Bat rabies in urban centers in Chile

One hundred and five rabies isolates obtained from domestic animals and insectivorous bats in Chile between 1977 and 1998 were molecularly characterized by limited sequence analysis of their nucleoprotein genes. These isolates were compared with viruses isolated from known domestic and wildlife rabies reservoirs in the Americas to identify potential reservoirs of rabies in Chile. The phylogenetic analyses showed that none of the Chilean isolates segregated with viruses from the terrestrial reservoirs. No non-rabies lyssaviruses were found in this study. The Chilean samples were not related to viruses of the sylvatic cycle maintained by the common vampire bat (Desmodus rotundus) in Latin America. Five genetic variants were identified from insectivorous bats in Chile. The Brazilian free-tailed bat (Tadarida brasiliensis) was identified as the reservoir for the rabies genetic variant most frequently isolated in the country between 1977 and 1998. The close association of a group of viruses obtained from a domestic dog (Canis familiaris), Brazilian free-tailed bats, and a red bat (Lasiurus borealis) with viruses maintained by Lasiurus spp. in North America implicated species of this genus as the possible reservoirs of this particular genetic variant in Chile. Reservoirs for the other three variants remain unknown.     

A comparative study of rabies virus isolates from hematophagous bats in Brazil

The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.     

Presence of rabies neutralizing antibodies in wild carnivores following an outbreak of bovine rabies.

In an outbreak of bovine rabies in Argentina, a study was made of vampire bats (Desmodus rotundus) and wild carnivores. Rabies antibody rates of high prevalence were found in the bats, foxes (Dusicyon gymnocercus) and skunks (Conepatus chinga). The outbreak was part of an extensive continuing epizootic of vampire transmitted bovine rabies which may have also involved other vectors in the area of this study. Consumption of dead and dying bats by the carnivores is the suggested means of passage of rabies virus from vampire bats to foxes and skunks. Given optimum conditions it is conceivable that some outbreaks in carnivores may begin in this way.     

New Genotype of Dengue Type 3 Virus Circulating in Brazil and Colombia Showed a Close Relationship to Old Asian Viruses

Dengue type 3 genotype V viruses have been recently detected in Brazil and Colombia. In this study, we described another Brazilian isolate belonging to this genotype. Phylogenetic analysis including dengue type 3 viruses isolated worldwide showed that Brazilian and Colombian viruses were closely related to viruses isolated in Asia more than two decades ago. The characteristic evolutionary pattern of dengue type 3 virus cannot explain the close similarity of new circulating viruses with old viruses. Further studies are needed to confirm the origin of the new dengue type III genotype circulating in Brazil and Colombia.     

Standardization of rabies virus genome amplification for its use in molecular epidemiology studies

In order to improve the diagnosis and typification of rabies viruses at the Instituto Nacional de Salud National Reference Laboratory for rabies, we standardized techniques for the amplification of a 902 nucleotide DNA fragment, complementary to a selected region of the rabies virus genomic RNA. This region codes for a segment of both the glycoprotein and protein L, and contains the G-L intergenic noncoding region known as Pseudogen Psi. The standardized techniques included: 1) biological amplification of rabies viruses by intracerebral mouse inoculation; 2) total RNA extraction from the brains of infected mice, 3) RT-PCR amplification of a 902 nucleotide DNA fragment complementary to the selected RNA region. The study sample consisted of 30 rabies virus strains isolated from dogs and selected from the virus bank of the Virology Laboratory. Due to their simplicity the methods described have several advantages when compared to methods reported in previous papers. The technology proposed is a precise complement to rabies diagnosis techniques, and can be applied to the identification of phylogenetic relations among rabies isolates, and, therefore, it is also used to identify rabies transmission dynamics and geographical distribution.     

Experimental rabies virus infection of big brown bats (Eptesicus fuscus)

A captive colony of adult Big Brown Bats (Eptesicus fuscus) was experimentally infected with a rabies virus (RABV) variant isolated from the salivary glands of a naturally infected Big Brown Bat and passaged once through murine neuroblastoma cell culture. Bats were divided into 11 groups, which were composed of one to three noninfected and one to three infected individuals each. Twenty of 38 animals were infected intramuscularly into both left and right masseter muscles; they received a total of 10(3.2) median mouse intracerebral lethal dose (MICLD50) of Big Brown Bat RABV variant. Experimental outcome after viral exposure was followed in the bats for 140 days postinoculation (PI). Of 20 infected bats, 16 developed clinical rabies, and the mean incubation period was 24 days (range: 13-52 days). Three infected bats never seroconverted and succumbed early to infection (13 days). Four infected bats that survived until the end of the experiment without any signs of disease maintained detectable antibody titers until the third month PI, peaking between days 13 and 43, and consequent drop-off below the threshold for detection occurred by day 140. Limited excretion of virus in saliva of infected bats during the clinical course of disease was observed in two individuals on days 13 and 15 PI (<24 hr prior to onset of clinical illness). No bat-to-bat transmission of RABV to noninfected bats was detected.     

Molecular Epidemiology of Rabies Viruses Circulating in Two Rabies Endemic Provinces of Laos, 2011–2012: Regional Diversity in Southeast Asia

BackgroundAlthough rabies is endemic in Laos, genetic characterization of the viruses in this country is limited. There are growing concerns that development in the region may have increased transport of dog through Laos for regional dog meat consumption, and that this may cause spillover of the viruses from dogs brought here from other countries. This study was therefore undertaken to evaluate the current rabies situation and the genetic characteristics of rabies viruses currently circulating in Laos.MethodsWe determined the rate of rabies-positive samples by analyzing data from animal samples submitted to the Lao Ministry of Agriculture and Forestry’s National Animal Health Centre rabies laboratory from 2004 through 2011. Twenty-three rabies-positive samples were used for viral genetic characterization. Full genome sequencing was performed on two rabies viruses.ResultsRabies-positive samples increased substantially from 40.5% in 2004 to 60.2% in 2009 and continued at this level during the study period. More than 99% of the samples were from dogs, followed by cats and monkeys. Phylogenetic analyses showed that three rabies virus lineages belonging to the Southeast Asian cluster are currently circulating in Laos; these are closely related to viruses from Thailand, Cambodia and Vietnam. Lineages of the circulating Laos rabies viruses diverged from common ancestors as recently as 44.2 years and as much as 55.3 years ago, indicating periodic virus invasions.ConclusionThere is an increasing trend of rabies in Laotian animals. Similar to other rabies-endemic countries, dogs are the main viral reservoir. Three viral lineages closely related to viruses from neighboring countries are currently circulating in Laos. Data provide evidence of periodic historic exchanges of the viruses with neighboring countries, but no recent invasion.     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

Epidemic and maintenance of rabies in chinese ferret badgers (Melogale moschata) indicated by epidemiology and the molecular signatures of rabies viruses

An epidemic of Chinese ferret badger-associated human rabies was investigated in Wuyuan county, Jiangxi province and rabies viruses isolates from ferret badgers in different districts in Jiangxi and Zhejiang provinces were sequenced with their nucleotides and amino acids and aligned for epidemiological analysis. The results showed that the human rabies in Wuyuan are only associated with ferret badger bites; the rabies virus can be isolated in a high percentage of ferret badgers in the epidemic areas in Jiangxi and Zhejiang provinces; the isolates share the same molecular features in nucleotides and have characteristic amino acid signatures, i.e., 2 sites in the nucleoprotein and 3 sites in the glycoprotein, that are distinct from virus isolates from dogs in the same region. We conclude that rabies in Chinese ferret badgers has formed an independent transmission cycle and ferret badgers may serve as another important rabies reservoir independent of dog rabies in China.     

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background

Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.

Methods

Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.

Results

In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.

Conclusion

This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

     

Detection of rabies virus antibodies in fruit bats (Eidolon helvum) from Nigeria

Fruit bats (Eidolon helvum) were collected from three different localities in Western Nigeria: Oti, Ibadan and Idanre. Fifty serum samples were analyzed using a modified rapid fluorescent focus inhibition technique against rabies, Mokola, Lagos bat and Duvenhage viruses. Twenty-five brain samples were screened for rabies and related lyssavirus antigens by direct fluorescent antibody microscopy and by an indirect fluorescent antibody technique with rabies anti-nucleocapsid monoclonal antibodies. All brain samples were negative. Two serum samples had relatively high anti-rabies activity; no neutralizing activity was detected against Mokola, Lagos bat or Duvenhage viruses.     

Surveillance and epidemiologic mapping of monoclonal antibody-defined rabies variants in Florida

Brain tissues from 128 rabid animals from Florida in 1987 and 1988 were analyzed with monoclonal antibodies and cases were mapped by species and antigenic variant. The single variant found in terrestrial animals was distinguished easily from the variety of antigenic variants identified for infected bats, and there was no evidence of transmission of rabies between bats and terrestrial animals. The raccoon (Procyon lotor) appeared to be the sole maintenance source for terrestrial animal rabies in Florida.     

Spatial and temporal dynamics of rabies virus variants in big brown bat populations across Canada: footprints of an emerging zoonosis

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.     

Genetic analysis of rabies virus isolates in the Philippines

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.     

Bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses?

An increasingly asked question is ''can we confidently link bats with emerging viruses?''. No, or not yet, is the qualified answer based on the evidence available. Although more than 200 viruses - some of them deadly zoonotic viruses - have been isolated from or otherwise detected in bats, the supposed connections between bats, bat viruses and human diseases have been raised more on speculation than on evidence supporting their direct or indirect roles in the epidemiology of diseases (except for rabies). However, we are convinced that the evidence points in that direction and that at some point it will be proved that bats are competent hosts for at least a few zoonotic viruses. In this review, we cover aspects of bat biology, ecology and evolution that might be relevant in medical investigations and we provide a historical synthesis of some disease outbreaks causally linked to bats. We provide evolutionary-based hypotheses to tentatively explain the viral transmission route through mammalian intermediate hosts and to explain the geographic concentration of most outbreaks, but both are no more than speculations that still require formal assessment.     

Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control

Despite extensive culling of common vampire bats in Latin America, lethal human rabies outbreaks transmitted by this species are increasingly recognized, and livestock rabies occurs with striking frequency. To identify the individual and population-level factors driving rabies virus (RV) transmission in vampire bats, we conducted a longitudinal capture-recapture study in 20 vampire bat colonies spanning four regions of Peru. Serology demonstrated the circulation of RV in vampire bats from all regions in all years. Seroprevalence ranged from 3 to 28 per cent and was highest in juvenile and sub-adult bats. RV exposure was independent of bat colony size, consistent with an absence of population density thresholds for viral invasion and extinction. Culling campaigns implemented during our study failed to reduce seroprevalence and were perhaps counterproductive for disease control owing to the targeted removal of adults, but potentially greater importance of juvenile and sub-adult bats for transmission. These findings provide new insights into the mechanisms of RV maintenance in vampire bats and highlight the need for ecologically informed approaches to rabies prevention in Latin America.     

Spatial expansions and travelling waves of rabies in vampire bats

A major obstacle to anticipating the cross-species transmission of zoonotic diseases and developing novel strategies for their control is the scarcity of data informing how these pathogens circulate within natural reservoir populations. Vampire bats are the primary reservoir of rabies in Latin America, where the disease remains among the most important viral zoonoses affecting humans and livestock. Unpredictable spatiotemporal dynamics of rabies within bat populations have precluded anticipation of outbreaks and undermined widespread bat culling programs. By analysing 1146 vampire bat-transmitted rabies (VBR) outbreaks in livestock across 12 years in Peru, we demonstrate that viral expansions into historically uninfected zones have doubled the recent burden of VBR. Viral expansions are geographically widespread, but severely constrained by high elevation peaks in the Andes mountains. Within Andean valleys, invasions form wavefronts that are advancing towards large, unvaccinated livestock populations that are heavily bitten by bats, which together will fuel high transmission and mortality. Using spatial models, we forecast the pathways of ongoing VBR epizootics across heterogeneous landscapes. These results directly inform vaccination strategies to mitigate impending viral emergence, reveal VBR as an emerging rather than an enzootic disease and create opportunities to test novel interventions to manage viruses in bat reservoirs.     

Detection and genetic characterization of rabies virus from human patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site III in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level. Foundation items: The project was supported by the Grants (2003BA712A08-02 and 2004BA718B03) from the Chinese Ministry of Science and Technology.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.     

家养犬、猫及野生动物中狂犬病病毒流行病学调查及我国不同类型狂犬病疫苗免疫效果观测研究

选择我国狂犬病高发地区(湖南、贵州)、低发地区(江苏、武汉)和无狂犬病报告地区(沈阳)等三种不同类型的地区,开展针对家养犬、猫和啮齿类动物,以及蝙蝠等野生动物的狂犬病病毒带毒率的流行病学调查,对分离到的病毒株在抗原型别、基因变异以及与现行疫苗是否匹配等方面进行分析、比较研究。结果显示:我国狂犬病的主要宿主动物为犬,捕获犬总带毒率为2.56%;犬中狂犬病病毒的监测点最高阳性检出率达到20.0%;啮齿类动物及蝙蝠等野生动物在本次研究未检测到狂犬病病毒。我国自主研制成功的以aG株和CTN株为毒种的现行人用狂犬病疫苗,经应用研究发现,CTN株的糖蛋白基因与本研究分离到的街毒核酸序列更接近。为进一步评价现行疫苗的有效性,在5个观察点分别以aG株和CTN株毒种生产的疫苗接种两组人群(每组各约50人),结果显示均未出现中、强度反应,疫苗免疫后第7d和14d产生的中和抗体分别达到0.49IU/mL~0.52IU/mL和6.7IU/mL~7.53IU/mL;抗体阳转率分别为45.1%~47.9%和100%,表明这些疫苗具有良好的安全性和保护效果。