Alphaviral equine encephalomyelitis (Eastern, Western and Venezuelan).

Summary Alphaviral equine encephalomyelitis is a mosquito-borne infection that causes severe neurological disease and fatalities in horses and humans in the Americas. Consequently, the equine alphaviruses (Eastern, Western and Venezuelan) are of considerable concern worldwide and are notifiable to the World Organisation for Animal Health. In addition, these diseases are considered a potent potential biological weapon, emphasising the need to develop an effective vaccine. Alphaviral equine encephalomyelitis is caused by Eastern equine encephalomyelitis virus (EEEV), Western equine encephalomyelitis virus (WEEV) or Venezuelan equine encephalomyelitis virus (VEEV), which are related members of the Alphavirus genus in the Togaviridae family. Although related, the three viruses are genetically and antigenically distinct. The disease is characterised by fever, anorexia, depression and clinical signs of encephalomyelitis, and may be fatal in up to 90% of cases, for both humans and horses, particularly in the case of EEE. Surviving horses develop lifelong immunity but may have permanent neuropathology. The aim of this paper is to analyse the scientific information available on the evolution of EEE, WEE and VEE, and any potential vaccines.

Control of canine rabies in developing countries: key features and animal welfare implications.

Over 90% of human deaths from rabies worldwide are caused by dog bites. Mass vaccination, along with the effective control of dog populations, has been used successfully in industrialised countries to control this disease. A lower success rate in developing countries is due to a number of factors, including vaccination campaigns that do not cover a sufficient number of animals or reach all communities, and a wide biodiversity that increases the number of reservoirs of the rabies virus. Educational programmes are needed, which focus on the commitment involved when acquiring a domestic animal, stating clearly what is required to provide it with a good quality of life. New technologies developed in the industrialised world will not always be successful in less developed countries. Approaches must be adapted to the particular conditions in each country, taking cultural and socio-economic issues into account. Authorities must promote research on dog population dynamics, the development of non-invasive methods to control dog populations and the most efficient, stable and low-cost options for vaccination. Under the One Health model, it is hoped that dog-transmitted human rabies will be accorded high priority as a zoonosis by human health authorities, international authorities and donor agencies to support ambitious eradication goals, particularly those being set in South-East Asia. Well-designed and adequately resourced vaccination programmes, based on the World Organisation for Animal Health (OIE) guidelines, will have significant animal welfare benefits, due to the availability of improved vaccines (in terms of efficacy, duration of immunity, ease of administration and lower cost), advances in dog population management and the more widespread implementation of the OIE Guidelines on Stray Dog Control. Animal welfare benefits include not only the elimination of pain and suffering caused by the clinical disease itself, but also the avoidance of the indirect impact of inhumane culling when methods are used that have not been approved by the OIE.

The coelomic fluid of the sea urchin Tripneustes depressus shows antiviral activity against Suid herpesvirus type 1 (SHV-1) and rabies virus (RV).

Several studies have reported that molecules extracted from invertebrates have activity against different viruses, even against those that do not infect these organisms in their environment. One of the main mechanisms against pathogens in these organisms is the production of antimicrobial peptides. The objective of this study was to determine whether the coelomic fluid (CF) of the sea urchin Tripneustes depressus has activity against Suid herpesvirus type 1 (SHV-1) and/or rabies virus (RV). We tested the antiviral activity of CF in neutralizing assays and observed 50% inhibition against SHV-1 lytic plaque formation using 33 µg of CF, whereas 21 µg CF was sufficient to obtain more than 90% inhibition for RV. Cytotoxicity to MDBK and BHK-21 cells was found with whole CF yet was eliminated by heating at 56 or 72 °C (even when using 50 µg of heat-inactivated CF supernatant [SN or thermostable fraction]), and SN retained the antiviral effect. In both cases, the antiviral effect was direct and thermostable (SN 56 and 72 °C), and the best inhibition was observed when CF + virus was incubated prior to the addition of the cells. Therefore, the coelomic fluid of T. depressus has antiviral activity against SHV-1 and RV that is direct and stable at 72 °C. We suggest that further assays should be performed using more accurate methods to characterize new molecules with antiviral activity that may result in new drugs.

Experimental infection of Artibeus intermedius bats with serotype-2 dengue virus.

Dengue fever is caused by a flavivirus that primarily infects humans and Aedes sp. mosquitoes. However, viral replication in wild animals other than non-human primates has been scarcely studied. In this report, the susceptibility of Artibeus intermedius frugivorous bat to serotype-2 dengue virus (DENV-2) infection was tested. Twenty-three bats were intraperitoneally inoculated with different viral loads of DENV-2 (New Guinea-C strain). Forty-three percent of the infected bats developed bruises on the chest or on the wings. Histological analyses showed structural alterations in the spleen and bleeding in liver and intestine, but the virus was not detected by RT-PCR in any of the analyzed tissues, and it was found in only one bat (kidney) by semi-nested RT-PCR. In sera, the viral RNA was detected by semi-nested RT-PCR in 39% of bats, but only 8% of bats seroconverted. Overall, these data indicate that DENV-2 replicates poorly in these bats, suggesting they are not suitable hosts to this virus.

Anti-baculovirus activity in a protein extracted from the exoskeleton of Pleuroncodes planipes [Decapoda: Galatheidae].

Antiviral activity (99.5% inhibition) against the Autographa californica polyhedrosis nuclear virus AcNPV+GFP was shown by a polypeptide of approximately 10 kDa, isolated from the exoskeleton of Pleuroncodes planipes, the pelagic red crab. This thermo-stable polypeptide retained its anti-viral properties after being exposed to 76 °C for 30 min and showed no apparent cytotoxic effect. Its anti-viral activity was observed when incubated with the virus, previous to the inoculation of cells. Using Tandem Mass Spectrometry (LC/ESI-MS/MS), this polypeptide showed sequence identity to a fragment of a myohemeritrin-like metalloprotein found in the Scoloplos armiger sea worm (VFYANLDEEHK).

New rabies virus variant found during an epizootic in white-nosed coatis from the Yucatan Peninsula.

In February 2008, three white-nosed coatis (Nasua narica) were found dead in a recreational park in Cancun, Mexico. The diagnosis of rabies virus (RABV) infection was confirmed by direct immunofluorescence test. The phylogenetic analysis performed with the complete RABV nucleoprotein gene positioned this isolate close to a sequence of a human rabies case reported during 2008 from Oaxaca, Mexico, sharing 93% similarity. In turn, these two variants are related to another variant found in rabid Tadarida brasiliensis mexicana bats across North America. Anti-RABV neutralizing activity (1.3 IU/ml) was found in the serum of one white-nosed coati captured with another five that cohabited with the dead animals. Enhanced rabies surveillance and pathogenesis studies should be conducted in coatis and insectivorous bats of the region to clarify the role of these species as potential emergent or long-term unidentified RABV reservoirs.

Dengue virus in Mexican bats.

Individuals belonging to five families, 12 genera, and 19 different species of bats from dengue endemic areas in the Gulf and Pacific coasts of Mexico were examined by ELISA, RT-PCR, and for the presence of dengue virus (DV) NS1 protein. Nine individuals from four species were seropositive by ELISA: three insectivorous, Myotis nigricans (four positives/12 examined), Pteronotus parnellii (3/19), and Natalus stramineus (1/4), and one frugivorous Artibeus jamaicensis (1/35) (12.86% seroprevalence in positive species). DV serotype 2 was detected by RT-PCR in four samples from three species (all from the Gulf coast - rainy season): two frugivorous, A. jamaicensis (2/9), and Carollia brevicauda (1/2), and one insectivorous, M. nigricans (1/11). The latter was simultaneously positive for NS1 protein. DV RT-PCR positive animals were all antibody seronegative. M. nigricans showed positive individuals for all three tests. This is the first evidence suggesting the presence of DV in bats from Mexico.

Rabies DNA vaccination by the intranasal route in dogs.

A DNA vaccine, using a pCl-neo plasmid encoding the glycoprotein gene of a Mexican isolate of rabies virus, was developed to induce long-lasting protective immunity against rabies virus in dogs. The aim of this work was to evaluate the intranasal (IN) vaccination route in mice and dogs. Mice and dogs were immunized via the intramuscular (IM) and IN routes. Mice received 50 microg of DNA vaccine, a booster on day 30, using the same doses and routes, and on day 90 they were challenged. Dogs received 100 microg of DNA vaccine, with a booster on day 180, and immune responses were studied on day 210. Virus-neutralizing antibodies were detected in blood sera (up to 0.5 IU) in animals immunized via the IN route and when the animals were submitted to a booster, the levels of neutralizing antibodies increased. Animals vaccinated via the IM route presented higher neutralizing antibody titres than those vaccinated IN. Control groups lacked anti-rabies antibodies. On day 90, mice were challenged. From these, a 100 % of the IM vaccinated mice, and an 80 % of the IN vaccinated mice survived the challenge. No animals from the control group survived. Dogs revealed significant virus-neutralizing antibody titres (up to 0.5 IU) on day 30 and, after booster, on day 210 neutralizing antibody titre was higher than 1.8 IU. The main advantage of using DNA vaccines over traditional live ones is that there is no contamination with viruses that could be disseminated in the environment and reproduced in susceptible animals. This study demonstrated that pGQH was succesful when administrated via the IN route. IN vaccination seems attractive due to its easy application and mucosal protection. This form of vaccination could also be advantageous in domestic animal vaccination campaigns, for it is less stressful than the parenteral route (no painful shots).

Could the homologous sequence of anti-inflammatory pentapeptide (MLIF) produced by Entamoeba histolytica in the N protein of rabies virus affect the inflammatory process?

Amebiasis and rabies are public health problems, and they have in common a poor inflammatory effect in the target organs that they affect. In the GenBank, it was found that the anti-inflammatory peptide monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica homologates 80%, with a fragment of the N protein of the rabies virus. We speculated if the N protein could contribute to the scant inflammatory reaction produced by rabies virus in central nervous system. The N protein was obtained and studied in vitro and in vivo. The N protein, as MLIF, inhibited the respiratory burst in human mononuclear phagocytes (43%, p<0.05), but in contrast to MLIF, it increased chemotaxis and it did not significantly inhibit delayed hypersensitivity skin reaction to 1-chloro-2-4-dinitrobenzene in guinea pigs. Therefore, the full peptide sequence has to be present or it has to be cleaved-free from the large recombinant N protein molecule (55 kDa) to become active.

Salivary excretion of rabies virus by healthy vampire bats.

Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (10(6) MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion.

[Genetic diversity of Lyssavirus]. / Diversité génétique des Lyssavirus.

Rabies is a zoonosis of which the archaeological agents belong to the Lyssavirus species. Seven genotypes are known to exist. The isolates are grouped according to a) their geographical origin, b) historical context, c) type of vector. In parts of the world where several epidemic cycles exist, practical tools have been developed in order to distinguish between them more easily. Vaccine strains--all of which have been elaborated from genotype 1--offer little or no protection against the other genotypes. It is thus necessary to work on extending vaccine coverage.

Bat lyssavirus infections.

Bats, which represent approximately 24% of all known mammalian species, frequently act as vectors of lyssaviruses. In particular, insectivorous bats play an important role in the epidemiology of rabies and some rabies-like viruses, while the haematophagous vampire bats are the major wildlife vector for rabies in Latin America. In contrast, the role of fruit bats (flying foxes) in the epidemiology of the recently discovered Australian bat lyssavirus is only just emerging. Information on the pathogenesis of lyssaviruses in bats is scarce. However, in general, mortality in bats infected via a natural route appears to be low, and seroconversion occurs in many of those that survive. While transmission of rabies from an infected bat may be via a bite, other routes are apparently also possible. Methods for the diagnosis of bat lyssavirus infections in bats and terrestrial mammals (including humans) are similar to the classical procedures for rabies. Measures for the prevention and control of these diseases are also similar to those for rabies, although additional innovative methods have been tested, specifically to control vampire bat rabies.

Molecular characterization of rabies virus isolates from Mexico: implications for transmission dynamics and human risk.

Twenty-eight samples from humans and domestic and wild animals collected in Mexico between 1990 and 1995 were characterized by using anti-nucleoprotein monoclonal antibodies and limited sequence analysis of the nucleoprotein gene. The variants of rabies viruses identified in these samples were compared with other isolates from Mexico and the rest of the Americas to establish epidemiologic links between cases and outbreaks and to increase the understanding of rabies epidemiology in the Western Hemisphere. Antigenic and genetic diversity was found in all samples from dogs and dog-related cases, suggesting a long-term endemic situation with multiple, independent cycles of virus transmission. Two isolates from bobcats were antigenically and genetically homologous to the rabies variant circulating in the Arizona gray fox population, indicating a wider distribution of this variant than previously reported. Rabies isolates from skunks were unrelated to any variant analyzed in this study and represent a previously unrecognized cycle of rabies transmission in skunks in Baja California Sur. Two antigenic and genetic variants co-circulating in southern and eastern Mexico were found in viruses obtained from cases epidemiologically related to vampire bats. These results serve as a baseline for the better understanding of the molecular epidemiology of rabies in Mexico.

Improved serological response to human diploid cell rabies vaccine when given simultaneously with antirabies hyperimmune globulin.

BACKGROUND: The prevention of rabies in Mexico continues to be an important goal for the health sector. Although the prevalence of this disease continues to fall, between 1990 and 1995 a total of 238 cases were registered (an average of 40 cases annually), with a mean annual incidence of 0.04 cases per 100,000 inhabitants and a mortality of almost 100%, so that it is important to rely on highly effective vaccines with few side effects. The objective of this work was to evaluate seroconversion and tolerance to the human diploid cell antirabies vaccine administered to individuals with a history of exposure to rabies, to compare these results with those reported in the literature for the Fuenzalida vaccine, a rabies vaccine produced in the brain tissue of suckling mice, and to find the role antirabies hyperimmune gamma globulin plays in the concentration of post-vaccination antibody concentrations. METHODS: An analytical transverse study was carried out in 40 children and adults with a history of rabies exposure who were given a complete, five-dose intramuscular schedule of the human diploid cell rabies vaccine. Subjects were followed daily, and local and systemic signs and symptoms were recorded. Two blood samples (at baseline and at the end of the vaccination schedule) were taken and antibody titers against rabies glycoprotein, using the ELISA technique, were measured. RESULTS: Adverse side effects produced by the human diploid cell antirabies vaccine, such as frequency of pain, erythema, itching, and regional adenopathy were fewer than those reported in the literature for the Fuenzalida vaccine (p < 0.05), and of induration and local pain (p < 0.05) in relation to the latter vaccine. All patients seroconverted, producing geometric mean antibody titers of 6.22 IU/mL, an arithmetic mean titer of 9.66 IU/mL with a SD of 9.1 IU/mL. The level of tolerance to the diploid cell vaccine was good and its adverse effects were minimal and fewer than those reported for the Fuenzalida rabies vaccine. Patients receiving the diploid cell vaccine plus antirabies hyperimmune gamma globulin developed higher antibody titers (measured by ELISA test) at the end of the vaccination schedule than those only receiving the vaccine. CONCLUSIONS: These results are important in order to achieve an adequate and opportune level of protection provided by prophylactic vaccines to patients with exposure to rabies.

Immunization of dogs with a DNA vaccine induces protection against rabies virus.

Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be effective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-effective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the first studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control.

Isolation of caprine arthritis encephalitis virus from goats in Mexico.

A lentivirus was isolated from 2 goats in Mexico that were seropositive to caprine arthritis encephalitis virus (CAEV) by the agar gel immunodiffusion (AGID) test. The lentivirus was identified as CAEV by the observation of giant multinucleated cells (syncytia) in goat synovial membrane (GSM) monolayers co-cultivated with blood mononuclear (BMN) cells from the seropositive goats, and by amplifying a DNA segment of the CAEV gag gene using the polymerase chain reaction (PCR) technique. Subsequently, cell supernatants from the GSM cells co-cultivated with BMN cells were used to infect 2 CAEV-seronegative goats. These goats seroconverted to CAEV as determined by the AGID test, and CAEV was re-isolated from these goats. One of the goats developed polyarthritis 8 mo after inoculation. Previous serological surveys indicate that infection with CAEV is prevalent among goats in Mexico. To our knowledge this is the first report of CAEV isolation in Mexico. Because of globalization of markets and increased trading among nations, the rapid identification and reporting of diseases such as CAEV are important to prevent the dissemination of these diseases.

Discrimination between epidemiological cycles of rabies in Mexico.

BACKGROUND: The design of efficient rabies control programs within a geographic area requires an appropriate knowledge of the local epidemiological cycles. In Latin America, there is a geographical overlap of the two main epidemiological cycles: (a) the terrestrial cycle, where the dog is the main terrestrial vector and the principal cause of human transmission; and (b) the aerial cycle, in which the vampire bat Desmodus rotundus is representative in Mexico. This bat is the major sylvatic rabies vector transmitting rabies to cattle. The purpose of this study was to distinguish between the epidemiological cycles of rabies virus (aerial and terrestrial) circulating in Mexico, using restriction fragment length polymorphism (RFLP). METHODS: Thirty positive rabies isolates were obtained from different species (including humans, domestic, and wildlife animals) and geographical regions. The methodology included the extraction of RNA, and synthesis of cDNA, PCR, and RFLP using four restriction endonucleases. To determine the aerial cycle, BsaW I and BsrG I were utilized, and for terrestrial cycle, BamH I and Stu I. Most of the samples belonged to the aerial and terrestrial cycles, except for two skunk isolates from Northwestern Mexico, which were not cut by any of the enzymes. RESULTS: Three different migration patterns were detected: (a) the first was observed in six amplicons, which were cut by BsaW I and BsrG I (aerial cycle); (b) 19 amplified samples were digested with BamH I and Stu I enzymes (terrestrial cycle); and (c) two skunk isolates from Northwest Mexico, were not cut by any of the enzymes utilized in the experiments (hypervariable cycle). CONCLUSIONS: This concludes that RFLP can be used for the classification of rabies field samples in epidemiological studies. Moreover, it has demonstrated its usefulness, not only for differentiating between the main epidemiological rabies cycles present in Mexico, but also to detect new cycles in wildlife species.

Investigation of rabies virus strains in Mexico with a panel of monoclonal antibodies used to classify Lyssavirus.

The purpose of this study was to evaluate the ability of a panel of eight antinucleocapsid monoclonal antibodies developed in Europe to assess rabies virus strains isolated from various animal species in geographically diverse areas of Mexico. Fifty-one rabies-positive brain tissue samples from animals and humans were examined. Material from these samples was used to infect mice, whose brain tissue was subsequently tested by indirect immunofluorescence using the monoclonal antibodies described above. The study did not turn up any strong evidence of Lyssavirus other than rabies virus, but did find four antigenic variants differing from the classic rabies virus serotype. Samples of these latter were sent to the Pasteur Institute in Paris for confirmation. Overall, the antibody panel was deemed useful for rapid typing of rabies virus in Mexico. It also appears possible that autochthonous antigenic variations are now appearing in strains of the virus found in Mexico, which could explain some of the failures observed with certain vaccines. These circumstances appear to create a need for producing antinucleocapsid monoclonal antibodies with strains of rabies virus indigenous to the area.

Inhibition of three alphaherpesviruses (herpes simplex 1 and 2 and pseudorabies virus) by heparin, heparan and other sulfated polyelectrolytes.

The first step of the herpes virus infection is the attachment to heparan sulfate molecules on the cellular membrane. In order to improve the characterization of this phenomenon, we compared the inhibitory effect of six sulfated polyelectrolytes (PE): heparin, heparan, low molecular weight heparin, chondroitin, dextran and protamine on plaque formation by pseudorabies virus (PRV) were compared. The PE with the highest antiherpetic effect was heparin, followed by dextran sulfate. Heparan sulfate, which has been proposed as the initial receptor of herpes virus on the cell surface showed an effect 100-fold lower than heparin. Comparative inhibition curves of heparin and heparan sulfate against three herpes viruses: herpes simplex virus 1 (HSV-1), HSV 2 and PRV showed similar kinetics of inhibition of plaque formation, suggesting these viruses could share similar cell adsorption mechanisms.

[The evaluation of a monoclonal antibody panel for Lyssavirus typing in Mexico]. / Evaluación de una serie de anticuerpos monoclonales para tipificar Lyssavirus en México.

The purpose of this study was to evaluate the ability of a panel of eight antinucleocapsid monoclonal antibodies developed in Europe to identify different strains of rabies virus isolated from a variety of animal species from diverse geographic areas in Mexico. Fifty-one virus-positive samples of brain tissue from various animal species and humans were studied. Material from these samples was used to infect mice, whose brains were later tested by indirect immunofluorescence, using the monoclonal antibodies described above. Strains of the virus that showed antigenic variations were sent to the Pasteur Institute in Paris for confirmation of the results. No mouse brain sample showed a pattern of antigenic reactivity that indicated the presence of a Lyssavirus other than the classic rabies virus. However, four antigenic variations from serotype 1 of classic rabies were found. The panel of antibodies was judged to be useful for the rapid classification of rabies virus in Mexico. It is possible that autochthonous antigenic variations are appearing among strains circulating in that country, a scenario that could explain some of the failures observed with certain vaccines. For this reason, there is a need to produce antinucleocapsid monoclonal antibodies with strains of rabies virus indigenous to the area.