Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil

In Latin America, rabies cases related to frugivorous bats have been reported since 1930's. Recently, two viruses isolated from Artibeus lituratus were proved to be vampire bat variants by monoclonal antibodies panels [2], but their genetic information is not well known. In this report, four rabies viruses were isolated from frugivorous bats (Artibeus spp.) in Brazil and their nucleoprotein gene sequences were determined. These isolates were found to be genotype 1 of lyssavirus and showed the maximum nucleotide sequence homology of 97.6-99.4% with vampire bat-related viruses in Brazil [6]. These results indicate that the Brazilian frugivorous bat rabies viruses in this study are closely related to vampire bat-related viruses that play a main role in rabies virus transmission to livestock in Brazil.     

Geographical distribution of vampire bat-related cattle rabies in Brazil

Seventy-seven rabies virus (RV) isolates originating from Brazilian cattle were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat-related RV group, were further subdivided into nine genetic subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by mountain ranges. In addition, separation of the groups in mountainous regions was correlated with altitude. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.     

Virus isolation from saliva and salivary glands of cattle naturally infected with paralytic rabies

The infectivity of saliva, salivary and mammary glands, muscle, lung, kidney and liver of 87 cattle infected with paralytic rabies (positive viral isolation from brains) was studied. Fifty percent dilutions of saliva and tissue samples were inoculated intracerebrally into 10- to 15-day-old mice. Viral isolation in mice was confirmed by direct rabies fluorescent-antibody test and the antigenic variant of the isolates characterized by monoclonal antibodies. Rabies virus was isolated from 4.6% of salivary glands and from 1.6% of saliva samples. The rest of the peripheral tissues were negative. Cerebral and peripheral isolates belonged to vampire-bat antigenic variants. These results indicate that cattle infected by vampire bats may be a source of infection for man. The infection risk would depend on the type of contact between rabid cattle and man.     

Molecular epidemiological analysis of bat rabies viruses in Brazil

A molecular epidemiological analysis was performed in 19 rabies viruses (RVs) isolated from haematophagous, frugivorous and insectivorous bats, in Sao Paulo, Brazil. The authors carried out RT-PCR for amplification of the RV nucleoprotein (N) gene, and determined 1,335 nucleotide sequences of N gene by direct sequencing method. Phylogenetic analysis, which was based on the N gene of Brazilian RV isolates identified presently and previously, revealed that RVs isolated from bats were genetically divided into four lineages had a tendency to depend on the host bat species. The first lineage consisted mainly of haematophagous bat (Desmodus rotundus) isolates, including frugivorous bat (Artibeus spp.) isolates. Other three lineages consisted of insectivorous bat isolates; mainly Eptesicus spp., Molossus spp. and Nyctinomops spp. isolates, respectively. These results indicate a possibility of that there are bat species-specific RV variants in Brazil.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Genetic analysis of phosphoprotein and matrix protein of rabies viruses isolated in Brazil

To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with Asp(42) and Glu(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants.     

The rabies viruses of bats

In the 1930s rabies was shown to affect blood-, insect- and fruit-eating bats. We have prepared anti-nucleocapsid monoclonal antibodies (MAbs) using Mokola and bat (Lagos, Duvenhage and Denmark) rabies viruses as immunogens. With these MAbs we have examined rabies viruses from vampire, insectivorous and frugivorous bats from the Americas, Africa, Europe and the Soviet Union and have compared them with isolates from terrestrial species including man. As well as confirming the findings of others with viruses of African and American bat origin, the results revealed the presence of a second biotype in European bats and demonstrated the presence of serotype 1 as well as serotype 4 viruses in bats of the Soviet Union.     

Experimental infection of Artibeus intermedius with a vampire bat rabies virus

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n = 35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n = 3), stomach (n = 1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.     

Determination and molecular analysis of the complete genome sequence of two wild-type rabies viruses isolated from a haematophagous bat and a frugivorous bat in Brazil

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and BR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined "vampire bat-related RABV lineage" which consisted of mainly D. rotundus- and A. lituratus-isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.     

An outbreak of vampire bat-transmitted rabies in cattle in northeastern Mexico.

An outbreak of bovine rabies occurred on a ranch when cattle were bitten by vampire bats. Microscopic lesions showed a nonsuppurative encephalitis with intracytoplasmic inclusion bodies. Immunohistochemistry demonstrated viral antigen in the brain, and monoclonal antibodies identified a serotype 1 (vampire strain) of the rabies virus.     

Molecular characterization of rabies virus isolates from Trinidad

Bovine rabies continues to be a serious problem facing the cattle industry in South and Central America. Although Trinidad played an important role in originally demonstrating the link between bats and bovine rabies, relatively little is known about rabies in Trinidad, an island 7miles off the coast of Venezuela. In order to obtain a more complete understanding of bovine rabies in the region, we report herein on a study undertaken in Trinidad to characterize isolates of rabies virus obtained from infected cattle. A portion of the nucleotide sequence of the nucleoprotein gene from six rabies virus isolates collected from bovine rabies from the years 1997, 1998 and 2000 was determined and compared both to themselves and the nucleotide sequence of other South American isolates. Results indicate that there are at least two independently evolving variants of rabies virus in Trinidad. The nucleotide sequence of either variant failed to match completely the sequence of South American isolates. However, the lack of South American isolates from coastal regions facing Trinidad leaves undetermined the question of South American influence on rabies in Trinidad. The results of this study helps complete the picture of bovine rabies in the South American region and provide basic information required locally for the creation of an effective rabies control and eradication strategy.     

Trends in national surveillance for rabies among bats in the United States (1993-2000)

OBJECTIVE: To describe surveillance trends and epidemiologic features of rabies in bats in the United States, focusing on 3 bat species primarily associated with variants of the rabies virus that affect humans. DESIGN: Retrospective study. ANIMALS: 31,380 bats. PROCEDURE: Data on rabies for bats identified to species and reported by state laboratories from 1993 to 2000 were analyzed, focusing on silver-haired, eastern pipistrelle, and Brazilian free-tailed bats. Categoric variables were derived from other provided information. RESULTS: Data were reported from 37 states during the study interval; complete species-specific data were not reported by any state for the entire interval. Bats primarily associated with rabies virus variants affecting humans were more likely to yield positive test results for rabies (22.7%), compared with all other bats (5.5%) in most seasons and from most regions of the United States. However, certain other bat species had higher percentages of positive results. Risk of positive results was highest in the fall and highest among bats originating in the southwestern United States. CONCLUSIONS AND CLINICAL RELEVANCE: Increased risk of rabies among certain groups of bat species was consistently found across seasons and most geographic regions of the United States. Results were in general agreement with those of previous studies conducted within smaller geographic regions. There are ongoing efforts to improve surveillance of rabies in bats, although surveillance is incomplete in some regions.     

Clinical, pathological and antigenic aspects of bovine viral diarrhea virus (BVDV) type 2 isolates identified in Brazil

Nucleotide sequencing and phylogenetic analysis of Brazilian bovine viral diarrhea virus (BVDV) field isolates identified four viruses belonging to the genotype 2. Comparison of 5' UTR sequences from these isolates to those of North American BVDV type 2 revealed genomic variations that correlated with the geographic origins of the isolates. Two of the Brazilian type 2 viruses were isolated from clinical cases of gastroenteric/respiratory disease and two were isolated from healthy bovine fetuses. The clinical cases affected young animals (8- and 18-months-old) and were characterized by diarrhea, respiratory signs, extensive oral and digestive tract erosions, conjunctival and vulvar congestion, occasional digestive bleeding and vulvar and heart petechial hemorrhage. Antigenic analysis of these isolates with a panel of 10 monoclonal antibodies revealed marked antigenic differences in the major envelope glycoprotein, gp53/E2, compared to standard laboratory and vaccine BVDV strains. In addition, virus-specific antisera raised to Brazilian BVDV type 2 viruses displayed very low serological cross-reactivity with standard BVDV type 1 strains. Differences up to 64-fold in cross-neutralization titers were observed between BVDV type 1 and Brazilian BVDV type 2 isolates. The identification of BVDV type 2 among Brazilian cattle may have important implications for epidemiological studies, diagnostic and immunization strategies. Furthermore, the low neutralizing activity of BVDV type 1 antisera against the recently identified Brazilian BVDV type 2 isolates raises the question about the degree of protection conferred by BVDV vaccines, most of them based on a single type 1 strain.     

Zoonotic pathogens isolated from wild animals and environmental samples at two California wildlife hospitals

During 2010, 48 states and Puerto Rico reported 6,154 rabid animals and 2 human rabies cases to the CDC, representing an 8% decrease from the 6,690 rabid animals and 4 human cases reported in 2009. Hawaii and Mississippi did not report any laboratory-confirmed rabid animals during 2010. Approximately 92% of reported rabid animals were wildlife. Relative contributions by the major animal groups were as follows: 2,246 raccoons (36.5%), 1,448 skunks (23.5%), 1,430 bats (23.2%), 429 foxes (6.9%), 303 cats (4.9%), 71 cattle (1.1 %), and 69 dogs (1.1 %). Compared with 2009, number of reported rabid animals decreased across all animal types with the exception of a 1 % increase in the number of reported rabid cats. Two cases of rabies involving humans were reported from Louisiana and Wisconsin in 2010. Louisiana reported an imported human rabies case involving a 19-year-old male migrant farm worker who had a history of a vampire bat (Desmodus rotundus) bite received while in Mexico. This represents the first human rabies case reported in the United States confirmed to have been caused by a vampire bat rabies virus variant. Wisconsin reported a human rabies case involving a 70-year-old male that was confirmed to have been caused by a rabies virus variant associated with tri-colored bats (Perimyotis subflavus).     

Serologic and genetic characterization of North American H3N2 swine influenza A viruses

The H3N2 subtype of influenza A viruses isolated from pigs in the United States and Canada has shown both genetic and antigenic diversity. The objective of this study was to determine the serologic and genetic characteristics of contemporary strains of these viruses. Genetic analysis of 18 reference strains and 8 selected strains demonstrated differences in 1% to 9% of the nucleotides of the hemagglutinin (HA) gene. Phylogenetic analysis of the HA gene revealed 3 genetic clusters, as well as divergence of cluster III viruses from a cluster III prototype virus (A/Swine/Illinois/21587/99). By means of 1-way cross-hemagglutination inhibition with antiserum against 5 field isolates and 3 vaccine viruses, most of 97 isolates tested could be placed in 1 of 3 serogroups. The several isolates that did not react with any antiserum were in genetic cluster III, which suggests that continuous antigenic drift in cluster III may have resulted in virus variants. The efficacy of commercial vaccines against these virus variants should be evaluated with vaccination and challenge studies.     

Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.     

Antigenic diversity of bovine viral diarrhea-mucosal disease (BVD-MD) viruses recently isolated from persistently infected cattle and mucosal disease, and serologic survey on bovine sera using antigenically different BVD-MD viruses

Twenty nine recent isolates of bovine viral diarrhea-mucosal disease (BVD-MD) virus, 17 from persistently infected cattle and 12 from mucosal disease, were compared antigenically with the reference strains by a serum neutralization test. The reference viruses were divided into 2 groups, tentatively designated as N and K, based on the antigenic relationships in the cross-neutralization test. Antigenic properties of recent isolates were considerably different with the sources of virus isolation. Seventeen isolates recovered from persistently infected cattle were divided into 3 groups in the neutralization test using antisera to the reference strains; 12 and 2 were considered as the possible members of groups K and N, respectively, and the others belonged to neither group. On the other hand, 10 of 12 isolates recovered from mucosal disease were considered as the possible members of group N, and the others were classified into neither group. Interestingly, none of BVD-MD viruses isolated from cases of mucosal disease belonged to group K. The results of serologic survey on sera collected from 713 cattle at the Hokkaido provinces in 1974 to 1988 indicated that infections of cattle with BVD-MD viruses other than group K were prominent before 1981. Cattle infected with group K BVD-MD virus were first detected in 1982, and increased in number thereafter. The results obtained in this study suggested that BVD-MD viruses with various antigenic properties spread widely among cattle herds, and also a possibility that clinical manifestations in cattle infected with BVD-MD viruses may differ with their antigenic properties.     

动物狂犬病流行毒株的抗原性差异分析

利用9株抗狂犬病病毒（Rabies virus,RABV）核蛋白单克隆抗体,通过间接免疫荧光方法对分离的34株传至F6代N2A细胞适应株进行了抗原差异分析。结果表明：这些RABV分离株存在抗原差异,根据病毒与单抗反应的类型,将所分析的病毒株分为6个抗原变异型。根据N基因系统发生分析将所分析的病毒株分为4个进化群,结果表明不同进化群病毒间的遗传差异与抗原分型没有直接的联系。