Integrating the landscape epidemiology and genetics of RNA viruses: rabies in domestic dogs as a model

Landscape epidemiology and landscape genetics combine advances in molecular techniques, spatial analyses and epidemiological models to generate a more real-world understanding of infectious disease dynamics and provide powerful new tools for the study of RNA viruses. Using dog rabies as a model we have identified how key questions regarding viral spread and persistence can be addressed using a combination of these techniques. In contrast to wildlife rabies, investigations into the landscape epidemiology of domestic dog rabies requires more detailed assessment of the role of humans in disease spread, including the incorporation of anthropogenic landscape features, human movements and socio-cultural factors into spatial models. In particular, identifying and quantifying the influence of anthropogenic features on pathogen spread and measuring the permeability of dispersal barriers are important considerations for planning control strategies, and may differ according to cultural, social and geographical variation across countries or continents. Challenges for dog rabies research include the development of metapopulation models and transmission networks using genetic information to uncover potential source/sink dynamics and identify the main routes of viral dissemination. Information generated from a landscape genetics approach will facilitate spatially strategic control programmes that accommodate for heterogeneities in the landscape and therefore utilise resources in the most cost-effective way. This can include the efficient placement of vaccine barriers, surveillance points and adaptive management for large-scale control programmes

Rabies virus cross-reactive murine T cell clones: analysis of helper and delayed-type hypersensitivity function.

Three T cell clones derived from rabies virus-immunized BALB/c mice were analysed for specificity and function. The clones proved to be broadly cross-reactive by responding to different rabies virus isolates (PM, ERA, CVS, HEP) and other representatives of the genus Lyssavirus, like the Duvenhage-6 (DUV6) and Mokola (MOK) viruses. The clones detected three different epitopes: an epitope expressed on the matrix protein (M) shared by PM, HEP, MOK and DUV6 viruses (clone AA8), an epitope expressed on the M-protein shared by PM, ERA, CVS, HEP and MOK viruses (clone 35A) and finally an epitope expressed on the glycoprotein (G-protein) shared by PM, ERA, CVS, HEP and MOK viruses (clone BG2). Antigen recognition of all clones proved to be MHC-restricted and they all displayed the CD4+ CD8- phenotype. Intravenous inoculation of the T cells in syngeneic mice, which had been injected intracutaneously in the ear with HEP virus, resulted in a localized DTH reaction characteristic for TH1 cells. In vitro, the clones were able to provide help to rabies virus-primed B cells, resulting in the production of virus-specific antibodies directed against all the four structural proteins of rabies virus. Further analysis of this antibody response revealed that part of it was directed against antigenic determinants of the G-protein which induce virus neutralizing antibody

The Specificity of Rabies Virus RNA Encapsidation by Nucleoprotein

AbstractRabies virus nucleoprotein (N) encapsidates negative-strand genomic RNAin vivo,and this RNA–N complex, together with the nominal viral phosphoprotein (P) and RNA polymerase (L), forms the active cytoplasmic ribonucleoprotein (RNP) complex in virus-infected cells and the RNP core in virus particles. The RNP complex is capable of initiating viral RNA transcription and replicationin vivoandin vitro.To obtain insight into the events leading to the formation of the RNA–N complex, we have investigated the interaction between rabies virus N and the positive-strand leader RNA transcript. Binding studies revealed that recombinant N binds preferentially to rabies virus leader RNA and that N binding to leader RNA was 5 to 10 times stronger than to nonleader RNA. Encapsidation of leader RNA by N could be competetively inhibited by unlabeled leader RNA but not by nonleader RNA. Furthermore, N protein encapsidation of nonleader RNA but not the leader RNA was inhibited when P was simultaneously added into the encapsidation reaction, indicating that P helps confer the specificity of leader RNA encapsidation by N. The initiation signal for leader RNA encapsidation by N has been mapped to nucleotides 20–30 of the RNA sequence which is A rich. Studies with N-deletion mutants indicate that the intact N is required to encapsidate RNA, since deletion of amino acid residues from either the N- or the C-terminus of N abolishes the ability of N to encapsidate leader RNA

Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

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

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

AbstractRabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus

An immune stimulating complex (iscom) subunit rabies vaccine protects dogs and mice against street rabies challenge.

Dogs and mice were immunized with either a rabies glycoprotein subunit vaccine incorporated into an immune stimulating complex (ISCOM) or a commercial human diploid cell vaccine (HDCV) prepared from a Pitman Moore (PM) rabies vaccine strain. Pre-exposure vaccination of mice with two intraperitoneal (i.p.) doses of 360 ng ISCOM or 0.5 ml HDCV protected 95% (38/40) and 90% (36/40) of mice, respectively, against a lethal intracerebral (i.c.) dose with challenge virus strain (CVS). One 360 ng i.p. dose of ISCOM protected 87.5% (35/40) of mice against i.c. challenge with CVS. Three groups of five dogs were vaccinated intramuscularly (i.m.) with 730 ng of rabies ISCOM prepared from either the PM or the CVS rabies strains, and they resisted lethal street rabies challenge. Postexposure treatment of mice with three or four 120 ng i.m. doses of ISCOM protected 90% (27/30) and 94% (45/48), respectively, of mice inoculated in the footpad with street rabies virus, but three doses of HDCV conferred no protection. When four doses of HDCV were administ

Emerging Pattern of Rabies Deaths and Increased Viral Infectivity

Most human rabies deaths in the United States can be attributed to unrecognized exposures to rabies viruses associated with bats, particularly those associated with two infrequently encountered bat species (Lasionycteris noctivagans and Pipistrellus subflavus). These human rabies cases tend to cluster in the southeastern and northwestern United States. In these regions, most rabies deaths associated with bats in nonhuman terrestrial mammals are also associated with virus variants specific to these two bat species rather than more common bat species; outside of these regions, more common bat rabies viruses contribute to most transmissions. The preponderance of rabies deaths connected with the two uncommon L. noctivagans and P. subflavus bat rabies viruses is best explained by their evolution of increased viral infectivity