Abortive vampire bat rabies infections in Peruvian peridomestic livestock

Rabies virus infections normally cause universally lethal encephalitis across mammals. However, ‘abortive infections’ which are resolved prior to the onset of lethal disease have been described in bats and a variety of non-reservoir species. Here, we surveyed rabies virus neutralizing antibody titers in 332 unvaccinated livestock of 5 species from a vampire bat rabies endemic region of southern Peru where livestock are the main food source for bats. We detected rabies virus neutralizing antibody titers in 11, 5 and 3.6% of cows, goats and sheep respectively and seropositive animals did not die from rabies within two years after sampling. Seroprevalence was correlated with the number of local livestock rabies mortalities reported one year prior but also one year after sample collection. This suggests that serological status of livestock can indicate the past and future levels of rabies risk to non-reservoir hosts. To our knowledge, this is the first report of anti-rabies antibodies among goats and sheep, suggesting widespread abortive infections among livestock in vampire bat rabies endemic areas. Future research should resolve the within-host biology underlying clearance of rabies infections. Cost-effectiveness analyses are also needed to evaluate whether serological monitoring of livestock can be a viable complement to current monitoring of vampire bat rabies risk based on animal mortalities alone

"Natively Unfolded" VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

Polyprotein processing is a major strategy used by many plant and animal viruses to maximize the number of protein products obtainable from a single open reading frame. In Sesbania mosaic virus, open reading frame-2 codes for a polyprotein that is cleaved into different functional proteins in cis by the N-terminal serine protease domain. The soluble protease domain lacking 70-amino-acid residues from the N terminus ($\triangle$N70Pro, where Pro is protease) was not active in trans. Interestingly, the protease domain exhibited trans-catalytic activity when VPg (viral protein genome-linked) was present at the C terminus. Bioinformatic analysis of VPg primary structure suggested that it could be a disordered protein. Biophysical studies validated this observation, and VPg resembled "natively unfolded" proteins. CD spectral analysis showed that the $\triangle$N70Pro-VPg fusion protein had a characteristic secondary structure with a 230 nm positive CD peak. Mutation of Trp-43 in the VPg domain to phenylalanine abrogated the positive peak with concomitant loss in cis- and trans-proteolytic activity of the $\triangle$N70Pro domain. Further, deletion of VPg domain from the polyprotein completely abolished proteolytic processing. The results suggested a novel mechanism of activation of the protease, wherein the interaction between the natively unfolded VPg and the protease domains via aromatic amino acid residues alters the conformation of the individual domains and the active site of the protease. Thus, VPg is an activator of protease in Sesbania mosaic virus, and probably by this mechanism, the polyprotein processing could be regulated in planta

A high throughput neutralization test based on GFP expression by recombinant rabies virus.

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers

Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine.

Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an in silico antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-luc, negative control) in mice and big brown bats (Eptesicus fuscus). Mice vaccinated and boosted intradermally with 1 x 107 plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x107 PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x108 PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-luc and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, in silico designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carriers

Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information. Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information. Virus-like particles (VLPs), formed by the structural elements of viruses, have received considerable attention over the past two decades. The number of reports on newly obtained VLPs has grown proportionally with the systems developed for the expression of these particles. When expressed in a suitable heterologous system, viral structural proteins involved in capsid or envelope formation often self-assemble into VLPs in the absence of other viral components usually required for virus assembly, such as multiple structural or nonstructural proteins and viral genomes. Protein–protein interactions in VLPs are relatively strong and can result in the formation of stable structures. Several experiments have been reported that may help answer questions regarding the requirements for VLP formation. Knowledge on the assembly process of VLPs is crucial to define the usefulness of such particles for the presentation of their own or foreign epitopes as carriers for transiently expressed proteins as a means of vaccine production. The aim of the present chapter is to outline recent achievements in two important fields of research brought about by the availability of VLPs produced in a foreign host. These are (1) the requirements for VLP assembly and (2) the use of VLPs as carriers for foreign epitopes. To date, reviews in these areas have mainly focused on results obtained with a specific virus genus or family of viruses (1, 2, 3, 4 and 5) and the reader is advised to refer to these reviews for complementary information

Rabies Outbreak in Captive Big Brown Bats (Eptesicus fuscus) Used in a White-nose Syndrome Vaccine Trial

An outbreak of rabies occurred in a captive colony of wild-caught big brown bats (Eptesicus fuscus). Five of 27 bats exhibited signs of rabies virus infection 22–51 d after capture or 18–22 d after contact with the index case. Rabid bats showed weight loss, aggression, increased vocalization, hypersalivation, and refusal of food. Antigenic typing and virus sequencing confirmed that all five bats were infected with an identical rabies virus variant that circulates in E. fuscus in the US. Two bats with no signs of rabies virus infection were seropositive for rabies virus-neutralizing antibodies; the brains of these bats had no detectable viral proteins by the direct fluorescence antibody test. We suspect bat-to-bat transmission of rabies virus occurred among our bats because all rabies-infected bats were confined to the cage housing the index case and were infected with viruses having identical sequences of the entire rabies nucleoprotein gene. This outbreak illustrates the risk of rabies virus infection in captive bats and highlights the need for researchers using bats to assume that all wild bats could be infected with rabies virus

An ELISA-based method for detection of rabies virus nucleoprotein-specific antibodies in human antemortem samples.

Rabies is a fatal encephalitic disease in humans and animals caused by lyssaviruses, most commonly rabies virus (RABV). Human antemortem diagnosis of rabies is a complex process involving multiple sample types and tests for the detection of antibodies, antigen (protein), and nucleic acids (genomic RNA). Serological diagnosis of human rabies includes the detection of either neutralizing or binding antibodies in the cerebrospinal fluid (CSF) or serum samples from unimmunized individuals without prior rabies vaccination or passive immunization with purified immunoglobulins. While neutralizing antibodies are targeted against the surface-expressed glycoprotein (G protein), binding antibodies to viral antigens are predominantly against the nucleoprotein (N protein), although there can be antibodies against all RABV-expressed proteins. To determine N protein-specific antibody responses in the CSF and serum during RABV infection, we developed an enzyme-linked immunosorbent assay (ELISA) with purified recombinant N protein expressed in E. coli. N protein-specific immunoglobulin (Ig) subtypes IgG and IgM were detected in the CSF or serum of previously diagnosed human rabies cases. In addition, anti-N protein seroconversion was demonstrated over the course of illness in individual rabies cases. We compared the N protein ELISA results to those of an indirect fluorescent antibody (IFA) test, the current binding antibody assay used in diagnosis, and show that our ELISA is consistent with the IFA test. Sensitivity and specificity of the N protein ELISA ranged from 78.38-100% and 75.76-96.77% with respect to the IFA results. Our data provide evidence for the use of an N protein ELISA as an additional option for the detection of RABV-specific IgG or IgM antibodies in human CSF or serum specimens

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carriers

Virus-Like Particles: Models for Assembly Studies and Foreign Epitope Carrier

Protection of bats <i>(Eptesicus fuscus)</i> against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine

<div><p>Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an <i>in silico</i> antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-<i>luc</i>, negative control) in mice and big brown bats (<i>Eptesicus fuscus</i>). Mice vaccinated and boosted intradermally with 1 x 10⁷ plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x10⁷ PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x10⁸ PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-<i>luc</i> and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, <i>in silico</i> designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife.</p></div

Rabies neutralizing antibody levels in mice following vaccination with various RCN-vectored rabies vaccines.

<p>Serum titers of rabies neutralizing antibodies (IU/ml) in mice 45 days post vaccination with RCN-MoG, RCN-IRES-MoG, or RCN-G. No significant differences were detected between groups (P = 0.399).</p