RESUMO
Disease control programs aim to constrain and reduce the spread of infection. Human disease interventions such as wildlife vaccination play a major role in determining the limits of a pathogen's spatial distribution. Over the past few decades, a raccoon-specific variant of rabies virus (RRV) has invaded large areas of eastern North America. Although expansion into Canada has been largely prevented through vaccination along the US border, several outbreaks have occurred in Canada. Applying phylogeographic approaches to 289 RRV whole-genome sequences derived from isolates collected in Canada and adjacent US states, we examined the processes underlying these outbreaks. RRV incursions were attributable predominantly to systematic virus leakage of local strains across areas along the border where vaccination has been conducted but also to single stochastic events such as long-distance translocations. These results demonstrate the utility of phylogeographic analysis of pathogen genomes for understanding transboundary outbreaks.
Assuntos
Surtos de Doenças , Genoma Viral , Vacina Antirrábica/administração & dosagem , Vírus da Raiva/genética , Raiva/epidemiologia , Raiva/prevenção & controle , Vacinação/veterinária , Administração Oral , Animais , Encéfalo/patologia , Encéfalo/virologia , Canadá/epidemiologia , Humanos , Filogenia , Filogeografia , RNA Viral/genética , Raiva/transmissão , Raiva/virologia , Vírus da Raiva/classificação , Vírus da Raiva/isolamento & purificação , Guaxinins/virologia , Análise de Sequência de DNA , Estados Unidos/epidemiologiaRESUMO
Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular reservoir host. Active management of these host vectors is needed to minimize the negative impacts of this disease, and an understanding of the immune response to RV infection aids strategies for host vaccination. Current knowledge of immune responses to RV infection comes primarily from rodent models in which an innate immune response triggers activation of several genes and signalling pathways. It is unclear, however, how well rodent models represent the immune response of natural hosts. This study investigates the innate immune response of a primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The extent and temporal course of this response during RRV infection was analysed using genes predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor-3; TNF receptor). We found that RRV activated components of the innate immune system, with changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that natural reservoirs of rabies may not mimic the immune response triggered in rodent models, highlighting the need for further studies of infection in primary hosts.
Assuntos
Modelos Animais de Doenças , Imunidade Inata , Vírus da Raiva/fisiologia , Raiva , Guaxinins/imunologia , Animais , Humanos , Interferons/genética , Interferons/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Raiva/genética , Raiva/imunologia , Raiva/virologia , Vírus da Raiva/imunologia , Guaxinins/virologiaRESUMO
Rabies kills approximately 60,000 humans each year, with deaths mostly occurring in developing countries, where rabies lyssavirus (RABV) variants are maintained in dog populations [...].
Assuntos
Doenças do Cão , Lyssavirus , Vírus da Raiva , Raiva , Humanos , Animais , Cães , Raiva/prevenção & controle , Raiva/veterinária , Lyssavirus/genética , Vírus da Raiva/genéticaRESUMO
Rabies spreads in both Arctic (Vulpes lagopus) and red foxes (Vulpes vulpes) throughout the Canadian Arctic but limited wildlife disease surveillance, due to the extensive landmass of the Canadian north and its small widely scattered human population, undermines our knowledge of disease transmission patterns. This study has explored genetic population structure in both the rabies virus and its fox hosts to better understand factors that impact rabies spread. Phylogenetic analysis of 278 samples of the Arctic lineage of rabies virus recovered over 40 years identified four sub-lineages, A1 to A4. The A1 lineage has been restricted to southern regions of the Canadian province of Ontario. The A2 lineage, which predominates in Siberia, has also spread to northern Alaska while the A4 lineage was recovered from southern Alaska only. The A3 sub-lineage, which was also found in northern Alaska, has been responsible for virtually all cases across northern Canada and Greenland, where it further differentiated into 18 groups which have systematically evolved from a common predecessor since 1975. In areas of Arctic and red fox sympatry, viral groups appear to circulate in both hosts, but both mitochondrial DNA control region sequences and 9-locus microsatellite genotypes revealed contrasting phylogeographic patterns for the two fox species. Among 157 Arctic foxes, 33 mitochondrial control region haplotypes were identified but little genetic structure differentiating localities was detected. Among 162 red foxes, 18 control region haplotypes delineated three groups which discriminated among the Churchill region of Manitoba, northern Quebec and Labrador populations, and the coastal Labrador locality of Cartwright. Microsatellite analyses demonstrated some genetic heterogeneity among sampling localities of Arctic foxes but no obvious pattern, while two or three clusters of red foxes suggested some admixture between the Churchill and Quebec-Labrador regions but uniqueness of the Cartwright group. The limited population structure of Arctic foxes is consistent with the rapid spread of rabies virus subtypes throughout the north, while red fox population substructure suggests that disease spread in this host moves most readily down certain independent corridors such as the northeastern coast of Canada and the central interior. Interestingly the evidence suggests that these red fox populations have limited capacity to maintain the virus over the long term, but they may contribute to viral persistence in areas of red and Arctic fox sympatry.
Assuntos
Raposas/classificação , Raposas/genética , Vírus da Raiva/patogenicidade , Animais , Canadá , DNA Mitocondrial/genética , Genótipo , Repetições de Microssatélites/genética , Filogenia , Vírus da Raiva/genéticaRESUMO
Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.
RESUMO
Phylogenetic analysis of a collection of rabies viruses that currently circulate in Canadian big brown bats (Eptesicus fuscus) identified five distinct lineages which have emerged from a common ancestor that existed over 400 years ago. Four of these lineages are regionally restricted in their range while the fifth lineage, comprising two-thirds of all specimens, has emerged in recent times and exhibits a recent demographic expansion with rapid spread across the Canadian range of its host. Four of these viral lineages are shown to circulate in the US. To explore the role of the big brown bat host in dissemination of these viral variants, the population structure of this species was explored using both mitochondrial DNA and nuclear microsatellite markers. These data suggest the existence of three subpopulations distributed in British Columbia, mid-western Canada (Alberta and Saskatchewan) and eastern Canada (Quebec and Ontario), respectively. We suggest that these three bat subpopulations may differ by their level of female phylopatry, which in turn affects the spread of rabies viruses. We discuss how this bat population structure has affected the historical spread of rabies virus variants across the country and the potential impact of these events on public health concerns regarding rabies.
Assuntos
Quirópteros/genética , Quirópteros/virologia , Genética Populacional , Filogenia , Vírus da Raiva/genética , Animais , Teorema de Bayes , Canadá , Núcleo Celular/genética , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Geografia , Interações Hospedeiro-Patógeno , Desequilíbrio de Ligação , Repetições de Microssatélites , Análise de Componente Principal , RNA Viral/genética , Raiva/epidemiologia , Análise de Sequência de DNA , Fatores de Tempo , Zoonoses/virologiaRESUMO
In this study, the complete genomic sequence of a rabies virus isolate HN10, recovered from brain tissue of a rabid patient in China, was determined. This is the first Chinese street isolate that has been fully characterized. The overall organization of this virus is typical of that observed for all other rabies viruses. Alignments of amino acid sequences of the phosphoprotein, glycoprotein and large protein of HN10 with those of other rabies viruses were used to examine the extent of conservation of known functional regions. Phylogenetic analysis using either the complete or partial genomic sequence of HN10 determined that this isolate is most closely associated with viruses previously shown to circulate in Guangxi and Hunan provinces. In addition, of all vaccine strains used for comparison, the attenuated Chinese vaccine strain CTN181 is most closely related to HN10.
Assuntos
Genoma Viral , Vírus da Raiva/genética , Raiva/virologia , Sequência de Aminoácidos , Sequência de Bases , Encéfalo/virologia , China , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Filogenia , RNA Viral/análise , RNA Viral/genética , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Proteínas Virais/análiseRESUMO
To improve timely ante-mortem human rabies diagnosis, methods to detect viral RNA by TaqMan-based quantitative reverse transcriptase polymerase chain reactions (qRT-PCRs) have been developed. Three sets of two primers and one internal dual-labeled probe for each primer set that target distinct conserved regions of the rabies virus N gene were designed and evaluated. Using a collection of 203 isolates representative of the world-wide diversity of rabies virus, all three primers/probe sets were shown to detect a wide range of rabies virus strains with very few detection failures; the RABVD1 set in particular was the most broadly reactive. These qRT-PCR assays were shown to be quantitative over a wide range of viral titer and were 100-1,000 times more sensitive than nested RT-PCR; however, both the standard and real-time PCR methods yielded concordant results when used to test a collection of archived human suspect samples. The qRT-PCR assay was employed to monitor virus load in the saliva of a rabies virus-infected patient undergoing the Milwaukee treatment protocol. However in this case it would appear that reduction of the viral load in the patient's saliva over time did not appear to correlate well with clearance of viral components from the brain.
Assuntos
Vírus da Raiva/isolamento & purificação , Raiva/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Primers do DNA/genética , Humanos , Masculino , Nucleoproteínas/genética , RNA Viral/genética , Vírus da Raiva/genética , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
A subpopulation of the arctic fox lineage of rabies virus has circulated extensively in red fox populations of Ontario, Canada, between the 1960s and 1990s. An intensive wildlife rabies control program, in which field operations were initiated in 1989, resulted in elimination of the disease in eastern Ontario. However in southwestern Ontario, as numbers of rabid foxes declined the proportion of skunks confirmed to be infected with this rabies virus variant increased and concerted control efforts targeting this species were employed to eliminate the disease. Since 2012 no cases due to this viral variant were reported in southwestern Ontario until 2015 when a single case of rabies due to the arctic fox variant was reported in a bovine. Several additional cases have been documented subsequently. Since routine antigenic typing cannot discriminate between the variants which previously circulated in Ontario and those from northern Canada it was unknown whether these recent cases were the result of a new introduction of this variant or a continuation of the previous enzootic. To explore the origins of this new outbreak whole genome sequences of a collection of 128 rabies viruses recovered from Ontario between the 1990s to the present were compared with those representative of variants circulating in the Canadian north. Phylogenetic analysis shows that the variant responsible for current cases in southwestern Ontario has evolved from those variants known to circulate in Ontario previously and is not due to a new introduction from northern regions. Thus despite ongoing passive surveillance the persistence of wildlife rabies went undetected in the study area for almost three years. The apparent adaptation of this rabies virus variant to the skunk host provided the opportunity to explore coding changes in the viral genome which might be associated with this host shift. Several such changes were identified including a subset for which the operation of positive selection was supported. The location of a small number of these amino acid substitutions in or close to protein motifs of functional importance suggests that some of them may have played a role in this host shift.
Assuntos
Raposas/virologia , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Zoonoses/transmissão , Animais , Animais Selvagens , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/virologia , Mephitidae/virologia , Ontário/epidemiologia , Filogenia , Raiva/epidemiologia , Raiva/transmissão , Raiva/virologia , Vírus da Raiva/classificação , Vírus da Raiva/genética , Vírus da Raiva/fisiologia , Zoonoses/epidemiologia , Zoonoses/virologiaRESUMO
ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41â¯days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6â¯h to 3â¯days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.
Assuntos
Animais Selvagens/imunologia , Imunogenicidade da Vacina , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Administração Oral , Animais , Anticorpos Antivirais/imunologia , Reservatórios de Doenças/virologia , Ensaio de Imunoadsorção Enzimática , Raposas , Imunização , Raiva/transmissão , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/genética , Guaxinins , Ratos , Sigmodontinae , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
The entire genome of a mid-Atlantic raccoon strain rabies virus (RRV) isolated in Canada was sequenced; this is the second North American wildlife rabies virus isolate to be fully characterized. The overall organization and length of the genome was similar to that of other lyssaviruses. The nucleotide sequence identity of the raccoon strain ranged between 32.7% and 85.0% when compared to other lyssaviruses, while the deduced amino acid sequence identity ranged between 22.9% and 94.2% with the nucleoprotein and polymerase being the most conserved. Notable features of RRV include the phosphoprotein's four amino acid extension compared to most other rabies viruses, and a nucleotide substitution immediately prior to the normal start codon that results in an additional methionine at the beginning of the L protein. This is the first report of the RRV L gene sequence and its 2128 amino acid product. Rates of non-synonymous and synonymous nucleotide changes within the lyssavirus L gene identified the conserved blocks II, III and IV as being most constrained. Analysis of L gene codon substitution patterns favoured models that supported positive selection, but only one site, corresponding to Leu62 of the RRV L protein, was identified as being under weak positive selection.
Assuntos
Genoma Viral , RNA Viral/genética , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Canadá , Sequência Conservada , Ordem dos Genes , Dados de Sequência Molecular , Mutação Puntual , Guaxinins/virologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sintenia , Proteínas Virais/genéticaRESUMO
The mongoose is the principal reservoir for rabies on the island of Puerto Rico. This report describes a molecular epidemiological study of representative rabies viruses recovered from the island in 1997. Two closely related but distinct variants circulating in regionally localised parts of the island were identified. The lack of a monophyletic relationship of these viruses suggests that two independent incursions of rabies onto the island have occurred. Both of these Puerto Rican variants were closely related to a variant, known as the north central skunk strain, currently circulating in North American skunk populations and all are members of the cosmopolitan rabies lineage spread during the colonial period. However, the Puerto Rican viruses are clearly distinct from those presently circulating in mongooses in Cuba and which are epidemiologically closely linked to the Mexican dog rabies virus. This study clearly establishes the distinct origins of the rabies viruses now circulating on these two Caribbean islands.
Assuntos
Estudos Epidemiológicos , Herpestidae/virologia , Vírus da Raiva/genética , Raiva/epidemiologia , Raiva/virologia , Animais , Epidemiologia Molecular , Porto Rico/epidemiologia , RNA Viral/genética , Raiva/veterinária , Vírus da Raiva/classificação , Vírus da Raiva/isolamento & purificação , Zoonoses/virologiaRESUMO
Human rabies virus vaccine strain CTN181 from China was sequenced. The overall length of the genome was 11,923 nucleotides (nt), comprising a leader sequence of 58 nt, nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609 nt, glycoprotein (G) gene of 1575 nt, RNA-dependent RNA polymerase (RdRp, L) gene of 6387 nt, and a trailer region of 70 nt. The five monocistrons are separated by intergenic regions (IGRs) of 2, 5, 5 and 24 nucleotides (nt), respectively. Two obvious differences between CTN181 and the other rabies virus vaccine strains were (1) the putative stop/polyadenylation signal of the G gene has only one poly (A) tract for CTN181, and (2) the start of the open reading frame for L has two repeats of ATG for CTN181. Both were similar to the SHBRV-18 (silver-haired bat-associated RV strain 18) strain. In addition, some mutations and new functional regions were discovered that are presumed crucial to the function of leader region and L protein. There is an equal role for all five genes in the phylogenetics of rabies virus.
Assuntos
Genoma Viral , Vacina Antirrábica , Vírus da Raiva/classificação , Vírus da Raiva/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , China , Primers do DNA , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Humanos , Dados de Sequência Molecular , Filogenia , Padrões de Referência , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China.
Assuntos
Antígenos Virais/genética , Glicoproteínas/genética , Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/virologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sequência de Bases , China , Cervos , Cães , Evolução Molecular , Feminino , Genótipo , Glicoproteínas/química , Glicosilação , Humanos , Dados de Sequência Molecular , Filogenia , Raiva/veterinária , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/químicaRESUMO
Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs.
Assuntos
Surtos de Doenças , Genoma Viral , Vírus da Raiva/genética , Raiva/veterinária , Guaxinins/virologia , Zoonoses/epidemiologia , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Ontário/epidemiologia , Filogenia , Polimorfismo de Nucleotídeo Único , Raiva/epidemiologia , Raiva/transmissão , Raiva/virologia , Vírus da Raiva/classificação , Vírus da Raiva/patogenicidade , Estudos Retrospectivos , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
While large-scale dog vaccination campaigns have significantly reduced urban rabies throughout Mexico, reports of sylvatic rabies, including cases of spill-over of bat strains into livestock and humans, are increasing. To improve knowledge of these epidemiological trends, 64 Mexican rabies virus isolates from various host species, have been characterized. Phylogenetic analysis at the viral P locus identified distinct viral strains associated with terrestrial reservoirs (dog, skunk and fox/bobcat) and a variant associated with the insectivorous bat, T. brasiliensis, consistent with prior reports. Of the two distinct clades of viruses associated with the vampire bat reservoir, one comprised just four specimens and formed an outlying group to all other vampire bat rabies isolates including those from South America and the Caribbean, a finding consistent with the early emergence of the vampire bat reservoir in Mexico. Antigenic variation of the vampire bat specimens did not correlate with the main genetic groupings; moreover complete N gene sequence analysis of selected specimens indicated limited variation within the encoded nucleoprotein that could form the basis of antigenic variation. A single isolate recovered from a cat represents a new viral variant not previously identified in North America that probably circulates in a species of insectivorous bat.
Assuntos
Quirópteros/virologia , Reservatórios de Doenças , Epidemiologia Molecular , Vírus da Raiva/genética , Raiva/veterinária , Raiva/virologia , Sequência de Aminoácidos , Animais , Variação Antigênica/genética , Geografia , Humanos , México/epidemiologia , Chaperonas Moleculares , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas/genética , Filogenia , RNA Viral/genética , Raiva/epidemiologia , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Proteínas Estruturais Virais/genéticaRESUMO
Genetic-variant analysis of rabies viruses provides the most sensitive epidemiologic tool for following the spread and persistence of these viruses in their wildlife hosts. Since its introduction by a southern epizootic movement that began in the far north, the arctic fox (AFX) strain of Rabies virus has been enzootic in Ontario for almost 50 y. Prior genetic studies identified 4 principal genetic variants (ONT.T1 to ONT.T4) that were localized to different regions of the province; furthermore, these viruses could be distinguished from the variant circulating in northern regions of Quebec, Newfoundland, and arctic zones, ARC.T5. Despite an intensive provincial control program undertaken over the last decade that involved aerial distribution of baits laden with rabies vaccine to combat fox rabies throughout the enzootic zone of Ontario, pockets of rabies activity persist. Re-evaluation of the genetic characteristics of the viral variants circulating in these areas of persistence has been undertaken. These data demonstrate that the recent outbreaks are, with 1 exception, due to persistence of the regional variant first identified in the area in the early 1990s. In contrast, the disease in the Georgian Bay area is a consequence of the incursion of a variant previously found further south. An outbreak that occurred in northern Ontario north and west of North Bay and in the neighboring border areas of Quebec in 2000-2001 was due to renewed incursion of the ARC.T5 variant from more northerly areas.
Assuntos
Surtos de Doenças/veterinária , Raposas/virologia , Variação Genética , Vírus da Raiva/genética , Raiva/veterinária , Animais , Animais Selvagens/virologia , Ontário/epidemiologia , Filogenia , RNA Viral/análise , Raiva/epidemiologia , Raiva/virologia , Vírus da Raiva/classificaçãoRESUMO
The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus.
Assuntos
Complexo Antígeno-Anticorpo/química , Antígenos Virais/genética , Epitopos/genética , Glicoproteínas/genética , Mutação , Vírus da Raiva/genética , Proteínas do Envelope Viral/genética , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Antígenos Virais/química , Antígenos Virais/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Feminino , Expressão Gênica , Glicoproteínas/química , Glicoproteínas/imunologia , Evasão da Resposta Imune/genética , Camundongos , Testes de Neutralização , Raiva/patologia , Raiva/virologia , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Análise de Sequência de RNA , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqMan)MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5' Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.
Assuntos
Doenças dos Bovinos/diagnóstico , Vírus da Imunodeficiência Bovina/isolamento & purificação , Infecções por Lentivirus/veterinária , Reação em Cadeia da Polimerase/métodos , Infecções por Retroviridae/veterinária , Spumavirus/isolamento & purificação , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/virologia , Sondas de DNA , Corantes Fluorescentes , Genes Virais/genética , Genes env/genética , Genes pol/genética , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/virologia , Sondas Moleculares , Dados de Sequência Molecular , Provírus/genética , Provírus/isolamento & purificação , Infecções por Retroviridae/diagnóstico , Infecções por Retroviridae/virologia , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
BACKGROUND: On September 30, 2000, staff at the Canadian Food Inspection Agency's Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease. The boy had been exposed to a bat in late August 2000, while vacationing with his family in the Quebec countryside. METHODS: Antemortem specimens taken from the patient were sent to the Canadian Food Inspection Agency laboratory for rabies diagnosis. Samples included saliva, eye secretions, corneal impressions, cerebral spinal fluid and skin. Specimens were examined by direct immunofluorescence microscopy, and results were confirmed using molecular biological techniques. Virus strain identification was performed by both genetic methods and phenotypic analysis with monoclonal antibodies. RESULTS: Initial results from direct immunofluorescence staining indicated that rabies virus was present in the skin biopsy specimen but not in the corneal impressions. This diagnosis of rabies was confirmed by polymerase chain reaction product analysis from several of the submitted specimens. Virus isolation from postmortem samples was not possible because fresh brain tissue was not available. Rabies virus was isolated from saliva and was determined to be similar to a variant that circulates in populations of silver-haired bats. INTERPRETATION: Intravitam diagnosis of rabies in humans is very dependent on the samples submitted for diagnosis and the method used for testing. Upon first examination, only skin specimens were positive for rabies virus antigen; using polymerase chain reaction analysis, only saliva yielded positive results for rabies virus genome. Extensive testing and retesting of specimens submitted for rabies diagnosis in humans must be done to avoid false negative results.