Communicating the risks of handling bats: analysing approaches used by Australian stakeholders in the context of Australian bat lyssavirus.

Australian bat lyssavirus (ABLV) is a member of the Lyssavirus genus of the Rhabdoviridae family and is found in Australian bat species. It is of public health concern because of the rabies-like syndrome it causes in humans, resulting in government health and wildlife agencies using varied communication approaches to inform targeted audiences about zoonotic risks associated with handling bats. Despite these warnings, the number of reports of human-bat interactions remains high. This paper details a survey conducted to analyse the approaches utilised by a range of stakeholders to educate and communicate warnings to their target audiences. The survey focused on identifying the target audiences, communication methods used, along with the message frequency, content, and perceived effectiveness. Analysis of the top three messages delivered by stakeholders revealed that over half were information-focused messages and over a third, instruction-focused. Stakeholders identified the need to balance messaging about bat handling risks with information regarding the vulnerable status of bats and their environmental significance. Whilst the most common and (perceived) effective method of communication was one-on-one discussions, it was also identified to be ineffective for targeting mass audiences leading stakeholders to recognise the need to adapt to more efficient means of communication. The outcomes of this study may be useful to improve risk communication strategies regarding ABLV in Australia.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines - viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.

Herbal active small molecule as an immunomodulator for potential application on resistance of common carp against SVCV infection.

Herbal immunomodulators are an important part of prevention and control on viral diseases in aquaculture because of their propensity to improve immunity in fish. The present study was conducted to evaluate the immunomodulatory effect and antiviral activity of a synthesized derivative (serial number: LML1022) against spring viremia of carp virus (SVCV) infection in vitro and in vivo. The antiviral data suggested that LML1022 at 100 µM significantly inhibited the virus replication in epithelioma papulosum cyprini (EPC) cells, and may completely inhibit the infectivity of SVCV virion particles to fish cells by affecting the viral internalization. The results in the related stability of water environments also demonstrated that LML1022 had an inhibitory half-life of 2.3 d at 15 °C, which would facilitate rapid degradation of LML1022 in aquaculture application. For in vivo study, the survival rate of SVCV-infected common carp was increased 30% at least under continuous oral injection of LML1022 at 2.0 mg/kg for 7 d treatment. Additionally, pretreatment of LML1022 on fish prior to SVCV infection also obviously reduced the viral loads in vivo as well as an improved survival rate, showing that LML1022 was potential as an immunomodulator. As an immune response, LML1022 significantly upregulated the immune-related gene expression including IFN-γ2b, IFN-I, ISG15 and Mx1, indicating that its dietary administration may improve the resistance of common carp against SVCV infection.

Development of a recombinant adenovirus-vectored vaccine against both infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.

Recovery of Siniperca chuatsi rhabdovirus from cloned cDNA.

Siniperca chuatsi rhabdovirus (SCRV) is an important pathogen that infects mandarin fish. A reverse genetics system is an important technical platform for virus research. In this study, the minigenome in which the enhanced green fluorescent protein gene is flanked by the viral genomic ends of SCRV and transcribed using a T7 promoter-terminator cassette was constructed. Co-transfection of the minigenome construct with SCRV-supporting plasmids of N, P, and L in BSRT7 cells resulted in the expression of the reporter gene. Transcription of a positive-strand RNA copy from cDNA of the SCRV genome along with the viral N, P, and L proteins resulted in the recovery of infectious SCRV in cells. Viral titre up to 108 PFU/ml was achieved. Recombinant SCRV was verified by the detection of a unique restriction site engineered into the SCRV genome. The phenotypes of the recombinant SCRV and the parental virus were evaluated by plaque size, replication kinetics in vitro, and pathogenicity in vivo. The recovered SCRV from cDNA showed similar phenotypes compared to the parental virus. The established reverse genetics system is of great significance and value for the functional genome study of SCRV and for laying a foundation for the development of the viral vector and SCRV vaccine.

Specificity of DNA Vaccines against the Genogroup J and U Infectious Hematopoietic Necrosis Virus Strains Prevalent in China.

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In addition to the common genogroup J IHNV, genogroup U has been newly discovered in China. However, there is no effective DNA vaccine to fight against this emerging genogroup U IHNV in China. In this study, DNA vaccines encoding the IHNV viral glycoprotein (G) gene of the GS2014 (genogroup J) and BjLL (genogroup U) strains isolated from northern China were successfully developed, which were identified by restriction analysis and IFA. The expression of the Mx-1 gene and G gene in the spleens and muscles of the injection site as well as the titers of the serum antibodies were measured to evaluate the vaccine efficacy by RT-qPCR and ELISA. We found that DNA vaccine immunization could activate Mx1 gene expression and upregulate G gene expression, and the mRNA levels of the Mx1 gene in the muscles were significantly higher than those in the spleens. Notably, DNA vaccine immunization might not promote the serum antibody in fish at the early stage of immunization. Furthermore, the efficacy of the constructed vaccines was tested in intra- and cross-genogroup challenges by a viral challenge in vivo. It seemed that the DNA vaccines were able to provide great immune protection against IHNV infection. In addition, the genogroup J IHNV-G DNA vaccine showed better immune efficacy than the genogroup U IHNV-G or divalent vaccine, which could provide cross-immune protection against the genogroup U IHNV challenge. Therefore, this is the first study to construct an IHNV DNA vaccine using the G gene from an emerging genogroup U IHNV strain in China. The results provide great insight into the advances of new prophylactic strategies to fight both the genogroup J and U IHNV in China.

Taiwan Bat Lyssavirus: In Vitro and In Vivo Assessment of the Ability of Rabies Vaccine-Derived Antibodies to Neutralise a Novel Lyssavirus.

Rabies is a neglected tropical disease. The prototype virus, the rabies virus, still causes tens of thousands of human fatalities annually. Rabies is one member of the genus Lyssavirus. The burden of other lyssaviruses is unclear. The continued emergence of novel lyssaviruses means that assessment of vaccine efficacy against these viruses is critical, as standard rabies vaccines are not efficacious against all lyssaviruses. Taiwan bat lyssavirus (TWBLV) was first reported in 2018 following isolation from Japanese house bats. Since the initial detection and genetic characterisation, no attempts have been made to antigenically define this virus. Due to the inaccessibility of the wildtype isolate, the successful generation of a live recombinant virus, cSN-TWBLV, is described, where the full-length genome clone of the RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of TWBLV. In vitro and in vivo characterization of cSN-TWBLV was undertaken and demonstrated evidence for cross-neutralisation of cSN-TWBLV with phylogroup I -specific sera and rabies virus standard sera. For neutralisation equivalent to 0.5 IU/mL of WHO and World Organisation of Animal Health (WOAH) sera against CVS, 0.5 IU/mL of WOAH sera and 2.5 IU/mL of WHO sera were required to neutralise cSN-TWBLV. In addition, specific sera for ARAV and EBLV-1 exhibited the highest neutralising antibody titres against cSN-TWBLV, compared to other phylogroup I-specific sera.

Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

How modular protein nanoparticles may expand the ability of subunit anti-viral vaccines: The spring viremia carp virus (SVCV) case.

Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election system to minimise stress and the vaccination costs associated to injection. However, administration of prophylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminary i.p injection challenge. The G3 domain of SVCV glycoprotein G was overexpressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or ifngr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and ifngr1 (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality.

Dual-Targeting Polymer Nanoparticles Efficiently Deliver DNA Vaccine and Induce Robust Prophylactic Immunity against Spring Viremia of Carp Virus Infection.

Spring viremia of carp virus (SVCV) is highly contagious and lethal to most cyprinid fish, causing serious economic losses to the carp aquaculture industry. Although DNA vaccines can generate long-term humoral and cellular immune responses, which provide protective immunity against SVCV, the major drawback of DNA vaccines is their low immunogenicity in clinical tests. Here, we construct a dual-targeted polymer DNA vaccine delivery platform (MCS-PCHG) by using mannosylated chitosan to encapsulate the poly(d,l-lactide-co-glycolide)-loaded DNA vaccine containing the heavy-chain CH3 region (CH3) of common carp IgM and the antigenic domain (G131c). The developed nanovaccine delivery platform showed good biocompatibility in vivo and in vitro. With the modification of the mannose moiety and the modification of CH3, the constructed MCS-PCHG could efficiently activate the maturation of antigen-presenting cells. Moreover, we observe significantly high level of immune-related genes expression, serum antigen-specific IgM, SVCV-neutralizing antibody titers in fish vaccinated with MCS-PCHG. Next, the protective efficacy of MCS-PCHG was further evaluated by challenge test. The highest survival rate (ca. 84%) was observed in fish vaccinated with MCS-PCHG after challenging with SVCV. This study presents a novel design for smart, dual-targeted polymer nanoparticles, which are inherently biocompatible, promising for targeted vaccine delivery. IMPORTANCE Spring viremia of carp virus (SVCV) affects global cyprinid fish farming industry, with no available commercial vaccine. Herein, we developed a dual-targeting polymer nanovaccine (MCS-PCHG) by using mannose and common carp IgM heavy chain CH3 region (CH3) as antigen presenting cell (APCs) recognition moiety, attaining the effective delivery of antigen. This dual-targeting polymer vaccine can efficiently activate the APCs, and further induce robust and durable adaptive immune response with good protection against SVCV infection. Our study provides valuable theoretical basis for developing efficient vaccine against infectious diseases in aquaculture.

Quinoline, with the active site of 8-hydroxyl, efficiently inhibits Micropterus salmoides rhabdovirus (MSRV) infection in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is an significant pathogen that causes high mortality and related economic losses in bass aquaculture. There is no effective or approved therapy to date. In this study, we evaluated the anti-MSRV effects of 22 quinoline derivatives in grass carp ovary (GCO) cells. Among these compounds, 8-hydroxyquinoline exhibited valid inhibition in decreasing MSRV nucleoprotein gene expression levels of 99.3% with a half-maximal inhibitory concentrations (IC50 ) value of 4.66 µM at 48 h. Moreover, 8-hydroxyquinoline significantly enhanced a protective effect in GCO cells by reducing the cytopathic effect (CPE). By comparing the anti-MSRV activity of 22 quinoline derivatives, we found that 8-hydroxyquinoline possessed the efficient active site of 8-hydroxyl and inhibited MSRV infection in vitro. For in vivo studies, 8-hydroxyquinoline via intraperitoneal injection exhibited an antiviral effect in MSRV-infected largemouth bass by substantially enhancing the survival rate by 15.0%. Importantly, the viral loads in the infected largemouth bass notably reduced in the spleen on the third days post-infection. Overall, 8-hydroxyquinoline was considered to be an efficient agent against MSRV in aquaculture.

Immersion immunization of common carp with bacterial ghost-based DNA vaccine inducing prophylactic protective immunity against spring viraemia of carp virus.

The interactive applications of immunization route, vaccine type and delivery vectors are emerging as a key area of research within the field of mass immunization in fishery production. In an effort to improve DNA vaccine's immune efficiency in large-scale immunization, a promising bacterial ghost-loaded DNA vaccine was constructed based on Escherichia coli DH5α. In common carp was investigated the immune response to immersion immunization via related indicator analysis, and the challenge test of spring viraemia of carp virus (SVCV) was carried out. The result indicated that BG-loaded DNA vaccine induced higher serum antibody level than naked pEG-G. Simultaneously, the immunophysiological indicators and genes change at the more advanced levels in the BG/pEG-G immune group. At the treatment concentration of 20 mg/L of the BG/pEG-G group, IgM and IgZ expressions in vivo were markedly increased by 21.62 times and 6.91 times, respectively, and the relative percentage survival reached the peak of 59.57%. This study paves the way for future aquatic animal vaccine research, which aimed to develop the highly effective immersion vaccine system by delivery vectors, with the ultimate aim to prevent and restrict SVCV in actual production.

Optimization of immunization procedure for SWCNTs-based subunit vaccine with mannose modification against spring viraemia of carp virus in common carp.

Immersion vaccination of single-walled carbon nanotubes loaded with mannose-modified glycoprotein (SWCNTs-MG) vaccine has been proved to be effective in preventing spring viraemia of carp virus (SVCV). Immunization procedure has immense consequence on the immune effect of the immersion vaccine. However, immunization procedure optimization for SWCNTs-MG vaccine against SVCV has not been reported. In this study, accordingly, a full-factor experiment was designed to optimize the immunization procedure of SWCNTs-MG vaccine by three aspects of vaccine dose (30 mg/L, 40 mg/L and 50 mg/L), immunization density (8 fish L-1 , 24 fish L-1 and 48 fish L-1 ) and immunization time (6, 12 and 24 hr). Furthermore, we used the immunization group (A1B2C1, 30 mg/L, 24 fish L-1 and 6 hr) in the previous study as a positive control (PC) to evaluate the immunization effect optimized conditions from the expression of immune-related genes and relative percentage survival (RPS). At 28 days post-vaccination (DPV), common carps were intraperitoneal injected SVCV challenged test indicated that the A1B2C2 group (30 mg/L, 24 fish L-1 , 12 hr) displayed superiority of protective efficacy compare with other groups and the RPS with 77.9%, which was 15.6% higher than the PC group of RPS with 62.3%. Moreover, the expression of immune-related genes such as IL-10, CD4 and MHC-II was also significantly higher than PC group. The specific experimental flow chart is shown in Figure 1. Conclusively, these results demonstrated that vaccine dose, immunization density and immunization time are 30 mg/L, 24 fish L-1 and 12 hr, which is the more appropriate immunization programme with juvenile carp for SWCNTs-MG vaccine. This study provides a profitable reference for improving the immune efficiency of aquatic immersion vaccine. [Figure: see text].

FIP200 restricts RNA virus infection by facilitating RIG-I activation.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

PEG-modified subunit vaccine encoding dominant epitope to enhance immune response against spring viraemia of carp virus.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) can infect almost all fish of cyprinids, which bring huge economic losses to aquaculture. Glycoprotein (G), as the most important antigenic determinant protein of SVCV, is widely considered as an effective method against SVCV. In our previous study, we found that G3 (131 aa) is the potential dominant antigen epitope that induces strong immune responses similar to G protein (510 aa). Here, in order to further improve the immune effect, we reported a subunit vaccine (PEG-G3) constructed by PEG-modified dominant epitope protein (G3). The results of serum antibody production, enzyme activities and immune-related genes expression showed that PEG-G3 induces significantly stronger immune protective responses against SVCV than G3. PEG modification significantly increased the serum antibody level of the vaccine, which increased significantly after immunization and reached the peak at 21 day post-vaccination. T-AOC and AKP activities in the lowest concentration group (5 µg) of PEG-G3 were significantly higher than those in the highest concentration group (20 µg) of G3. In PEG-G3 group, the expression of almost all genes increased at least 4 times compared with the control group. After 14-day challenge, the RPS (relative percentage survival) of the highest concentration of PEG-G3 group was 53.6%, while that of G3 group is 38.9%. Therefore, this work shows that PEG modification and dominant epitope screening may be effective methods to improve the immune protective effect of vaccines and to resist the infection of aquatic animal viral diseases.

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation.

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

Lagos Bat Virus, an Under-Reported Rabies-Related Lyssavirus.

Lagos bat virus (LBV), one of the 17 accepted viral species of the Lyssavirus genus, was the first rabies-related virus described in 1956. This virus is endemic to the African continent and is rarely encountered. There are currently four lineages, although the observed genetic diversity exceeds existing lyssavirus species demarcation criteria. Several exposures to rabid bats infected with LBV have been reported; however, no known human cases have been reported to date. This review provides the history of LBV and summarizes previous knowledge as well as new detections. Genetic diversity, pathogenesis and prevention are re-evaluated and discussed.

Genetic and Antigenetic Characterization of the Novel Kotalahti Bat Lyssavirus (KBLV).

There is a growing diversity of bat-associated lyssaviruses in the Old World. In August 2017, a dead Brandt's bat (Myotis brandtii) tested positive for rabies and based on partial sequence analysis, the novel Kotalahti bat lyssavirus (KBLV) was identified. Because the bat was in an autolyzed state, isolation of KBLV was neither successful after three consecutive cell passages on cells nor in mice. Next generation sequencing (NGS) was applied using Ion Torrent ™ S5 technology coupled with target enrichment via hybridization-based capture (myBaits®) was used to sequence 99% of the genome, comprising of 11,878 nucleotides (nt). KBLV is most closely related to EBLV-2 (78.7% identity), followed by KHUV (79.0%) and BBLV (77.6%), supporting the assignment as phylogroup I lyssavirus. Interestingly, all of these lyssaviruses were also isolated from bat species of the genus Myotis, thus supporting that M. brandtii is likely the reservoir host. All information on antigenic and genetic divergence fulfil the species demarcation criteria by ICTV, so that we recommend KBLV as a novel species within the Lyssavirus genus. Next to sequence analyses, assignment to phylogroup I was functionally corroborated by cross-neutralization of G-deleted RABV, pseudotyped with KBLV-G by sera from RABV vaccinated humans. This suggests that conventional RABV vaccines also confer protection against the novel KBLV.