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1.  Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009 
PLoS Pathogens  2012;8(6):e1002786.
In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001–2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.
Author Summary
Host shifts of the rabies virus (RABV) from bats to carnivores are important for our understanding of viral evolution and emergence, and have significant public health implications, particularly for the areas where “terrestrial” rabies has been eliminated. In this study we addressed several rabies outbreaks in carnivores that occurred in the Flagstaff area of Arizona during 2001–2009, and caused by the RABV variant associated with big brown bats (Eptesicus fuscus). Based on phylogenetic analysis we demonstrated that each outbreak resulted from a separate introduction of bat RABV into populations of carnivores. No post-shift changes in viral genomes were detected under the positive selection analysis. Trying to answer the question why certain bat RABV variants are capable for host shifts to carnivores and other variants are not, we developed a convergent evolution analysis, and implemented it for multiple RABV lineages circulating worldwide. This analysis identified several amino acids in RABV proteins which may facilitate host shifts from bats to carnivores. Precise roles of these amino acids require additional investigations, using reverse genetics and animal experimentation. In general, our approach and the results obtained can be used for prediction of host shifts and emergence of other zoonotic pathogens.
PMCID: PMC3380930  PMID: 22737076
2.  A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test 
BioMed Research International  2014;2014:256175.
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.
PMCID: PMC4009295  PMID: 24822188
3.  Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein 
The Journal of General Virology  2013;94(Pt 2):284-292.
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.
PMCID: PMC3709617  PMID: 23100360
4.  A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories 
PLoS ONE  2013;8(3):e58372.
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.
PMCID: PMC3592807  PMID: 23520505
5.  Rabies Virus Infection Induces Type I Interferon Production in an IPS-1 Dependent Manner While Dendritic Cell Activation Relies on IFNAR Signaling 
PLoS Pathogens  2010;6(7):e1001016.
As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5−/− and RIG-I−/− mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I−/− cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1−/− mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.
Author Summary
Rabies virus (RABV) is a neurotropic RNA virus responsible for the deaths of the at least 40,000 to 70,000 individuals globally each year. However, the innate immune response induced by both wildtype and vaccine strains of RABV is not well understood. In this study, we assessed the pattern recognition receptors involved in the host immune response to RABV in bone marrow derived dendritic cells (DC). Our studies revealed that Toll like receptor (TLR) signaling is not required to induce innate responses to RABV. On the other hand, we see that IPS-1, the adaptor protein for RIG-I like receptor (RLR) signaling, is essential for induction of innate immune responses. Furthermore, we found that RIG-I and Mda-5, both RLRs, are able to induce DC activation and type I interferon production. This finding is significant as we can target unused pattern recognition receptors with recombinant RABV vaccine strains to elicit a varied, and potentially protective, immune response. Lastly, we show that IPS-1 plays an important role in mediating the pathogenicity of RABV and preventing RABV associated paralysis. Overall, this study illustrates that RLRs are essential for recognition of RABV infection and that the subsequent host cell signaling is required to prevent disease.
PMCID: PMC2908622  PMID: 20661430
6.  Lyssavirus Detection and Typing Using Pyrosequencing▿#‖ 
Journal of Clinical Microbiology  2011;49(5):1932-1938.
Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.
PMCID: PMC3122702  PMID: 21389152
7.  Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection 
Journal of Virology  2013;87(13):7608-7621.
Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.
PMCID: PMC3700271  PMID: 23637400
8.  Bat Rabies in Guatemala 
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.
Author Summary
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.
PMCID: PMC4117473  PMID: 25080103
9.  Adeno-Associated Viruses Serotype 2-Mediated RNA Interference Efficiently Inhibits Rabies Virus Replication In Vitro and In Vivo 
To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.
PMCID: PMC3942934  PMID: 23774028
AAV2 vectors; inhibit; N gene; RABV; siRNA
10.  Rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms 
Veterinary microbiology  2012;162(2-4):601-613.
Our previous studies have suggested that street and fixed rabies viruses (RABV) induce diseases in the mouse model via different mechanisms. In the present study, attempts were made to determine if it is the glycoprotein (G) that is responsible for the observed differences in the pathogenic mechanisms. To this end, an infectious clone from fixed virus B2c was established and used as a backbone for exchange of the G from street viruses. The rate of viral replication, expression of viral proteins, and the induction of innate immune responses were compared in cells or in mice infected with each of the viruses. Furthermore, the infiltration of inflammatory cells into the CNS and the enhancement of blood-brain-barrier (BBB) permeability were also compared. It was found that fixed viruses induced stronger innate immune responses (expression of chemokines, infiltration of inflammatory cells, and enhancement of BBB permeability) than street RABV or recombinant viruses expressing the G from street RABVs. Fixed viruses induce disease via an immune-mediated pathogenic mechanism while street viruses or recombinant viruses expressing the G from street RABVs induce diseases via a mechanism other than immune-mediated pathogenesis. Therefore, RABV G is an important determinant for the induction of innate immune responses and consequently the pathogenic mechanisms.
PMCID: PMC3568536  PMID: 23265241
Rabies virus; innate immune response; inflammation; blood brain barrier
11.  The origin and phylogeography of dog rabies virus 
The Journal of General Virology  2008;89(Pt 11):2673-2681.
Rabies is a progressively fatal and incurable viral encephalitis caused by a lyssavirus infection. Almost all of the 55 000 annual rabies deaths in humans result from infection with dog rabies viruses (RABV). Despite the importance of rabies for human health, little is known about the spread of RABV in dog populations, and patterns of biodiversity have only been studied in limited geographical space. To address these questions on a global scale, we sequenced 62 new isolates and performed an extensive comparative analysis of RABV gene sequence data, representing 192 isolates sampled from 55 countries. From this, we identified six clades of RABV in non-flying mammals, each of which has a distinct geographical distribution, most likely reflecting major physical barriers to gene flow. Indeed, a detailed analysis of phylogeographic structure revealed only limited viral movement among geographical localities. Using Bayesian coalescent methods we also reveal that the sampled lineages of canid RABV derive from a common ancestor that originated within the past 1500 years. Additionally, we found no evidence for either positive selection or widespread population bottlenecks during the global expansion of canid RABV. Overall, our study reveals that the stochastic processes of genetic drift and population subdivision are the most important factors shaping the global phylogeography of canid RABV.
PMCID: PMC3326349  PMID: 18931062
12.  Passive carriage of rabies virus by dendritic cells 
SpringerPlus  2013;2:419.
The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections.
PMCID: PMC3765594  PMID: 24024103
Rabies virus; Dendritic cells; Immune invasion
13.  Interferon Response and Viral Evasion by Members of the Family Rhabdoviridae 
Viruses  2009;1(3):832-851.
Like many animal viruses, those of the Rhabdoviridae family, are able to antagonize the type I interferon response and cause disease in mammalian hosts. Though these negative-stranded RNA viruses are very simple and code for as few as five proteins, they have been seen to completely abrogate the type I interferon response early in infection. In this review, we will discuss the viral organization and type I interferon evasion of rhabdoviruses, focusing on vesicular stomatitis virus (VSV) and rabies virus (RABV). Despite their structural similarities, VSV and RABV have completely different mechanisms by which they avert the host immune response. VSV relies on the matrix protein to interfere with host gene transcription and nuclear export of anti-viral mRNAs. Alternatively, RABV uses its phosphoprotein to interfere with IRF-3 phosphorylation and STAT1 signaling. Understanding the virus-cell interactions and viral proteins necessary to evade the immune response is important in developing effective vaccines and therapeutics for this viral family.
PMCID: PMC3185512  PMID: 21994572
rhabdovirus; interferon; rabies virus; vesicular stomatitis virus
14.  Recent Observations on Australian Bat Lyssavirus Tropism and Viral Entry 
Viruses  2014;6(2):909-926.
Australian bat lyssavirus (ABLV) is a recently emerged rhabdovirus of the genus lyssavirus considered endemic in Australian bat populations that causes a neurological disease in people indistinguishable from clinical rabies. There are two distinct variants of ABLV, one that circulates in frugivorous bats (genus Pteropus) and the other in insectivorous microbats (genus Saccolaimus). Three fatal human cases of ABLV infection have been reported, the most recent in 2013, and each manifested as acute encephalitis but with variable incubation periods. Importantly, two equine cases also arose recently in 2013, the first occurrence of ABLV in a species other than bats or humans. Similar to other rhabdoviruses, ABLV infects host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion facilitated by its single fusogenic envelope glycoprotein (G). Recent studies have revealed that proposed rabies virus (RABV) receptors are not sufficient to permit ABLV entry into host cells and that the unknown receptor is broadly conserved among mammalian species. However, despite clear tropism differences between ABLV and RABV, the two viruses appear to utilize similar endocytic entry pathways. The recent human and horse infections highlight the importance of continued Australian public health awareness of this emerging pathogen.
PMCID: PMC3939488  PMID: 24556791
rhabdovirus; Australian bat lyssavirus; rabies virus; viral entry; emerging; zoonosis; tropism; glycoprotein; endocytosis
15.  Enhancement of Blood-Brain Barrier Permeability and Reduction of Tight Junction Protein Expression Are Modulated by Chemokines/Cytokines Induced by Rabies Virus Infection 
Journal of Virology  2014;88(9):4698-4710.
Infection with laboratory-attenuated rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, which has been demonstrated to be an important factor for host survival, since it allows immune effectors to enter the central nervous system (CNS) and clear RABV. To probe the mechanism by which RABV infection enhances BBB permeability, the expression of tight junction (TJ) proteins in the CNS was investigated following intracranial inoculation with laboratory-attenuated or wild-type (wt) RABV. BBB permeability was significantly enhanced in mice infected with laboratory-attenuated, but not wt, RABV. The expression levels of TJ proteins (claudin-5, occludin, and zonula occludens-1) were decreased in mice infected with laboratory-attenuated, but not wt, RABV, suggesting that enhancement of BBB permeability is associated with the reduction of TJ protein expression in RABV infection. RABV neither infects the brain microvascular endothelial cells (BMECs) nor modulates the expression of TJ proteins in BMECs. However, brain extracts prepared from mice infected with laboratory-attenuated, but not wt, RABV reduced TJ protein expression in BMECs. It was found that brain extracts from mice infected with laboratory-attenuated RABV contained significantly higher levels of inflammatory chemokines/cytokines than those from mice infected with wt RABV. Pathway analysis indicates that gamma interferon (IFN-γ) is located in the center of the cytokine network in the RABV-infected mouse brain, and neutralization of IFN-γ reduced both the disruption of BBB permeability in vivo and the downregulation of TJ protein expression in vitro. These findings indicate that the enhancement of BBB permeability and the reduction of TJ protein expression are due not to RABV infection per se but to virus-induced inflammatory chemokines/cytokines.
IMPORTANCE Previous studies have shown that infection with only laboratory-attenuated, not wild-type, rabies virus (RABV) enhances blood-brain barrier (BBB) permeability, allowing immune effectors to enter the central nervous system (CNS) and clear RABV from the CNS. This study investigated the mechanism by which RABV infection enhances BBB permeability. It was found that RABV infection enhances BBB permeability by downregulation of tight junction (TJ) protein expression in the brain microvasculature. It was further found that it is not RABV infection per se but the chemokines/cytokines induced by RABV infection that downregulate the expression of TJ proteins and enhance BBB permeability. Blocking some of these cytokines, such as IFN-γ, ameliorated both the disruption of BBB permeability and the downregulation of TJ protein expression. These studies may provide a foundation for developing therapeutics for clinical rabies, such as medication that could be used to enhance BBB permeability.
PMCID: PMC3993813  PMID: 24522913
16.  The Roles of Chemokines in Rabies Virus Infection: Overexpression May Not Always Be Beneficial▿  
Journal of Virology  2009;83(22):11808-11818.
It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1α (MIP-1α), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1α further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.
PMCID: PMC2772667  PMID: 19740983
17.  ICAM-1-Based Rabies Virus Vaccine Shows Increased Infection and Activation of Primary Murine B Cells In Vitro and Enhanced Antibody Titers In-Vivo 
PLoS ONE  2014;9(1):e87098.
We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 103 focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV vaccine that requires only a minimum of virus.
PMCID: PMC3906113  PMID: 24489846
18.  Analysis of Adaptive Evolution in Lyssavirus Genomes Reveals Pervasive Diversifying Selection during Species Diversification 
Viruses  2014;6(11):4465-4478.
Lyssavirus is a diverse genus of viruses that infect a variety of mammalian hosts, typically causing encephalitis. The evolution of this lineage, particularly the rabies virus, has been a focus of research because of the extensive occurrence of cross-species transmission, and the distinctive geographical patterns present throughout the diversification of these viruses. Although numerous studies have examined pattern-related questions concerning Lyssavirus evolution, analyses of the evolutionary processes acting on Lyssavirus diversification are scarce. To clarify the relevance of positive natural selection in Lyssavirus diversification, we conducted a comprehensive scan for episodic diversifying selection across all lineages and codon sites of the five coding regions in lyssavirus genomes. Although the genomes of these viruses are generally conserved, the glycoprotein (G), RNA-dependent RNA polymerase (L) and polymerase (P) genes were frequently targets of adaptive evolution during the diversification of the genus. Adaptive evolution is particularly manifest in the glycoprotein gene, which was inferred to have experienced the highest density of positively selected codon sites along branches. Substitutions in the L gene were found to be associated with the early diversification of phylogroups. A comparison between the number of positively selected sites inferred along the branches of RABV population branches and Lyssavirus intespecies branches suggested that the occurrence of positive selection was similar on the five coding regions of the genome in both groups.
PMCID: PMC4246234  PMID: 25415197
rabies virus; molecular adaptation; positive selection; Chiroptera
19.  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.
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.
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.
Author Summary
The Lyssavirus genus presently comprises 12 species and two unapproved species with different antigenic characteristics. Rabies virus is detectable worldwide; Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus, and Ikoma lyssavirus circulate in Africa; European bat lyssavirus types 1 and 2, Irkut virus, West Caucasian bat virus, and Bokeloh bat lyssavirus are found in Europe; and Australian bat lyssavirus has been isolated in Australia. Only Aravan and Khujand viruses have been identified in central Asia. Bats are recognized as the most important reservoirs of lyssaviruses. In China, all lyssavirus isolates in previous reports have been rabies virus, mainly from dogs; none has been from bats. Recently, however, at least two bat-associated human rabies or rabies-like cases have been reported in northeast China. Therefore, we conducted a search for bat lyssaviruses in Jilin province, close to where the first bat-associated human rabies case was recorded. We isolated a bat lyssavirus, identified as an Irkut virus isolate with high pathogenicity in experimental mice. Our findings suggest that public warnings and medical education regarding bat bites in China should be increased, and that surveillance should be extended to provide a better understanding of Irkut virus ecology and its significance for public health.
PMCID: PMC3591329  PMID: 23505588
20.  Recent Emergence and Spread of an Arctic-Related Phylogenetic Lineage of Rabies Virus in Nepal 
Rabies is a zoonotic disease that is endemic in many parts of the developing world, especially in Africa and Asia. However its epidemiology remains largely unappreciated in much of these regions, such as in Nepal, where limited information is available about the spatiotemporal dynamics of the main etiological agent, the rabies virus (RABV). In this study, we describe for the first time the phylogenetic diversity and evolution of RABV circulating in Nepal, as well as their geographical relationships within the broader region. A total of 24 new isolates obtained from Nepal and collected from 2003 to 2011 were full-length sequenced for both the nucleoprotein and the glycoprotein genes, and analysed using neighbour-joining and maximum-likelihood phylogenetic methods with representative viruses from all over the world, including new related RABV strains from neighbouring or more distant countries (Afghanistan, Greenland, Iran, Russia and USA). Despite Nepal's limited land surface and its particular geographical position within the Indian subcontinent, our study revealed the presence of a surprising wide genetic diversity of RABV, with the co-existence of three different phylogenetic groups: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade. This observation suggests at least two independent episodes of rabies introduction from neighbouring countries. In addition, specific phylogenetic and temporal evolution analysis of viruses within the Arctic-related clade has identified a new recently emerged RABV lineage we named as the Arctic-like 3 (AL-3) sub-clade that is already widely spread in Nepal.
Author Summary
Rabies is endemic in most Asian countries and represents a serious public health issue, with an estimated 31,000 people dying each year of this disease. The majority of human cases are transmitted by domestic dogs, which act as the principal reservoir host and vector. However, molecular epidemiology and evolutionary dynamics of the main etiological agent, the rabies virus (RABV), remains largely unappreciated in some regions such as in Nepal. Based on a subset of 24 new Nepalese isolates collected from 2003 to 2011 and representative RABV strains at a global scale, phylogenetic analysis based on the complete nucleoprotein and glycoprotein genes sequences revealed the presence of a surprising wide genetic diversity of RABV circulating in this country. The presence of three different co-existing phylogenetic groups was identified: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade, namely Arctic-like (AL)-1, lineage a (AL-1a), and AL-3. Among these clusters, the AL-3 sub-clade appears as the major Nepalese phylogroup which emerged relatively recently in this country, within the last 30 years. These data has raised some concerns about the exchange of RABV between different countries, and provided key elements for implementation of effective control measures of rabies in Nepal.
PMCID: PMC3836727  PMID: 24278494
21.  Evolutionary History of Rabies in Ghana 
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.
Author Summary
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.
PMCID: PMC3071360  PMID: 21483707
22.  Antibody Quality and Protection from Lethal Ebola Virus Challenge in Nonhuman Primates Immunized with Rabies Virus Based Bivalent Vaccine 
PLoS Pathogens  2013;9(5):e1003389.
We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.
Author Summary
Ebola virus (EBOV) has been associated with outbreaks in human and nonhuman primate populations since 1976. With a fatality rate approaching 90%, EBOV is one of the most lethal infectious diseases in humans. The increased frequency of EBOV outbreaks along with its potential to be used as a bioterrorism agent has dramatically strengthened filovirus vaccine research and development. While there are currently no approved vaccines or post exposure treatments available for human use, several vaccine candidates have shown to protect nonhuman primates from lethal EBOV challenge. Our primary focus is to develop vaccine candidates to protect humans and endangered wildlife species at risk of infection in Africa. Here, we evaluated the efficacy and immunogenicity of our dual vaccines against EBOV and rabies virus (RABV) in rhesus macaques. Our live replication-competent vaccine provided 100% protection following EBOV challenge while the replication-deficient and inactivated candidates provided 50% protection. Interestingly, protection is dependent on the quality of the antibodies rather than the quantity. All three RABV-based EBOV vaccines did induce antibody levels necessary for protection from RABV infection. These results encourage the further development of these novel dual vaccines directed against two of the most lethal viral diseases.
PMCID: PMC3667758  PMID: 23737747
23.  Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison 
The Journal of General Virology  2008;89(Pt 9):2204-2213.
Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag–pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40 % of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100 % concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or β-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 μl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies.
PMCID: PMC2886951  PMID: 18753230
24.  Temporal Dynamics of European Bat Lyssavirus Type 1 and Survival of Myotis myotis Bats in Natural Colonies 
PLoS ONE  2007;2(6):e566.
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 (R0 = 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.
PMCID: PMC1892799  PMID: 17593965
25.  Early Diagnosis of Irkut Virus Infection Using Magnetic Bead-Based Serum Peptide Profiling by MALDI-TOF MS in a Mouse Model 
Early diagnosis is important for the prompt post-exposure prophylaxis of lyssavirus infections. To diagnose Irkut virus (IRKV) infection during incubation in mice, a novel method using magnetic bead-based serum peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been established. For this test, serum peptides were concentrated by adsorption to and elution from the magnetic bead-based weak cation ion exchanger. Mass spectrograms obtained by MALDI-TOF MS were analyzed using ClinProTools bioinformatics software. Construction of the diagnostic model was performed using serum samples from mice infected with IRKV and rabies virus (RABV) BD06, Flury-LEP, and SRV9 (as controls). The method accurately diagnosed sera 2, 4 and 8 days after IRKV and RABV infections. The sensitivity, specificity, and total accuracy of diagnosis were 86.7%, 95.2%, and 92.9%, respectively. However, IRKV could not be differentiated from RABV 1 day after infection. The results of the present study indicate that serum peptide profiling by MALDI-TOF MS is a promising technique for the early clinical diagnosis of lyssavirus infections and needs to be further tested in humans and farm animals.
PMCID: PMC4013557  PMID: 24670473
lyssavirus; Irkut virus; rabies; early diagnosis; serum peptide profiling

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