV amount was fixed. The ascites 4F12 exhibited 100% inhibition against AKAV infection in BHK-21 cells at dilutions up to 1:8, while maintaining \u0026gt; 50% inhibitory activity at dilutions as high as 1:64 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003e3.2 Determination of LD100 for AKAV infection in suckling Kunming mice\u003c/h2\u003e\u003cp\u003ePrevious study has shown that the AKAV strain TJ2016 can kill the suckling Kunming mice by IC or IP route [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, we aim to study whether the NAb 4F12 can protect the Kunming mice against AKAV lethal challenge. Therefore, we did a pre-trial to determine the minimum LD100 of AKAV in 7-day-old Kunming mice by each inoculation route. As the results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed, the minimum LD100 of the TJ2016 strain in suckling Kunming mice was 10\u003csup\u003e0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mouse by IC route or 10\u003csup\u003e6\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mouse by IP route, which was used for the subsequent animal experiment.\u003c/p\u003e\u003ch2\u003e3.3 NAb 4F12 protects Kunming mice against AKAV lethal challenge\u003c/h2\u003e\u003cp\u003eIn preliminary experiments, we found that administering 4F12 at 24 hours before AKAV infection provided stronger protection compared to administration at 24 hours post-infection (data not shown). Therefore, only prophylactic administration was employed in this experiment. Here, 4F12 showed great protection from clinical symptoms and mortality. In the IP infection groups, 66.67% (4/6) of mice in the IP-4F12-100 group and 83.33% (5/6) of mice in the IP-4F12-200 group survived until the end of the trial (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the IC infection groups, survival rates of 50% (3/6), 66.67% (4/6), and 66.67% (4/6) were observed in AKAV-infected mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results demonstrate a dose-dependent increase in survival rates among AKAV-infected mice with escalating doses of 4F12.\u003c/p\u003e\u003cp\u003eNo clinical symptoms were observed in any mouse surviving until the end of the experiment in any group. While neurological symptoms such as tremors, ataxia, paddling movements, or paralysis were observed in all the mice that died or were euthanized before the end of the experiment in all the groups. After performing clinical scoring for all mice in each group, we found that the average clinical symptom scores in groups IP-4F12-100 and IP-4F12-200 were significantly lower than those in group IP-AKAV at 7 ~ 14 dpi; the scores in group IC-4F12-100 were significantly lower than those in group IC-AKAV at 7 ~ 14 dpi; and the scores in group IC-4F12-200 were significantly lower than those in group IC-AKAV at 3 ~ 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). For average daily weight gain (ADWG), no significant differences were observed between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F).\u003c/p\u003e\u003ch2\u003e3.4 Detection of AKAV in brains of the inoculated mice\u003c/h2\u003e\u003cp\u003eTo determine whether AKAV was present in the brain tissues of the inoculated mice, we detected the AKAV-N gene in brain tissue samples using RT-PCR and quantified the viral load by a microtitration infectivity assay. RT-PCR results showed that positive bands with 649 bp were only observed in the brains of the mice that died or were euthanized before the end of the experiment in all the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Consistent with the RNA detection results, viral titer determination demonstrated that replication-competent AKAV particles were detected exclusively in the brain tissues of the mice that died or were euthanized before the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 NAb 4F12 protects against AKAV invasion into brain tissues and prevents virus-induced brain lesions in intraperitoneally infected mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince no AKAV was detected in the surviving mice of groups IP-4F12-100 and IP-4F12-200, we hypothesized that the pre-administration of 4F12 prevented the invasion of intraperitoneally injected AKAV into brain tissues. To further validate this hypothesis,\u003c/p\u003e\u003cp\u003eBrain tissues of these surviving mice were subjected to IHC analysis and HE staining. Brains of mice in groups IP-DMEM and 4F12-control were used as the negative control, and brains of mice in the IP-AKAV group were used as the positive control. As expected, neither AKAV antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B) nor microscopic brain lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D) were observed in the brain tissues of these surviving mice. While viral antigens in the neurons as well as the lesions, including perivascular cuffing (PVC) of lymphocytes and macrophages, glial nodules (GN) consisting of microglial cells, and tissue liquefaction with loose structure, were present in the brains of the mice in the IP-AKAV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6 NAb 4F12 inhibits AKAV replication in brain tissues and prevents virus-induced brain lesions in intracranially infected mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether 4F12 pretreatment could suppress AKAV replication and prevent virus-induced brain lesions following IC inoculation. Histopathology examinations were performed on the brain tissues of the surviving mice from groups IC-4F12-50, IC-4F12-100, and IC-4F12-200. Brains of mice in groups IC-DMEM and IC-AKAV served as the negative and positive controls, respectively. As a result, the AKAV antigen was only detected in brains from the IC-AKAV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Besides, brain lesions such as PVC and tissue liquefaction with loose structure were observed exclusively in the IC-AKAV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). Histopathological analysis revealed no apparent abnormalities in the surviving mice from Groups IC-4F12-50, IC-4F12-100, and IC-4F12-200 relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eVaccination with the live attenuated vaccines has proven to be the most effective way to prevent Akabane disease. However, there are still some safety concerns regarding these vaccines. Additionally, existing live attenuated vaccines might not cover newly emerged AKAV strains arising from genetic mutations or recombination. Developing a safe and broadly applicable passive antibody treatment strategy could be a promising alternative. Studies on various viruses, including SARS-CoV-2, MERS-CoV, and EBV, have demonstrated that NAbs exhibit excellent prophylactic or therapeutic efficacy in animal models [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe Gc protein, which can mediate cell attachment and membrane fusion, as well as induce the production of NAbs to neutralize the infectivity of corresponding viruses, is very closely related to the pathogenicity and immunogenicity of bunyaviruses. Recent studies on bunyavirus, such as Rift Valley fever virus (RVFV), Schmallenberg virus (SBV), Crimean-Congo Hemorrhagic Fever Virus (CCHFV), Hantaviruses, and Severe fever with thrombocytopenia syndrome virus (SFTSV), have proved that the NAbs directed against part or the whole of the Gc protein hold great promise for the development of bunyavirus antiviral therapies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our previous study, three mAbs targeting the truncated Gc protein (aa991\u0026thinsp;~\u0026thinsp;1232) of AKAV were generated and were identified to possess \u003cem\u003ein vitro\u003c/em\u003e neutralizing activity. All of the mAbs were mapped to the same linear epitope, \u003csup\u003e1134\u003c/sup\u003eSVQSFDGKL\u003csup\u003e1142\u003c/sup\u003e, which is highly conserved across different genotypes of AKAV strains [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this present study, we selected one of the NAbs, 4F12, with the highest neutralizing titer, to analyze its ability to protect against AKAV infection in a mouse model. The NAb 4F12 was shown to reduce and prevent AKAV-induced morbidity and mortality (from 100\u0026ndash;16.67% ~ 50%) via IC or IP inoculation in suckling Kunming mice upon prophylactic administration. The brain tissues of all the surviving mice showed no detectable AKAV RNA, viral particles, or antigens and exhibited no AKAV-induced pathological alterations. Given that 4F12 recognizes a highly conserved linear epitope, we speculate that this NAb may confer broad-spectrum protection against different AKAV genotypes, possibly even against emerging variants, and this epitope could serve as a target for the development of new broadly protective AKAV vaccines. However, since we do not have other AKAV strains in stock, it is currently impossible to conduct the relevant animal experiments to verify this. Although the mouse model is a classic and commonly used system for evaluating drug or vaccine efficacy, validation in large animal models (such as cattle or sheep) remains necessary before 4F12 can be applied clinically.\u003c/p\u003e\u003cp\u003eOur recent study [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], as well as the positive controls in this study, showed that the AKAV strain TJ2016 can kill the suckling Kunming mice by both IC and IP routes, and AKAV particles can be detected in the brains of the dead mice. While in this study, we found that preemptive administration of 4F12 not only prevented intraperitoneally injected AKAV from invading the brain but also suppressed the replication of intracranially inoculated AKAV viral particles, ultimately achieving viral clearance. Whether 4F12 employs the same mechanism to counteract AKAV invading through different routes remains unclear. The mechanistic basis for these observations requires further investigation. Some of the antibodies can prevent viral entry into cells or induce lysis of infected cells through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The ADCC and CDC effects of NAb 4F12 should be further explored.\u003c/p\u003e\u003cp\u003eIn addition to Gc-specific NAbs, Gn-derived NAbs represent an equally crucial component of the immune response. NAbs targeting SFTSV Gn protein can elicit a robust humoral response and inhibit the viral infection, which can serve as promising therapeutic drugs for treating SFTSV infection [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Future studies should further investigate (1) the therapeutic efficacy of AKAV Gn-specific NAbs, (2) the combinatorial effects of Gc- and Gn-targeting NAbs, and (3) the underlying protective mechanisms mediated by these antibodies.\u003c/p\u003e\u003cp\u003eOur study revealed a positive correlation between 4F12 ascites administration and improved survival rates in AKAV-infected mice. However, the unpurified nature of the ascites fluid precluded precise antibody dosage determination, representing a key limitation. Future studies should employ purified antibodies with graded dose regimens to obtain more accurate results. Furthermore, although the increased survival rate strongly demonstrates 4F12's protective efficacy, we were unable to analyze antibody levels or viral load dynamics in blood due to the impracticality of collecting blood samples from 7-day-old Kunming suckling mice.\u003c/p\u003e\u003cp\u003eAltogether, our study demonstrates that the NAb 4F12, targeting a highly conserved epitope, provides potent protection against lethal AKAV infection in a mouse model. These findings not only offer a novel therapeutic option for AKAV infection but also provide critical insights for the design of new broadly protective AKAV vaccines.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthis statements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies were carried out in strict accordance with the experimental animal care and use guidelines of Beijing Animal Control Committee. All experimental protocols were reviewed and approved by the Animal Welfare Ethics Committee of Beijing MDKN Biotechnology Co., LTD., with approval No. MDKN-2024-104. All efforts were made to minimize animal suffering. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data associated with this study are included in the paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2022YFD1800505), the Fundamental Research Funds of Chinese Academy of Quality and Inspection \u0026amp; Testing (2024JK002), and the Beijing Natural Science Foundation (6254044).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJJ.W.: Conceptualization, methodology, formal analysis, funding acquisition, writing—original draft preparation.\u0026nbsp;RY.Y. and F.W.: investigation, methodology. CY.F. and XM.L.: funding acquisition, formal analysis. DJ.C. and SQ.W.: supervision, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoverdale OR, Cybinski DH, St George TD. Congenital abnormalities in calves associated with akabane virus and aino virus. Aust Vet J. 1978 1978 Mar;54(3):151-52. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\u0026amp;db=pubmed\u0026amp;dopt=Abstract\u0026amp;list_uids=687271\u0026amp;query_hl=1 doi: 10.1111/j.1751-0813.1978.tb05538.x\u003c/li\u003e\n\u003cli\u003eKurogi H, Inaba Y, Goto Y, Miura Y, Takahashi H. Serologic evidence for etiologic role of akabane virus in epizootic abortion-arthrogryposis-hydranencephaly in cattle in japan, 1972-1974. 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Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\u0026amp;db=pubmed\u0026amp;dopt=Abstract\u0026amp;list_uids=39147753\u0026amp;query_hl=1 doi: 10.1038/s41467-024-51108-z\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Akabane virus, neutralizing antibody, mouse model, protective efficacy","lastPublishedDoi":"10.21203/rs.3.rs-6963641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6963641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAkabane virus (AKAV) infection is associated with arthrogryposis-hydranencephaly syndrome in ruminants. Current commercialized AKAV live attenuated vaccines have safety concerns and cannot fully protect against all genotypes or emerging strains. Thus, developing a new type of vaccine or treatment is urgently required. The neutralizing antibodies (NAbs) directed against the Gc protein can efficiently neutralize the corresponding \u003cem\u003eBunyavirales\u003c/em\u003e viruses. We previously generated three NAbs against the Gc protein of AKAV that collectively recognize a highly conserved epitope among diverse AKAV genotypes and established a mouse model of AKAV infection. Here, our objective was to evaluate the protective efficacy of one of the produced NAbs, 4F12, against AKAV infection using the mouse model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSuckling Kunming mice were first intraperitoneally administered varying doses of the NAb 4F12, followed by intraperitoneal (IP) or intracerebral (IC) challenge with a lethal dose of AKAV. Clinical symptoms, body weight, and mortality were then monitored and recorded daily for 14 days. The AKAV RNA, viral particles, antigens distribution, and microscopic lesions in the brain tissues of the experimental mice were analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAll mice that did not receive 4F12 pretreatment died before the experimental endpoint, regardless of the AKAV challenge routes. While a dose-dependent survival increase (50% ~ 83.33%) was observed in 4F12-pretreated mice, with higher antibody concentrations conferring greater protection against both IC and IP AKAV challenges. Moreover, all mice that survived AKAV challenge due to 4F12 pretreatment showed complete absence of AKAV RNA, viral particles, and antigens in brain tissues, with no observable virus-associated brain lesions.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eWe proved that the NAb 4F12 could reduce the AKAV-induced mortality in mice. 4F12 is a promising candidate suitable for clinical development as an AKAV therapeutic. The highly conserved epitope recognized by 4F12 provides critical insights for the design of new broadly protective AKAV vaccines.\u003c/p\u003e","manuscriptTitle":"A neutralizing antibody protects Kunming mice against AKAV lethal challenge","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 16:47:26","doi":"10.21203/rs.3.rs-6963641/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adff81d0-1b48-43ed-b9d9-421235f13424","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-29T15:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-30 16:47:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6963641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6963641","identity":"rs-6963641","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}