Detection of lyssavirus antigen and antibody levels among apparently healthy and suspected rabid dogs in South-Eastern Nigeria

Objectives

Domestic dogs are the main reservoir of rabies virus (RABV) infection in Nigeria, thus surveillance of rabies in dog populations is crucial in order to understand the patterns of spread of infection and ultimately devise an appropriate rabies control strategy. This study determined the presence of lyssavirus antigen in brain tissues and anti-rabies antibodies in sera of apparently healthy and suspected-rabid dogs slaughtered for human consumption at local markets in South-Eastern Nigeria.

Results

Our findings demonstrated that 8.3% (n?=?23) of brain tissues were lyssavirus positive and 2.5% (n?=?25) of sera had rabies antibody levels as percentage blocking of 70% and above correlating with a cut-off value?≥?0.5 IU/mL in the fluorescent antibody neutralization test. There was an inverse correlation between lyssavirus positivity and rabies antibody levels confirming that infected individuals most often do not develop virus neutralizing antibodies to the disease. The low percentage of rabies antibodies in this dog population suggests a susceptible population at high risk to RABV infection. These findings highlight a huge challenge to national rabies programs and subsequent elimination of the disease from Nigeria, considering that majority of dogs are confined to rural communal areas, where parenteral dog vaccination is not routinely undertaken.

     

Enhanced Passive Bat Rabies Surveillance in Indigenous Bat Species from Germany - A Retrospective Study

In Germany, rabies in bats is a notifiable zoonotic disease, which is caused by European bat lyssaviruses type 1 and 2 (EBLV-1 and 2), and the recently discovered new lyssavirus species Bokeloh bat lyssavirus (BBLV). As the understanding of bat rabies in insectivorous bat species is limited, in addition to routine bat rabies diagnosis, an enhanced passive surveillance study, i.e. the retrospective investigation of dead bats that had not been tested for rabies, was initiated in 1998 to study the distribution, abundance and epidemiology of lyssavirus infections in bats from Germany. A total number of 5478 individuals representing 21 bat species within two families were included in this study. The Noctule bat (Nyctalus noctula) and the Common pipistrelle (Pipistrellus pipistrellus) represented the most specimens submitted. Of all investigated bats, 1.17% tested positive for lyssaviruses using the fluorescent antibody test (FAT). The vast majority of positive cases was identified as EBLV-1, predominately associated with the Serotine bat (Eptesicus serotinus). However, rabies cases in other species, i.e. Nathusius'' pipistrelle bat (Pipistrellus nathusii), P. pipistrellus and Brown long-eared bat (Plecotus auritus) were also characterized as EBLV-1. In contrast, EBLV-2 was isolated from three Daubenton''s bats (Myotis daubentonii). These three cases contribute significantly to the understanding of EBLV-2 infections in Germany as only one case had been reported prior to this study. This enhanced passive surveillance indicated that besides known reservoir species, further bat species are affected by lyssavirus infections. Given the increasing diversity of lyssaviruses and bats as reservoir host species worldwide, lyssavirus positive specimens, i.e. both bat and virus need to be confirmed by molecular techniques.     

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.     

A reduced panel of anti-nucleocapsid monoclonal antibodies for bat rabies virus identification in Europe

A reduced panel of 4 anti-nucleocapsid monoclonal antibodies (mAb) was set up to distinguish viruses of terrestrial mammal origin from viruses of bat origin in Europe. Four additional mAb were necessary to identify each one of the four serotypes of lyssavirus. These 8 mAb were selected out of 25 mAb secreted by hybridomas obtained form mice immunized with either serotype 1 lyssavirus (rabies virus PV4) or serotype 3 lyssavirus (Mokola). They were screened with 32 viruses representative of the four lyssavirus serotypes and the two types of European bat lyssavirus. The panel was tested by immunofluorescence assay with 25 cell-culture-adapted European wildlife isolates and in routine rabies identification with 65 rabid animal brain smears, Two isolates from Eptesicus serotinus in France were identified as European bat lyssavirus 1 with the reduced panel.     

Investigation of the evolutionary history of the lyssaviruses

Dear Editor, We report the results of evolutionary history estimation of the lyssaviruses based on an analysis of the Glycoprotein (G) sequences gene using the BEAST software package.The most recent common ancestor (TMRCA) of all the lyssavirus strains was estimated to be approximately 5030 years (95％ HPD 3988-6069 years),and there was a significant spread of the rabies virus throughout the world range in the last 200 years,consistent with significant time points in development and migration of human civilizations.We speculate that increased and expansion of human migration during this time period may have promote the increase in lyssavirus diversity.In addition,evidence of host switching in lyssavirus history from the Chiroptera to the Carnivora orders was also identified.     

Temporal dynamics of European bat Lyssavirus type 1 and survival of Myotis myotis bats in natural colonies

Many emerging RNA viruses of public health concern have recently been detected in bats. However, the dynamics of these viruses in natural bat colonies is presently unknown. Consequently, prediction of the spread of these viruses and the establishment of appropriate control measures are hindered by a lack of information. To this aim, we collected epidemiological, virological and ecological data during a twelve-year longitudinal study in two colonies of insectivorous bats (Myotis myotis) located in Spain and infected by the most common bat lyssavirus found in Europe, the European bat lyssavirus subtype 1 (EBLV-1). This active survey demonstrates that cyclic lyssavirus infections occurred with periodic oscillations in the number of susceptible, immune and infected bats. Persistence of immunity for more than one year was detected in some individuals. These data were further used to feed models to analyze the temporal dynamics of EBLV-1 and the survival rate of bats. According to these models, the infection is characterized by a predicted low basic reproductive rate (R(0) = 1.706) and a short infectious period (D = 5.1 days). In contrast to observations in most non-flying animals infected with rabies, the survival model shows no variation in mortality after EBLV-1 infection of M. myotis. These findings have considerable public health implications in terms of management of colonies where lyssavirus-positive bats have been recorded and confirm the potential risk of rabies transmission to humans. A greater understanding of the dynamics of lyssavirus in bat colonies also provides a model to study how bats contribute to the maintenance and transmission of other viruses of public health concern.     

Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 10^0.1 to 10^4.1 median tissue culture infectious dose (TCID₅₀). More bats died due to the development of rabies after the middle dose (10^2.1 TCID₅₀, 4/4 bats) than after lower (10^1.1, 2/4; 10^1.1, 2/4) or higher (10^3.1, 2/4; 10^4.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 10^2.1 TCID₅₀ of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats.     

Bat rabies surveillance in France: first report of unusual mortality among serotine bats

Background

Rabies is a fatal viral encephalitic disease that is caused by lyssaviruses which can affect all mammals, including human and bats. In Europe, bat rabies cases are attributed to five different lyssavirus species, the majority of rabid bats being attributed to European bat 1 lyssavirus (EBLV-1), circulating mainly in serotine bats (Eptesicus serotinus). In France, rabies in bats is under surveillance since 1989, with 77 positive cases reported between 1989 and 2016.

Case presentation

In the frame of the bat rabies surveillance, an unusual mortality of serotine bats was reported in 2009 in a village in North-East France. Six juvenile bats from an E. serotinus maternity colony counting ~200 individuals were found to be infected with EBLV-1. The active surveillance of the colony by capture sessions of bats from July to September 2009 showed a high detection rate of neutralising EBLV-1 antibodies (≈ 50%) in the colony. Moreover, one out of 111 animals tested was found to shed viable virus in saliva, while lyssavirus RNA was detected by RT-PCR for five individuals.

Conclusion

This study demonstrated that the lyssavirus infection in the serotine maternity colony was followed by a high rate of bat rabies immunity after circulation of the virus in the colony. The ratio of seropositive bats is probably indicative of an efficient virus transmission coupled to a rapid circulation of EBLV-1 in the colony.

     

Raccoons (Procyon lotor) in Germany as potential reservoir species for Lyssaviruses

Raccoons can be found almost everywhere in Germany since their first successful introduction in 1934. Although the animal is a well-known reservoir species for rabies in the USA, during the last European fox rabies epizootic, only a few rabid raccoons were reported from Germany. In recent years, the raccoon population density has increased tremendously, especially in (semi) urban settings. Presently, Germany is free of terrestrial wildlife rabies. To assess the potential risk that the raccoon population in Germany could act as a reservoir species upon reemergence of rabies, the susceptibility of the local raccoon population was investigated. Wild-caught animals were inoculated with the most likely lyssavirus variants to infect the local population. It was shown that the raccoons were fully susceptible for a dog and raccoon rabies virus isolate. Also, five of six raccoons inoculated with a fox rabies virus isolate showed clinical signs. However, none of the raccoons infected with European Bat Lyssavirus type 1 succumbed to rabies; meanwhile, all these raccoons seroconverted. It is concluded that the highest risk for the raccoon population in Germany to become infected with lyssaviruses is through the importation of rabies infected dogs.     

Mitochondrial Dysfunction in Rabies Virus-Infected Human and Canine Brains

Neurochemical Research - Rabies is a fatal encephalitis caused by the Rabies lyssavirus (RABV). The presence of minimal neuropathological changes observed in rabies indicates that neuronal...     

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.     

Focus: Zoonotic Disease: Rabies in Costa Rica – Next Steps Towards Controlling Bat-Borne Rabies After its Elimination in Dogs

Rabies is an acute, progressive encephalitis caused by a lyssavirus, with the highest case fatality of any conventional infectious disease. More than 17 different lyssaviruses have been described, but rabies virus is the most widely distributed and important member of the genus. Globally, tens of thousands of human fatalities still occur each year. Although all mammals are susceptible, most human fatalities are caused by the bites of rabid dogs, within lesser developed countries. A global plan envisions the elimination of human rabies cases caused via dogs by the year 2030. The combination of prophylaxis of exposed humans and mass vaccination of dogs is an essential strategy for such success. Regionally, the Americas are well on the way to meet this goal. As one example of achievement, Costa Rica, a small country within Central America, reported the last autochthonous case of human rabies transmitted by a dog at the end of the 1970s. Today, rabies virus transmitted by the common vampire bat, Desmodus rotundus, as well as other wildlife, remains a major concern for humans, livestock, and other animals throughout the region. This review summarizes the historical occurrence of dog rabies and its elimination in Costa Rica, describes the current occurrence of the disease with a particular focus upon affected livestock, discusses the ecology of the vampire bat as the primary reservoir relevant to management, details the clinical characteristics of recent human rabies cases, and provides suggestions for resolution of global challenges posed by this zoonosis within a One Health context.     

Disease Risk Perception and Safety Practices: A Survey of Australian Flying Fox Rehabilitators

Interactions with flying foxes pose disease transmission risks to volunteer rehabilitators (carers) who treat injured, ill, and orphaned bats. In particular, Australian bat lyssavirus (ABLV) can be transmitted directly from flying foxes to humans in Australia. Personal protective equipment (PPE) and rabies vaccination can be used to protect against lyssavirus infection. During May and June 2014, active Australian flying fox carers participated in an online survey (SOAR: Survey Of Australian flying fox Rehabilitators) designed to gather demographic data, assess perceptions of disease risk, and explore safety practices. Responses to open-ended questions were analysed thematically. A logistic regression was performed to assess whether rehabilitators’ gender, use of PPE, threat perception, and years of experience predicted variation in their odds of being bitten or scratched. Eligible responses were received from 122 rehabilitators located predominantly on the eastern coast of Australia. Eighty-four percent of respondents were female. Years of experience ranged from <1 to 30 years (median 5 years). Respondents were highly educated. All rehabilitators were vaccinated against rabies and 94% received a rabies titre check at least every two years. Sixty-three percent of carers did not perceive viruses in flying foxes as a potential threat to their health, yet 74% of carers reported using PPE when handling flying foxes. Eighty-three percent of rehabilitators had received a flying fox bite or scratch at some point during their career. Carers provide an important community service by rescuing and rehabilitating flying foxes. While rehabilitators in this study have many excellent safety practices, including a 100% vaccination rate against rabies, there is room for improvement in PPE use. We recommend 1) the establishment of an Australia-wide set of guidelines for safety when caring for bats and 2) that the responsible government agencies in Australia support carers who rescue potentially ABLV-infected bats by offering compensation for PPE.     

Host switching in Lyssavirus history from the Chiroptera to the Carnivora orders

Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora and Chiroptera orders. We studied 36 carnivoran and 17 chiropteran lyssaviruses representing the main genotypes and variants. We compared their genes encoding the surface glycoprotein, which is responsible for receptor recognition and membrane fusion. The glycoprotein is the main protecting antigen and bears virulence determinants. Point mutation is the main force in lyssavirus evolution, as Sawyer's test and phylogenetic analysis showed no evidence of recombination. Tests of neutrality indicated a neutral model of evolution, also supported by globally high ratios of synonymous substitutions (d(S)) to nonsynonymous substitutions (d(N)) (>7). Relative-rate tests suggested similar rates of evolution for all lyssavirus lineages. Therefore, the absence of recombination and similar evolutionary rates make phylogeny-based conclusions reliable. Phylogenetic reconstruction strongly supported the hypothesis that host switching occurred in the history of lyssaviruses. Indeed, lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies, very likely following spillovers from bats. Using dated isolates, the average rate of evolution was estimated to be roughly 4.3 x 10(-4) d(S)/site/year. Consequently, the emergence of carnivoran rabies from chiropteran lyssaviruses was determined to have occurred 888 to 1,459 years ago. Glycoprotein segments accumulating more d(N) than d(S) were distinctly detected in carnivoran and chiropteran lyssaviruses. They may have contributed to the adaptation of the virus to the two distinct mammal orders. In carnivoran lyssaviruses they overlapped the main antigenic sites, II and III, whereas in chiropteran lyssaviruses they were located in regions of unknown functions.     

Isolation of Irkut Virus from a Murina leucogaster Bat in China

Background and objectives

Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined.

Results

We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV.

Conclusions

We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health.     

Identification of a Role for Nucleolin in Rabies Virus Infection

Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.     

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.