Comparative efficacies of three commercially available vaccines against West Nile Virus (WNV) in a short-duration challenge trial involving an equine WNV encephalitis model

We used a severe challenge model that produces clinical West Nile virus (WNV) disease to test the efficacy of three commercially available equine WNV vaccines in horses. Twenty-four healthy, WNV-seronegative horses of varying ages and genders were placed, in random and blind manner, into three trial groups consisting of eight horses each; two horses in each group received (i) an inactivated WNV vaccine (K-WN), (ii) a modified-live vaccine (CP-WN) containing the WNV prM and E proteins expressed by a canarypox vector, (iii) a live-chimera vaccine (WN-FV) containing WNV prM and E proteins expressed in a YF17D vector, or (iv) a diluent control. Challenge by this model caused grave neurological signs, viremia, moderate to severe histopathologic lesions in the brain and spinal cord, and an outcome of 0% survivorship in all six control horses. In contrast, challenge in horses at between 28 days postvaccination with the chimera vaccine and 56 days postvaccination with the commercial inactivated or modified-live vaccine resulted in 100% survivorship (protection from the onset of WNV encephalitis and viremia). Horses vaccinated with the live-chimera vaccine showed significantly fewer clinical signs than did the control horses (P 相似文献   

Immunological Correlates of Vaccination and Infection for Equine Herpesvirus 1

Equine herpesvirus 1 (EHV-1) induces a variety of disease manifestations, including respiratory disease, abortions, and myeloencephalopathy. Several vaccines are commercially available but could not previously be distinguished by serologic testing from infection with EHV-1 (or the closely related EHV-4). Currently available vaccines are not reliably protective against the severe manifestations of the disease, including fatal myeloencephalopathy. We determined immunological parameters that can differentiate vaccinated from previously infected animals by comparing humoral and cellular EHV-1-specific responses in clinically healthy horses 10 months after vaccination. Forty-seven horses with known histories of vaccination and infection were studied, including a group of horses that survived a severe neurological outbreak 5 years prior to vaccination. Results of serum virus neutralization (SN), serum IgG isotyping, and cytokine profiling of lymphocyte subsets were compared. IgG4/7 levels strongly correlated with virus neutralization (P < 0.0001). IgG1/3 and SN values distinguished vaccinated/outbreak-exposed (vacc/outbreak) horses from vaccinated horses (P < 0.05). EHV-1-specific gamma interferon (IFN-γ)-producing CD4⁺ (but not CD8⁺) T-cell numbers were also increased in vacc/outbreak horses, which distinguished them from vaccinated horses (P < 0.01). IFN-α secretion was similar between all groups and independent of previous exposure or vaccination. Our data suggest that IgG isotype responses to EHV-1 are more diverse under field conditions than is revealed by experimental studies and that the current modified-live virus (MLV) vaccine induces a more restricted IgG isotype response than does natural exposure to EHV-1. Since these parameters can be assessed in a high-throughput manner, they may prove useful in screening future vaccine candidates and assessing levels of protection.     

Mutations in the West Nile prM protein affect VLP and virion secretion in vitro

Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.     

Neutralizing monoclonal antibodies against Russian strain of the West Nile virus

We have developed a panel of 16 hybridomas secreting neutralizing monoclonal antibodies (Nt- MAbs) to Russian isolate (LEIV-Vlg99-27889-human) of the West Nile virus (WNV). Most of the Nt-Mabs were either IgG1 or IgG3 subtypes. Nine of the 16 neutralizing MAbs detected WNV protein E in Western blot. According to their Nt-activities, Western blot results and cross-reactivity, the MAbs were divided into four groups. Monoclonal antibodies from group I were able to neutralize WNV strains Vlg99-27889, Vlg00-27924, Hp-94, A-1640, A-72, Tur-2914, and Eg101. The Nt-activity of MAbs from groups II-IV towards these WNV strains was variable. Recombinant fragments E(1-180), E(1-321), and E(260-466) of protein E were used for preliminary mapping of domains recognized by Nt-MAbs. Only five Nt-MAbs were able to react with the recombinant polypeptides. The MAbs 9E2, 7G9, 11G3, and 7E6 from group Ia recognized Nt-epitope(s) between amino acids 321 and 466 of protein E and Nt-MAb 4F11 (group III) reacted with residues 1-180. This demonstrates that two discrete regions of protein E are involved in neutralization of WNV. Our data on immunochemical, biological activities of Nt-MAbs and mapping of Nt-epitopes using recombinant polypeptides suggest at least 13 different Nt-epitopes for WNV.     

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: effects of WN virus NS1 antibodies induced by inactivated WN vaccine

Antibodies to non-structural protein 1 (NS1) of West Nile virus (WNV) have been used to differentiate WNV infection from infection by serologically cross-reactive flaviviruses, including Japanese encephalitis virus (JEV), in horses. However, since the inactivated West Nile (WN) vaccine has been reported to induce NS1 antibodies, there is concern about the reliability of using NS1-based assays for testing vaccinated horses. Therefore, the effect of inactivated WN vaccine-induced antibodies on an epitope-blocking ELISA and complement-dependent cytotoxicity (CDC) assay were investigated. Both assays are based on NS1 antibodies and were established previously to differentiate WNV from JEV infections in horses. Groups of three horses were vaccinated with two or three doses of a commercial inactivated WN vaccine and NS1 antibodies were detected by a conventional ELISA after the second vaccination. Vaccine-induced NS1 antibodies were also detected by blocking ELISA and a CDC assay and affected the ability of these assays to differentiate WNV from JEV infections. However, the effect was less significant in the CDC assay, where use of a low serum concentration ensured effective differentiation. The more efficient detection of infection-induced antibodies over vaccine-induced antibodies by the CDC assay was potentially attributable to the different IgG isotype profiles of these antibodies.     

Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses

Summary Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced.     

Cross protection against lethal West Nile virus challenge in mice immunized with recombinant E protein domain III of Japanese encephalitis virus

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that cause severe encephalitic diseases with global impact. Cross protection among JEV and WNV has been previously described, and most cross reactive epitopes were identified within the domain II of E protein (EDII). In this study, the E protein domain III (EDIII) of JEV was successfully expressed in Escherichia coli, purified by a Ni-NTA column and characterized by Western blotting assay. Competitive inhibition assay showed that this recombinant JEV EDIII blocks the entry of JEV into BHK-21 cells. Mice immunized with the recombinant JEV EDIII developed high IgG and neutralizing antibodies titers against JEV. Most importantly, antibodies induced by JEV EDIII could neutralize WNV in vitro and partially protected mice against lethal WNV challenge. These results demonstrate that immunization with JEV EDIII induces cross-protective immunity against WNV infection, indicating a possible role of EDIII for the cross-protection among flavivirus.     

Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection.

Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freund's complete adjuvant were fully protected against 4.9 x 10(5) LD50 of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia virus.     

Evaluation of widely used diagnostic tests to detect West Nile virus infections in horses previously infected with St. Louis encephalitis virus or dengue virus type 2

Primary West Nile virus (WNV) infections can be diagnosed using a number of tests that detect infectious particles, nucleic acid, and specific IgM and/or IgG antibodies. However, serological identification of the infecting agent in secondary or subsequent flavivirus infections is problematic due to the extensive cross-reactivity of flavivirus antibodies. This is particularly difficult in the tropical Americas where multiple flaviviruses cocirculate. A study of sequential flavivirus infection in horses was undertaken using three medically important flaviviruses and five widely utilized diagnostic assays to determine if WNV infection in horses that had a previous St. Louis encephalitis virus (SLEV) or dengue virus type 2 (DENV-2) infection could be diagnosed. Following the primary inoculation, 25% (3/12) and 75% (3/4) of the horses mounted antibody responses against SLEV and DENV-2, respectively. Eighty-eight percent of horses subsequently inoculated with WNV had a WNV-specific antibody response that could be detected with one of these assays. The plaque reduction neutralization test (PRNT) was sensitive in detection but lacked specificity, especially following repeated flavivirus exposure. The WNV-specific IgM enzyme-linked immunosorbent assay (IgM ELISA) was able to detect an IgM antibody response and was not cross-reactive in a primary SLEV or DENV response. The WNV-specific blocking ELISA was specific, showing positives only following a WNV injection. Of great importance, we demonstrated that timing of sample collection and the need for multiple samples are important, as the infecting etiology could be misdiagnosed if only a single sample is tested.     

Baculovirus-expressed glycoprotein E (gE) of herpes simplex virus type-1 (HSV-1) protects mice against lethal intraperitoneal and lethal ocular HSV-1 challenge.

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) in Sf9 cells. The expressed gE migrated on gels as a double band with apparent molecular weights of 68 and 70 kDa. The recombinant gE was glycosylated based on its susceptibility to tunicamycin treatment and was transported to the membrane of Sf9 cells based on indirect immunofluorescence. Mice vaccinated with gE developed high serum titers of HSV-1-neutralizing antibodies based on plaque reduction assays. gE vaccination also induced a strong delayed type hypersensitivity (DTH) response to HSV-1. In addition, mice vaccinated with the recombinant gE were protected from both intraperitoneal and ocular lethal HSV-1 challenge. To our knowledge, this is the first report in which vaccination with gE was shown to induce high neutralizing antibody titers, a DTH response, or protection against lethal HSV-1 challenge.     

Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.     

Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis.

Four recombinant vaccinia viruses were constructed for expression of different portions of the 17D yellow fever virus (YFV-17D) open reading frame. A recombinant, vP869, expressing prM and E induced high titers of neutralizing and hemagglutination inhibiting antibodies in mice and was protective against intracranial challenge with the French neurotropic strain of YFV. Levels of protection were equivalent to those achieved by immunization with the YFV-17D vaccine virus. Recombinant vaccinia viruses expressing E and NS1, C prM, E, NS1, or only NS1 failed to protect mice against challenge with YFV despite eliciting antibodies to NS1. The vP869-infected HeLa cells produced a particulate extracellular hemagglutinin (HA) similar to that produced by YFV-infected cells, supporting previous studies with Japanese encephalitis virus (Mason et al., 1991), suggesting that the ability of recombinant vaccinia virus to produce extracellular HA particles is important for effective flavivirus immunity.     

Experimental Infection of Horses with an Attenuated Venezuelan Equine Encephalomyelitis Vaccine (Strain TC-83)

Ten horses (Equus caballus) were vaccinated with strain TC-83 Venezuelan equine encephalomyelitis (VEE) virus vaccine. Febrile responses and leukopenia due to a reduction of lymphocytes and neutrophils were observed in all animals. Viremias were demonstrable in eight horses, with a maximum of 10(3.5) median tissue culture infectious dose units per ml of serum in two horses. Clinical illness with depression and anorexia were observed in five horses. Neutralizing (N), hemagglutination-inhibiting, and complement-fixing antibodies to the vaccine virus were demonstrable by 5, 6.5, and 7 days, respectively, after vaccination. Differential titrations of serum to six VEE strains revealed high titers of N antibody to vaccine virus, moderate titers to the epizootic Trinidad donkey no. 1 strain (VEE antigenic subtype I, variant A) from which TC-83 was derived, and low titers to two other epizootic strains (subtype I, variants B and C) in all horses at 1 month after vaccination; some animals responded with low levels of N antibody to the enzootic viruses (subtype I, variants D and E). Fourteen months after vaccination, six animals with detectable N antibody were challenged with MF-8 (subtype I, variant B), an epidemic-epizootic strain isolated in 1969 from a man in Honduras. All horses resisted challenge with the equine pathogenic strain of VEE. Marked increases of N antibody in most horses were demonstrable to some VEE strains when tested 1 month after challenge.     

Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus.

Immunization with recombinant vaccinia viruses that specified the synthesis of Japanese encephalitis virus (JEV) glycoproteins protected mice from a lethal intraperitoneal challenge with JEV. Recombinants which coexpressed the genes for the structural glycoproteins, prM and E, elicited high levels of neutralizing (NEUT) and hemagglutination inhibiting (HAI) antibodies in mice and protected mice from a lethal challenge by JEV. Recombinants expressing only the gene for the nonstructural glycoprotein, NS1, induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of JEV. Antibodies to the NS3 protein in postchallenge sera, representing the degree of infection with challenge virus, were inversely correlated to NEUT and HAI titers and levels of protection. These results indicate that although vaccinia recombinants expressing NS1 can provide some protection from lethal JEV infection, recombinants expressing prM and E elicited higher levels of protective immunity.     

Targeting DNA vaccines to myeloid cells using a small peptide

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG‐P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T‐cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG‐P were able to restimulate vaccinia‐specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG‐P was able to prime a vaccinia‐specific T‐cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA‐vaccine encoding West Nile virus (WNV) prM and E proteins via RVG‐P elicited high titers of WNV‐neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG‐P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T‐cell and humoral immune responses.     

A West Nile virus CD4 T cell epitope improves the immunogenicity of dengue virus serotype 2 vaccines

Flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are among the most prevalent human disease-causing arboviruses world-wide. As they continue to expand their geographic range, multivalent flavivirus vaccines may become an important public health tool. Here we describe the immune kinetics of WNV DNA vaccination and the identification of a CD4 epitope that increases heterologous flavivirus vaccine immunogenicity. Lethal WNV challenge two days post-vaccination resulted in 90% protection with complete protection by four days, and was temporally associated with a rapid influx of activated CD4 T cells. CD4 T cells from WNV vaccinated mice could be stimulated from epitopic regions in the envelope protein transmembrane domain. Incorporation of this WNV epitope into DENV-2 DNA and virus-like particle vaccines significantly increased neutralizing antibody titers. Incorporating such potent epitopes into multivalent flavivirus vaccines could improve their immunogenicity and may help alleviate concerns of imbalanced immunity in multivalent vaccine approaches.     

登革Ⅰ型病毒E蛋白在293T细胞中的分泌表达

目的 分泌表达登革Ⅰ型病毒prM/E基因,为研究该蛋白的免疫学功能和特性奠定基础.方法 用RT-PCR法获得登革Ⅰ型病毒广州分离株全长prM/E基因,在prM基因前添加乙型脑炎病毒的信号肽或同时替换E基因羧基末端的20%为乙脑病毒E基因相应的部分,分别将其克隆入真核表达载体pcDNAS/FRT中,获得三种重组质粒DlprME-pc5,D1JsprME-pc5,D1JsprM80E20JE-pc5.用脂质体法分别将重组质粒DNA转入293T细胞,通过免疫荧光、Western印迹检测外源基因在真核细胞中的分泌表达.结果 用免疫荧光法检测到分别转染了三种重组质粒的293T细胞的胞质中均有登革Ⅰ型病毒蛋白的表达.Western印迹检测转染了D1prME-pc5重组质粒的293T细胞上清中没有特异蛋白条带,转染了经基因改造的重组质粒D1JsprME-pc5和D1JsprM80E20JE-pc5的细胞上清中均存在登革Ⅰ型病毒的特异蛋白条带.结论 转染了三种重组质粒的293T细胞均可表达登革Ⅰ型病毒prM/E蛋白,只有在prM基因前添加了信号肽的重组质粒转染后蛋白才获得分泌表达.     

重组登革病毒E蛋白结构域Ⅲ抑制登革病毒感染的初步研究

目的 了解原核表达的登革病毒(dengue virus, DV)的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法 在大肠埃希菌中表达1~4型登革病毒E蛋白结构域Ⅲ(EⅢ).重组蛋白纯化后,进行阻断DV-2感染BHK~21细胞试验.用重组蛋白制备免疫血清,检测抗体中和作用.结果 在大肠埃希菌中成功表达了1-4型登革病毒E蛋白结构域埃希菌,4型重组E蛋白结构域Ⅲ均能够阻断2型DV感染,4型重组蛋白的免疫血清均能中和2型DV,但中和抗体效价不同.结论 原核表达的登革病毒结构域Ⅲ可以直接抑制病毒感染,所产生的抗体具有中和作用.直接抑制和中和抗体均对同型病毒作用较强.     

Baboon model for West Nile virus infection and vaccine evaluation

Animal models that closely mimic the human condition are of paramount significance to study pathogenic mechanisms, vaccine and therapy scenarios. This is particularly true for investigations that involve emerging infectious diseases. Nonhuman primate species represent an alternative to the more intensively investigated rodent animal models and in a number of instances have been shown to represent a more reliable predictor of the human response to infection. West Nile virus (WNV) has emerged as a new pathogen in the Americas. It has a 5% fatality rate, predominantly in the elderly and immune compromised. Typically, infections are cleared by neutralizing antibodies, which suggests that a vaccine would be efficacious. Previously, only macaques had been evaluated as a primate model for WNV vaccine design. The macaques did not develop WNV disease nor express the full complement of IgG subclasses that is found in humans. We therefore explored baboons, which exhibit the similar four IgG subclasses observed in humans as a new model for WNV infection and vaccine evaluation. In this present report, we describe the experimental infection of baboons with WNV and test the efficacy of an inactivated WNV vaccination strategy. All experimentally infected animals developed transient viremia and subsequent neutralizing antibodies. Anti-WNV IgM antibodies peaked at 20 days post-infection. Anti-WNV IgG antibodies appeared later and persisted past 60 days. Prior vaccination with chemically inactivated virus induced neutralizing titers and a fast, high titer IgG recall response, which resulted in lower viremia upon challenge. This report is the first to describe the development of the baboon model for WNV experimental infection and the utility of this model to characterize the immunologic response against WNV and a candidate WNV vaccine.     

Respiratory and neurological disease in rabbits experimentally infected with equid herpesvirus 1

Equid herpesvirus type 1 (EHV-1) is an important pathogen of horses worldwide, associated with respiratory, reproductive and/or neurological disease. A mouse model for EHV-1 infection has been established but fails to reproduce some important aspects of the viral pathogenesis. Then, we investigated the susceptibility of rabbits to EHV-1 aiming at proposing this species as an alternative model for EHV-1 infection. Weanling rabbits inoculated intranasal with EHV-1 Kentucky D (10⁷ TCID₅₀/animal) shed virus in nasal secretions up to day 8–10 post-inoculation (pi), presented viremia up to day 14 pi and seroconverted to EHV-1 (virus neutralizing titers 4 to 64). Most rabbits (75%) developed respiratory disease, characterized by serous to hemorrhagic nasal discharge and mild to severe dyspnea. Some animals (20%) presented neurological signs as circling, bruxism and opisthotonus. Six animals died during acute disease (days 3–6); infectious virus and/or viral DNA were detected in the lungs, trigeminal ganglia (TG), olfactory bulbs (OBs) and cerebral cortex/brain (CC). Histological examination showed necrohemorrhagic, multifocal to coalescent bronchointerstitial pneumonia and diffuse alveolar edema. In two rabbits euthanized at day 50 pi, latent EHV-1 DNA was detected in the OBs. Dexamethasone administration at day 50 pi resulted in virus reactivation, demonstrated by virus shedding, viremia, clinical signs, and increase in VN titers and/or by detection of virus DNA in lungs, OBs, TGs and/or CC. These results demonstrate that rabbits are susceptible to EHV-1 infection and develop respiratory and neurological signs upon experimental inoculation. Thus, rabbits may be used to study selected aspects of EHV-1 biology and pathogenesis, extending and complementing the mouse model.