The First Detection of Koi Herpesvirus (CyHV3) in Migratory Wild Ducks in North America.

A viromics study on the intestinal contents of migratory wild ducks of the genera Anas, Mareca, Spatula, and Oxyura during their winter stay in Mexico showed the presence of the virus family Alloherpesviridae. The genus Cyprinivirus is part of this family and includes cyprinid herpesvirus 3 (CyHV3). This is the etiological agent of the lethal disease known as koi herpesvirosis, which affects different strains of Common Carp Cyrprinus carpio. In this study, samples of the contents of 87 wild duck intestines were analyzed by endpoint PCR, of which 7 samples were positive for the amplification of the TK gene fragment corresponding to CyHV3. These results contribute to the knowledge about the spread of this virus to other species of aquatic animals in areas where fish and ducks coexist.

Whole genome sequence phylogenetic analysis of four Mexican rabies viruses isolated from cattle.

Phylogenetic analysis of the rabies virus in molecular epidemiology has been traditionally performed on partial sequences of the genome, such as the N, G, and P genes; however, that approach raises concerns about the discriminatory power compared to whole genome sequencing. In this study we characterized four strains of the rabies virus isolated from cattle in Querétaro, Mexico by comparing the whole genome sequence to that of strains from the American, European and Asian continents. Four cattle brain samples positive to rabies and characterized as AgV11, genotype 1, were used in the study. A cDNA sequence was generated by reverse transcription PCR (RT-PCR) using oligo dT. cDNA samples were sequenced in an Illumina NextSeq 500 platform. The phylogenetic analysis was performed with MEGA 6.0. Minimum evolution phylogenetic trees were constructed with the Neighbor-Joining method and bootstrapped with 1000 replicates. Three large and seven small clusters were formed with the 26 sequences used. The largest cluster grouped strains from different species in South America: Brazil, and the French Guyana. The second cluster grouped five strains from Mexico. A Mexican strain reported in a different study was highly related to our four strains, suggesting common source of infection. The phylogenetic analysis shows that the type of host is different for the different regions in the American Continent; rabies is more related to bats. It was concluded that the rabies virus in central Mexico is genetically stable and that it is transmitted by the vampire bat Desmodus rotundus.

Prevalence of neutralizing antibodies against West Nile virus (WNV) in monkeys (Ateles geoffroyi and Alouatta pigra) and crocodiles (Crocodylus acutus and C. acutus-C. moreletti hybrids) in Mexico.

West Nile virus (WNV) is a mosquito-borne neurotropic viral pathogen maintained in an enzootic cycle between mosquitoes (vectors) and birds (natural hosts) with equids, humans, and other vertebrates acting as dead-end hosts. WNV activity in Mexico has been reported in several domestic and wild fauna and in humans, and the virus has been isolated from birds, mosquitoes, and humans. However, no serological studies have been conducted in monkeys, and only two in a limited number of crocodiles (Crocodylus moreletii). Here we present data on the prevalence of neutralizing antibodies against WNV in 53 healthy wild monkeys (49 Ateles geoffroyi and four Alouatta pigra), and 80 semi-captive healthy crocodiles (60 C. acutus and 20 C. acutus-C. moreletti hybrids) sampled during 2012. None of the monkey sera neutralized WNV, whereas 55% of the crocodile sera presented neutralizing antibodies against WNV. These results can contribute to the design of surveillance programmes in Mexico.

The continuous spread of West Nile virus (WNV): seroprevalence in asymptomatic horses.

West Nile virus (WNV) was probably introduced in southern and northern Mexico from the USA in two independent events. Since then, WNV activity has been reported in several Mexican states bordering the USA and the Gulf of Mexico, but disease manifestations seen there in humans and equids are quite different to those observed in the USA. We have analysed WNV seroprevalence in asymptomatic, unvaccinated equids from two Mexican states where no data had been previously recorded. WNV IgG antibodies were detected in 31.6% (91/288) of equine sera from Chiapas and Puebla states (53.3% and 8.0%, respectively). Analysis by plaque reduction neutralization test (PRNT) showed good specificity (99.4%) and sensitivity (84.9%) with the ELISA results. Further analyses to detect antibodies against three different flaviviruses (WNV, St Louis encephalitis virus, Ilheus virus) by haemagglutination inhibition (HI) tests on a subset of 138 samples showed that 53% of the 83 HI-positive samples showed specific reaction to WNV. These data suggest continuous expansion of WNV through Mexico.

Development of an edible rabies vaccine in maize using the Vnukovo strain.

The objective of this study was to obtain transgenic maize expressing the rabies virus glycoprotein (G) of the Vnukovo strain and to evaluate its immunogenicity in mice, by the oral route. The ubiquitin maize promoter fused to the whole coding region of the rabies virus G gene, and a constitutive promoter from cauliflowermosaic virus (CaMV)were used. Maize embryogenic callus were transformed with the above construct by biolistics. Regenerated maize plants were recovered and grown in a greenhouse. The presence of the G gene and its product was detected by PCR and western blot, respectively. The amount of G protein detected in the grains was approximately 1% of the total soluble plant protein. Transformed kernels containing 50 microg of G protein were given once by the oral route in adult mice (BALB-C strain). Challenge was undertaken at 90-days post-vaccination using a lethal dose of a vampire bat rabies virus (100 LD 50% in mice); vampire bats are one of the main reservoirs in Latin America. The edible vaccine induced viral neutralizing antibodies (VNA) which, protected mice 100% against challenge. The control group did not survive. The G protein of the Vnukovo strain expressed in transgenic maize may be considered as an oral immunogen against rabies, conferring cross-protection.

Detection of multiple strains of rabies virus RNA using primers designed to target Mexican vampire bat variants.

A reverse transcription-polymerase chain reaction (RT-PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 10(7) dilution using stock virus at an original titre of 10(7.5) LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%.

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.

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.

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.

[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.