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.

Antiviral effects of natural small molecules on aquatic rhabdovirus by interfering with early viral replication.

Spring viremia of carp virus (SVCV) is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range. To develop effective agents to control SVCV infection, we selected 16 naturally active small molecules to assess their anti-SVCV activity. Notably, dihydroartemisinin (DHA) (100 µmol/L) and (S, S)-(+)-tetrandrine (TET) (16 µmol/L) exhibited high antiviral effects in epithelioma papulosum cyprinid (EPC) cells, with inhibitory rates of 70.11% and 73.54%, respectively. The possible antiviral mechanisms were determined as follows: 1. Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells, suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted; 2. Although neither had an effect on viral adhesion, TET (but not DHA) interfered with SVCV entry into host cells (>80%), suggesting that TET may have an antiviral function in early viral replication. For in vivo study, both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%, significantly decreased viral load, and modulated the expression of antiviral-related genes, indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection. Overall, our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication, thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.

Dietary supplementation of exopolysaccharides from Lactobacillus rhamnosus GCC-3 improved the resistance of zebrafish against spring viremia of carp virus infection.

Spring viremia of carp virus (SVCV) can cause high mortality of fish. The aim of this study was to investigate the effects of Lactobacillus rhamnosus GCC-3 exopolysaccharides (GCC-3 EPS) on zebrafish (Danio rerio) infected with SVCV and elucidate the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.5% and 1% of GCC-3 EPS for 2 weeks. The results showed that supplementation of GCC-3 EPS significantly improved the survival rate of zebrafish compared with the control group. In addition, dietary 0.5% and 1% GCC-3 EPS significantly up-regulated the expression of genes related to type I interferon (IFN) antiviral immunity. Consistent with in vivo results, GCC-3 EPS significantly inhibited SVCV replication in zebrafish embryonic fibroblast (ZF4) cells while significantly increased the expression of type I IFN signaling pathway related genes. Furthermore, knocking down TANK-binding kinase 1 significantly blocked the antiviral effect of GCC-3 EPS. Dietary GCC-3 EPS improved gut microbiota, and the culture supernatant of GCC-3 EPS-associated microbiota significantly inhibited SVCV replication in ZF4 cells compared with the control-microbiota counterpart. In conclusion, our results indicate that dietary GCC-3 EPS can improve the resistance of zebrafish against SVCV infection, and the mechanism may involve enhanced type I interferon signaling.

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 µg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 µg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 µg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 µg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.

Ursolic acid from Prunella vulgaris L. efficiently inhibits IHNV infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100 mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72 h of UA on IHNV was 8.0 µM. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

Highly efficient inhibition of spring viraemia of carp virus replication in vitro mediated by bavachin, a major constituent of psoralea corlifonia Lynn.

As one of nine piscine viruses recognized by the International Office of Epizootics, spring viraemia of carp virus (SVCV) is an important pathogen bringing high mortality to cyprinids. Up to now, there is no approved therapy on SVCV, making them strong public health threat in aquaculture. In this study, the anti-SVCV activities of 12 plant crude extracts were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Psoralea corylifolia Linn. showed the highest inhibition on SVCV replication, with an inhibitory percentage of 67.98%. Further studies demonstrated that bavachin (BVN), one of the major constituents of Psoralea corylifolia Linn., was also highly effective to SVCV infection. The half maximal inhibitory concentrations (IC50) of BVN on SVCV glycoprotein and nucleoprotein expression were 0.46 (0.29-0.73) and 0.31 (0.13-0.55) mg/L, respectively. In addition, SVCV-induced apoptosis which may be negative to SVCV replication was inhibited by BVN. The apoptotic cells were decreased 21.42% for BVN compared with SVCV group. These results indicated that the inhibition of BVN on SVCV replication was, in some extent, via blocking SVCV induced apoptosis. Furthermore, cellular morphological damage induced by SVCV was also blocked by BVN treatment. Mechanistically, BVN did not affect SVCV infectivity and cannot be used for prevention of SVCV infection. Time-of-addition and viral binding assays revealed that BVN mainly inhibited the early events of SVCV replication but did not interfere with SVCV adsorption. In conclusion, BVN was considered to develop as a promising agent to treat SVCV infection.

Effects of Three Types of Inactivation Agents on the Antibody Response and Immune Protection of Inactivated IHNV Vaccine in Rainbow Trout.

Infectious hematopoietic necrosis virus (IHNV) infects salmonid fish, resulting in high mortality and serious economic losses to salmonid aquaculture. Therefore, an effective IHNV vaccine is urgently needed. To select an inactivation agent for the preparation of an effective IHNV vaccine, rainbow trout were immunized with mineral oil emulsions of IHNV vaccines inactivated by formaldehyde, binary ethylenimine (BEI), or ß-propiolactone (BPL). The fish were challenged 8 weeks after vaccination, and their IgM antibody response and relative percent survival (RPS) were evaluated. The results show that formaldehyde, BEI, and BPL abolished IHNV HLJ-09 infectivity within 24, 48, and 24 h at final concentrations of 0.2%, 0.02%, and 0.01%, respectively. The mean levels of specific IgM, both in serum and mucus (collected from the skin surface and gills), for the three immunized groups (from high to low) ranked as follows: the BPL group, BEI group, and formaldehyde group. From weeks 5 to 9, the mean log2 serum titers of IgM in the BPL group were significantly higher compared with those of the other groups (p < 0.05) during the 9 weeks of observation after vaccination (immunized at weeks 0 and6). Mucus OD490 values of the BPL group were significantly higher compared with those of the other groups (p < 0.05) when reaching their peak at weeks 5 and 8, but the difference between the formaldehyde and BEI groups was not significant (p > 0.05). The BPL-inactivated whole-virus vaccine had the greatest protective effect on the rainbow trout after challenge by an intraperitoneal injection of live IHNV, with an RPS rate of 91.67%, which was significantly higher compared with the BEI (83.33%) and formaldehyde (79.17%) groups. These results indicate that the BPL-inactivated IHNV oil-adjuvant vaccine was more effective than the formaldehyde- or BEI-inactivated vaccines. The results of this study provide an important foundation for further studies on inactivated IHNV vaccines.

An oral DNA vaccine against infectious haematopoietic necrosis virus (IHNV) encapsulated in alginate microspheres induces dose-dependent immune responses and significant protection in rainbow trout (Oncorrhynchus mykiss).

Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 µg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 µg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5 µg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100 µg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100 µg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our results demonstrated a significant increase in fish immune responses and resistance to an IHNV infection after the oral administration of increasing concentrations of a DNA vaccine against IHNV encapsulated into alginate microspheres.

Two variants of selenium-dependent glutathione peroxidase from the disk abalone Haliotis discus discus: Molecular characterization and immune responses to bacterial and viral stresses.

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases (AbSeGPxs) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus. The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3' untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPxs. The expression of AbSeGPxs mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPxs was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPxs to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial (Listeria monocytogenes and Vibrio parahaemolyticus) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics.

Infectious disease ecology has recently raised its public profile beyond the scientific community due to the major threats that wildlife infections pose to biological conservation, animal welfare, human health and food security. As we start unravelling the full extent of emerging infectious diseases, there is an urgent need to facilitate multidisciplinary research in this area. Even though research in ecology has always had a strong theoretical component, cultural and technical hurdles often hamper direct collaboration between theoreticians and empiricists. Building upon our collective experience of multidisciplinary research and teaching in this area, we propose practical guidelines to help with effective integration among mathematical modelling, fieldwork and laboratory work. Modelling tools can be used at all steps of a field-based research programme, from the formulation of working hypotheses to field study design and data analysis. We illustrate our model-guided fieldwork framework with two case studies we have been conducting on wildlife infectious diseases: plague transmission in prairie dogs and lyssavirus dynamics in American and African bats. These demonstrate that mechanistic models, if properly integrated in research programmes, can provide a framework for holistic approaches to complex biological systems.

Survival of the North American strain of viral hemorrhagic septicemia virus (VHSV) in filtered seawater and seawater containing ovarian fluid, crude oil and serum-enriched culture medium.

The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15 degrees C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.

Rapid detection of rabies and rabies-related viruses by RT-PCR and enzyme-linked immunosorbent assay.

A rapid detection method for the six established genotypes of rabies and rabies-related viral RNA using RT-PCR-ELISA is described. The detection of digoxigenin-labelled amplified products is performed by solution hybridization to two specific, biotin-labelled, capture probes, which are complementary to the inner region of the amplification products. The capture probe and amplified product hybrid are then immobilised on a streptavidin-coated microtitre plate, bound products are detected by an anti-DIG Fab fragment conjugated to peroxidase, and colorimetric reaction automatically measured. This method was up to 100-fold more sensitive than Southern blot hybridization, detecting 0.00002 TCID50/ml of a genotype 1, classical rabies virus strain. The complete detection methodology from RT-PCR to PCR-ELISA detection could be completed within 10 h. Using this procedure, we were 100% successful in detecting 60 isolates from a representative selection of the six established genotypes from all over the world. This test is a useful additional tool for the detection of the rabies and rabies-related viruses, which is easy to perform, rapid and highly sensitive.