European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (104 f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (103 f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G–matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.
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.
According to the World Health Organization rabies is considered both a neglected zoonotic and a tropical disease. The causative agents are lyssaviruses which have their primary reservoir in bats. Although bat rabies is notifiable in Germany, the number of submitted bats during routine surveillance is rarely representative of the natural bat population. Therefore, the aim of this study was to include dead bats from various sources for enhanced bat rabies surveillance. The results show that a considerable number of additional bat rabies cases can be detected, thus improving the knowledge on the frequency, geographical distribution and reservoir-association of bat lyssavirus infections in Germany. The overall proportion of positives was lower than during routine surveillance in Germany. While the majority of cases were found in the Serotine bat and characterized as European bat lyssavirus type 1 (EBLV-1), three of the four EBLV-2 infections detected in Germany were found in Myotis daubentonii during this study.
Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory.
The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result.
Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing.
In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.
Rabies, a fatal disease of animals and humans has been endemic in Grenada, West Indies, since the early 1900s. The small Indian mongoose, an introduced animal, is the most likely rabies reservoir, with spillover into domestic animals and humans. To control rabies, large numbers of mongooses were killed in the 1960s/1970s, but this effort did not alter long-term rabies dynamics. Vaccination of dogs, cats and livestock is efficient in protecting these animals, yet is not regularly undertaken. Post-exposure prophylaxis (PEP) in humans is routinely done and no human has died of rabies in Grenada since 1970. However, the threat of rabies and potential to adversely affect the tourism industry, are a burden on the Grenadian government and public. This study has re-evaluated the role of the mongoose in the maintenance of rabies in Grenada, and for the first time, the rabies virus circulating in Grenada has been described. Grenada offers optimal conditions for an oral rabies vaccination (ORV) program, being an island with strict live animal import controls, and a single wildlife rabies reservoir. Although further work is needed before an ORV campaign could be implemented, elimination of rabies from Grenada seems a realistic goal.
The delivery of therapeutic genes to the central nervous system (CNS) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the CNS. Important factors determining CNS targeting include tropism of the viral vectors and retrograde transport of the vector particles. Retrograde transport of equine anemia virus (EIAV)-based lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus RabERA strain from peripheral muscle to spinal motor neurons (MNs) was previously reported. Despite therapeutic effects achieved in mouse models of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the efficiency of this approach needs to be improved for clinical translation. To date there has not been a quantitative assessment of pseudotyped HIV-1-based lentiviral vectors to transduce MNs. Here, we describe quantitative tests to analyze the retrograde transport capacity of HIV-1 vectors pseudotyped with the G glycoprotein derived from Rabies and Rabies-related viruses (Lyssaviruses).
With a view toward optimizing the retrograde transport properties of HIV-1-based lentiviral vectors, we compared the glycoproteins from different enveloped viruses belonging to the Rhabdoviridae family, genus Lyssavirus, and evaluated their ability to transduce specific cell populations and promote retrograde axonal transport. We first tested the transduction performance of these pseudotypes in vitro in SH-SY5Y neuroblastoma cells, NSC-34 neuroblastoma-spinal cord hybrid cells, and primary mixed spinal cord and pure astrocyte cultures. We then analyzed the uptake and retrograde transport of these pseudotyped vectors in vitro, using Campenot chambers. Finally, intraneural injections were performed to evaluate the in vivo retrograde axonal transport of these pseudotypes.
Both the in vitro and in vivo studies demonstrated that lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus PV strain possessed the best performance and neuronal tropism among the vectors tested.
Our results indicate that HIV-1-based lentiviral vectors pseudotyped with the Rabies PV glycoprotein might provide important vehicles for CNS targeting by peripheral injection in the treatment of motor neuron diseases (MND), pain, and neuropathy.
The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.
rabies virus; eGFP; virus-neutralizing antibody (VNA); FAVN
In 1985, a bat researcher in Finland died of rabies encephalitis caused by European bat lyssavirus type 2 (EBLV-2), but an epidemiological study in 1986 did not reveal EBLV-infected bats. In 2009, an EBLV-2-positive Daubenton’s bat was detected. The EBLV-2 isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related. In order to assess the prevalence of EBLVs in Finnish bat populations and to gain a better understanding of the public health risk that EBLV-infected bats pose, a targeted active surveillance project was initiated.
Altogether, 1156 bats of seven species were examined for lyssaviruses in Finland during a 28–year period (1985–2012), 898 in active surveillance and 258 in passive surveillance, with only one positive finding of EBLV-2 in a Daubenton’s bat in 2009. In 2010–2011, saliva samples from 774 bats of seven species were analyzed for EBLV viral RNA, and sera from 423 bats were analyzed for the presence of bat lyssavirus antibodies. Antibodies were detected in Daubenton’s bats in samples collected from two locations in 2010 and from one location in 2011. All seropositive locations are in close proximity to the place where the EBLV-2 positive Daubenton’s bat was found in 2009. In active surveillance, no EBLV viral RNA was detected.
These data suggest that EBLV-2 may circulate in Finland, even though the seroprevalence is low. Our results indicate that passive surveillance of dead or sick bats is a relevant means examine the occurrence of lyssavirus infection, but the number of bats submitted for laboratory analysis should be higher in order to obtain reliable information on the lyssavirus situation in the country.
EBLV; Lyssavirus; Rabies; Seroprevalence
Among other Lyssaviruses, Daubenton’s and pond-bat-related European bat lyssavirus type 2 (EBLV-2) can cause human rabies. To investigate the diversity and evolutionary trends of EBLV-2, complete genome sequences of two Finnish isolates were analysed. One originated from a human case in 1985, and the other originated from a bat in 2009. The overall nucleotide and deduced amino acid sequence identity of the two Finnish isolates were high, as well as the similarity to fully sequenced EBLV-2 strains originating from the UK and the Netherlands. In phylogenetic analysis, the EBLV-2 strains formed a monophyletic group that was separate from other bat-type lyssaviruses, with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV). EBLV-2 showed limited diversity compared to RABV and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years, and EBLV-2 diverged from KHUV about 8000 years ago. In a phylogenetic tree of partial N gene sequences, the Finnish EBLV-2 strains clustered with strains from Central Europe, supporting the hypothesis that EBLV-2 circulating in Finland might have a Central European origin. The Finnish EBLV-2 strains and a Swiss strain were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the two Finnish strains appear to have evolved from a common ancestor during the last 200 years.
A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.
Since 1954, there have been in excess of 800 cases of rabies as a result of European Bat Lyssaviruses types 1 and 2 (EBLV-1, EBLV-2) infection, mainly in Serotine and Myotis bats respectively. These viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in Denmark, a stone marten in Germany and a cat in France. The purpose of this study was to investigate the susceptibility of foxes to EBLVs using silver foxes (Vulpes vulpes) as a model.
Our experimental studies have shown that the susceptibility of foxes to EBLVs is low by the intramuscular (IM) route, however, animals were sensitive to intracranial (IC) inoculation. Mortality was 100% for both EBLV-1 (~4.5 logs) and EBLV-2 (~3.0 logs) delivered by the IC route. Virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody (VNA) was limited (log(ED50) = 0.24–2.23 and 0.95–2.39 respectively for specific EBLV-1 and EBLV-2). Foxes were also susceptible, at a low level, to peripheral (IM) infection (~3.0 logs) with EBLV-1 but not EBLV-2. Three out of 21 (14.3%) foxes developed clinical signs between 14 and 24 days post-EBLV-1 infection. None of the animals given EBLV-2 developed clinical disease.
These data suggest that the chance of a EBLV spill-over from bat to fox is low, but with a greater probability for EBLV-1 than for EBLV-2 and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection.
As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans.
Human mortality from endemic canine rabies is estimated to be 55,000 deaths per year in Africa and Asia, yet rabies remains a neglected disease throughout most of these countries. More than 99% of human rabies cases are caused by infections resulting from a dog-bite injury. In the vast majority of human exposures to rabies, patients require post-exposure prophylaxis (PEP), which includes both passive (rabies immunoglobulin, RIG) and active immunization (rabies vaccine). The number of victims requiring PEP has increased exponentially in recent years, and human and equine RIG (HRIG and ERIG) were not sufficiently available in countries where canine rabies is endemic. Rabies virus-neutralizing monoclonal antibodies (MAbs) of mouse (Mo) origin have been identified as promising alternatives to HRIG and ERIG. We have developed and assessed both in vitro and in vivo unique mouse monoclonal antibody (MoMAb) cocktails, which are highly efficacious. Three novel combinations were shown to have an equal or superior efficacy to HRIG and therefore could be considered a potentially less expensive alternative for passive prophylactic use to prevent the development of rabies in humans, particularly where needed most in developing countries.
Bats have been proposed as major reservoirs for diverse emerging infectious viral diseases, with rabies being the best known in Europe. However, studies exploring the ecological interaction between lyssaviruses and their natural hosts are scarce. This study completes our active surveillance work on Spanish bat colonies that began in 1992. Herein, we analyzed ecological factors that might affect the infection dynamics observed in those colonies. Between 2001 and 2011, we collected and tested 2,393 blood samples and 45 dead bats from 25 localities and 20 bat species. The results for dead confirmed the presence of EBLV-1 RNA in six species analyzed (for the first time in Myotis capaccinii). Samples positive for European bat lyssavirus-1 (EBLV-1)–neutralizing antibodies were detected in 68% of the localities sampled and in 13 bat species, seven of which were found for the first time (even in Myotis daubentonii, a species to date always linked to EBLV-2). EBLV-1 seroprevalence (20.7%) ranged between 11.1 and 40.2% among bat species and seasonal variation was observed, with significantly higher antibody prevalence in summer (July). EBLV-1 seroprevalence was significantly associated with colony size and species richness. Higher seroprevalence percentages were found in large multispecific colonies, suggesting that intra- and interspecific contacts are major risk factors for EBLV-1 transmission in bat colonies. Although bat-roosting behavior strongly determines EBLV-1 variability, we also found some evidence that bat phylogeny might be involved in bat-species seroprevalence. The results of this study highlight the importance of life history and roost ecology in understanding EBLV-1–prevalence patterns in bat colonies and also provide useful information for public health officials.
Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre.
Influenza; pseudotype virus; viral screening; neutralizing antibody; H3N8; SRH; TMPRSS2
Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.
In this study we provide results of the first active and extensive surveillance effort for rabies virus (RABV) circulation among bats in Guatemala. The survey included multiple geographic areas and multiple species of bats, to assess the broader public and veterinary health risks associated with rabies in bats in Guatemala. RABV was isolated from vampire bats (Desmodus rotundus) collected in two different locations in Guatemala. Sequencing of the isolates revealed a closer relationship to Mexican and Central American vampire bat isolates than to South American isolates. The detection of RABV neutralizing antibodies in 11 species, including insectivorous, frugivorous, and sanguivorous bats, demonstrates viral circulation in both hematophagous and non-hematophagous bat species in Guatemala. The presence of bat RABV in rural communities requires new strategies for public health education regarding contact with bats, improved laboratory-based surveillance of animals associated with human exposures, and novel techniques for modern rabies prevention and control. Additionally, healthcare practitioners should emphasize the collection of a detailed medical history, including questions regarding bat exposure, for patients presenting with clinical syndromes compatible with rabies or any clinically diagnosed progressive encephalitis.
The monomer of influenza haemagglutinin is synthesized as a single polypeptide precursor that during maturation is cleaved by proteases into two active subunits. Other studies have demonstrated that the human Transmembrane Protease Serine 2 (TMPRSS2) can cleave the HA of human seasonal influenza viruses. Consequently, we have investigated the use of human Transmembrane Protease Serine 2 to produce high titre influenza haemmagglutinin (HA) lentiviral pseudotypes from Group 2 influenza viruses. Such pseudotypes represent powerful and safe tools to study viral entry and immune responses. Influenza pseudotype particles are obtained by co-transfecting human embryonic kidney HEK293T/17 cells using plasmids coding for the influenza HA, HIV gag-pol and a lentiviral vector incorporating firefly luciferase. However, in order to produce Group 2 pseudotypes, it was necessary to co-transfect a plasmid expressing the TMPRSS2 endoprotease, to achieve the necessary HA cleavage for infective particle generation. These lentiviral pseudotypes were shown to transduce HEK293T/17 cells with high efficiency. This demonstrates that TMPRSS2 is necessary for the functional activation, in vitro, of both the HA of human seasonal influenza and other Group 2 HA influenza strains. Additionally, we show that the Group 2 influenza pseudotype particles can be used as surrogate antigens in neutralization assays and are efficiently neutralized by corresponding influenza virus reference sera. These data demonstrate that the viral pseudotype system is a powerful method for serological surveillance of a wide range of influenza viruses.
Haemagglutinin; protease cleavage; antibody response; pseudotype serology
The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipavirus in the family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms, recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type (wt) HeV glycoprotein (G) gene. The RABV vector expressing the codon-optimized HeV G showed a 2- to 3-fold increase in incorporation compared to the RABV vector expressing wt HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vectors. To test the immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10 μg chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic.
IMPORTANCE Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study, we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV), expressing wild-type or codon-optimized HeV glycoprotein, with the goal of developing a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving post-exposure treatment. A person, disproportionately a child, dies of rabies every 20 minutes and the cost of rabies prevention exceeds $1 billion US dollars per year. The development of a single-dose human rabies vaccine would greatly reduce the burden of rabies globally by lowering the cost associated with rabies vaccination and saving lives. Understanding how B cells develop to produce protective virus neutralizing antibodies would greatly help to achieve the goal of developing a single-dose vaccine. In this report, we show that IL-21 is critical for the induction of primary vaccine-induced anti-RABV G antibody titers and that the effects of IL-21 are highly dependent on the dose of vaccine administered. In our model of rabies immunogenicity and protection, the lack of IL-21 receptor influenced the detection of B cells in germinal centers in lymph nodes or of plasma cells in bone marrow after immunization with low or high doses of vaccine, respectively. Overall, these preliminary results indicate that IL-21 has the potential to influence B cell development and functions in the context of rabies vaccine-induced immunity and protection.
The inflexibility of existing serological techniques for detection of rabies in surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r = 0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections.
Rabies virus; Lyssavirus; Africa; Pseudotype
Rabies virus (RABV) is enzootic throughout Africa, with the domestic dog (Canis familiaris) being the principal vector. Dog rabies is estimated to cause 24,000 human deaths per year in Africa, however, this estimate is still considered to be conservative. Two sub-Saharan African RABV lineages have been detected in West Africa. Lineage 2 is present throughout West Africa, whereas Africa 1a dominates in northern and eastern Africa, but has been detected in Nigeria and Gabon, and Africa 1b was previously absent from West Africa. We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected over an eighteen-month period (2007–2009). Phylogenetic analysis of the sequences obtained confirmed all viruses to be RABV, belonging to lineages previously detected in sub-Saharan Africa. However, unlike earlier reported studies that suggested a single lineage (Africa 2) circulates in West Africa, we identified viruses belonging to the Africa 2 lineage and both Africa 1 (a and b) sub-lineages. Phylogeographic Bayesian Markov chain Monte Carlo analysis of a 405 bp fragment of the RABV nucleoprotein gene from the 76 new sequences derived from Ghanaian animals suggest that within the Africa 2 lineage three clades co-circulate with their origins in other West African countries. Africa 1a is probably a western extension of a clade circulating in central Africa and the Africa 1b virus a probable recent introduction from eastern Africa. We also developed and tested a novel reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of RABV in African laboratories. This RT-LAMP was shown to detect both Africa 1 and 2 viruses, including its adaptation to a lateral flow device format for product visualization. These data suggest that RABV epidemiology is more complex than previously thought in West Africa and that there have been repeated introductions of RABV into Ghana. This analysis highlights the potential problems of individual developing nations implementing rabies control programmes in the absence of a regional programme.
Rabies virus (RABV) is widespread throughout Africa, with the domestic dog being the principal vector. Dog rabies is estimated to cause 24,000 human deaths per year in Africa, however, this estimate is still considered to be conservative. Two sub-Saharan African RABV lineages (Africa 1 and 2) are thought to circulate in western and central Africa. We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected from 2007 to 2009. In addition we developed and tested a novel molecular diagnostic assay for the detection of RABV, which offers an alternative RABV diagnostic tool for African laboratories. Our analysis of the genetic sequences obtained confirmed all viruses to be RABV, however, unlike previous studies we detected two sub-Saharan African RABV viruses (Africa 1 and 2) in this cohort, which included a single virus previously undetected in West Africa. We suggest that there has been repeated introduction of new RABVs into Ghana over a prolonged period from other West African countries and more recently from eastern Africa. These observations further highlight the problems of individual developing nations implementing rabies control programmes at a local, rather than regional level.
In Japan, the import
quarantine regulation against rabies has required from 2005 that dogs and cats should be
inoculated with the rabies vaccine and that the neutralizing antibody titer should be
confirmed to be at least 0.5 international units (IU)/ml. The fluorescent
antibody virus neutralization (FAVN) test is used as an international standard method for
serological testing for rabies. To achieve proper immunization of dogs and cats at the
time of import and export, changes in the neutralizing antibody titer after inoculation of
the rabies vaccine should be understood in detail. However, few reports have provided this
information. In this study, we aimed to determine evaluated, such changes by using sera
from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples
using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled
the necessary titer of neutralizing antibodies to be maintained in the long term. However,
inappropriate timing of blood sampling after vaccination could result in insufficient
detected levels of neutralizing antibodies.
canine; feline; rabies; vaccine
Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.
Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.
Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.
Currently available rabies post‐exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad‐spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non‐RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post‐vaccination antibody response.
human monoclonal antibody; lyssaviruses; post‐exposure prophylaxis; rabies; Immunology; Microbiology, Virology & Host Pathogen Interaction
United Kingdom, Ireland, Malta and Sweden maintain their national provisions for a transitional period regarding rules concerning rabies vaccination and individual serological test for rabies neutralizing antibodies. The purpose of vaccinating dogs against rabies is to establish pre-exposure immunity and protect individual animals from contracting rabies.
The aim of the study was to investigate factors associated with reaching the internationally accepted threshold antibody titre of 0.5 IU/mL after rabies vaccination of dogs.
The study was a prospective single cohort study including 6,789 samples from Swedish dogs vaccinated with commercially available vaccines in Sweden, and the dog's antibody responses were determined by the OIE approved FAVN test. Information on potential risk factors; breed, age, gender, date of vaccination, vaccine label and the number of vaccinations, was collected for each dog. Associations between the dependent variable, serological response ≥ 0.5 IU/mL or < 0.5 IU/mL and each of the potential risk factors were investigated using logistic regression analysis.
Of 6,789 vaccinated dogs, 6,241 (91.9%) had an approved test result of ≥ 0.5 IU/mL. The results of the multivariable logistic regression analysis showed that vaccinating with vaccine B reduced the risk of having antibody titres of < 0.5 IU/mL by 0.2 times compared with vaccination using vaccine A. Breed size was found significant as an interaction with number of vaccinations and age at vaccination as an interaction with day of antibody testing after last vaccination. In summary, larger breeds were at higher risk of having antibody titres of < 0.5 IU/mL but if vaccinated twice this risk was reduced. Moreover, there were a increased risk for dogs < 6 months of age and > 5 years of age to have antibody titres of < 0.5 IU/mL, but this was affected by number of days from vaccination till testing.
The probability of success of rabies vaccinations of dogs depends on type of vaccine used, number of rabies vaccinations, the breed size of the dog, age at vaccination, and number of days after vaccination when the antibody titres are tested. The need for a booster vaccination regimen is recommended for larger breeds of dog.
Dendritic cells (DCs) are the most efficient antigen-presenting cells, playing a key role in the adaptive immune responses to viral infections. Our studies demonstrate that wild-type (wt) rabies virus (RABV) does not activate DCs. Adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate virus neutralizing antibodies (VNA), or protect recipients against challenge. However, adoptive transfer of DCs primed with laboratory-attenuated RABV resulted in DC activation, production of VNA, and protection against challenge. In vitro studies with recombinant RABV (laboratory-attenuated RABV expressing the glycoprotein or the phosphoprotein from wt RABV) demonstrate that DC activation is dependent on the glycoprotein and involves the IPS-1 pathway. Furthermore, binding to and entry into DCs by wt RABV is severely blocked, and the copy number of de novo-synthesized leader RNA was two logs lower in DCs infected with the wt than in DCs treated with laboratory-attenuated RABV. However, transient transfection of DCs with synthesized leader RNA from either wt or attenuated RABV is capable of activating DCs in a dose-dependent manner. Thus, the inability of wt RABV to activate DCs correlates with its low level of the de novo-synthesized leader RNA.
IMPORTANCE Rabies remains a public health threat, with more than 55,000 fatalities each year around the world. Since DCs play a key role in the adaptive immune responses to viral infections, we investigated the ability of rabies virus (RABV) to activate DCs. It was found that the adoptive transfer of DCs primed with wt RABV did not activate DCs, stimulate VNA, or protect mice against lethal challenge. However, laboratory-attenuated RABV mediates the activation of DCs via the IPS-1 pathway and is glycoprotein dependent. We further show that wt RABV evades DC-mediated immune activation by inefficient binding/entry into DCs and as a result of a reduced level of de novo-synthesized leader RNA. These findings may have important implications in the development of efficient rabies vaccines.
Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality and quantity, which is expected to correlate with vaccine potency. In this study an antigen-capture electrochemiluminescent (ECL) assay was developed utilizing anti-RABV G monoclonal antibodies (MAb) to quantify RABV G. One MAb 2-21-14 was specific for a conformational epitope so that only immunogenic, natively-folded G was captured in the assay. A second MAb (62-80-6) that binds a linear epitope or MAb 2-21-14 was used for detection of RABV G. Vaccine efficacy was also assessed in vivo using pre-exposure vaccination of mice. Purified native RABV G induced a RABV neutralizing antibody (rVNA) response with a geometric mean titer of 4.2 IU/ml and protected 100% of immunized mice against RABV challenge, while an experimental vaccine with a lower quality and quantity of G induced a rVNA titer <0.05 IU/ml and protected <50% of immunized mice. These preliminary results support the hypothesis that in vivo immunogenicity may be predicted from the in vitro measurement of RABV G using an ECL assay. Based upon these results, the ECL assay may have utility in replacement of the NIH test.
Rabies virus; Vaccine; Glycoprotein; Electrochemiluminescent assay; Antigenicity; Immunogenicity