Denise A. Marston

Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century.

The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.

Ikoma Lyssavirus, Highly Divergent Novel Lyssavirus in an African Civet

Evidence in support of a novel lyssavirus was obtained from brain samples of an African civet in Tanzania. Results of phylogenetic analysis of nucleoprotein gene sequences from representative Lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species, designated Ikoma lyssavirus.

Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison

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.

Quantifying Antigenic Relationships among the Lyssaviruses

ABSTRACT All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses.

Paralytic rabies after a two week holiday in India

Rabies is an acute infection of the central nervous system (CNS) and caused by rabies virus or related members of the genus Lyssavirus , family Rhabdoviridae.1 The virus is usually transmitted through a dog bite and produces one of the most important viral encephalitides worldwide, with at least 40 000 deaths reported annually.2 However, it is rare in the United Kingdom, where just 12 cases have been reported since 19773: 11 were imported from overseas, and one occurred in a bat handler infected in Scotland with European bat lyssavirus type 2a.4 Most UK patients presented with furious rabies, which is characterised by hydrophobia and spasms. We report a case of paralytic rabies in a tourist after a two week holiday in Goa, India. Fig 2 Phylogenetic tree depicting the relation between the rabies virus sequence amplified from our patient (RV1964—boxed) and other viruses originating in Asia. The horizontal branch lengths represent the extent of difference between the strains (expressed as nucleotide substitutions per nucleotide site), and the closer viruses are on the tree, the more closely they are related A woman in her late 30s was admitted to her local general hospital under the orthopaedic surgeons, with lower back pain radiating to the left leg. The pain had started four days earlier, was severe and shooting in nature, and was getting worse. She had been seen twice in casualty in the preceding days, and by the time of admission she was unable to walk. She also had a headache and had vomited once. Three and a half months before admission, during a two week holiday to Goa, India, she had been bitten by a dog; she was walking in the street when a puppy on a lead nipped her on the left leg. There was a slight …

A universal real-time assay for the detection of Lyssaviruses

Highlights ► Universal real-time PCR primer pair demonstrated to hybridize to and detect each of the known Lyssaviruses (including Rabies virus) with greater sensitivity than a standard pan-Lyssavirus hemi-nested RT-PCR typically used. ► Target sequences of bat derived virus species unavailable for analysis (Aravan-, Khujand-, Irkut-, West Caucasian bat- and Shimoni bat virus) were synthesized to produce oligonucleotides and the synthetic DNA was used as a target for primer hybridization.

Immunovirological correlates in human rabies treated with therapeutic coma

A 37‐year‐old woman was admitted to hospital and over the next 5 days developed a progressive encephalitis. Nuchal skin biopsy, analyzed using a Rabies TaqMan© PCR, demonstrated rabies virus RNA. She had a history in keeping with exposure to rabies whilst in South Africa, but had not received pre‐ or post‐exposure prophylaxis. She was treated with a therapeutic coma according to the “Milwaukee protocol,” which failed to prevent the death of the patient. Rabies virus was isolated from CSF and saliva, and rabies antibody was demonstrated in serum (from day 11 onwards) and cerebrospinal fluid (day 13 onwards). She died on day‐35 of hospitalization. Autopsy specimens demonstrated the presence of rabies antigen, viral RNA, and viable rabies virus in the central nervous system. J. Med. Virol. 82:1255–1265, 2010.

Genetic Characterization of Highly Pathogenic Avian Influenza (H5N8) Virus from Domestic Ducks, England, November 2014

Genetic sequences of a highly pathogenic avian influenza (H5N8) virus in England have high homology to those detected in mainland Europe and Asia during 2014. Genetic characterization suggests this virus is an avian-adapted virus without specific affinity for zoonoses. Spatio-temporal detections of H5N8 imply a role for wild birds in virus spread.

Analysis of vaccine-virus-associated rabies cases in red foxes ( Vulpes vulpes ) after oral rabies vaccination campaigns in Germany and Austria

To eradicate rabies in foxes, almost 97 million oral rabies vaccine baits have been distributed in Germany and Austria since 1983 and 1986, respectively. Since 2007, no terrestrial cases have been reported in either country. The most widely used oral rabies vaccine viruses in these countries were SAD (Street Alabama Dufferin) strains, e.g. SAD B19 (53.2%) and SAD P5/88 (44.5%). In this paper, we describe six possible vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) detected during post-vaccination surveillance from 2001 to 2006, involving two different vaccines and different batches. Compared to prototypic vaccine strains, full-genome sequencing revealed between 1 and 5 single nucleotide alterations in the L gene in 5 of 6 SAD isolates, resulting in up to two amino acid substitutions. However, experimental infection of juvenile foxes showed that those mutations had no influence on pathogenicity. The cases described here, coming from geographically widely separated regions, do not represent a spatial cluster. More importantly, enhanced surveillance showed that the vaccine viruses involved did not become established in the red fox population. It seems that the number of reported vaccine virus-associated rabies cases is determined predominantly by the intensity of surveillance after the oral rabies vaccination campaign and not by the selection of strains.

Lyssavirus in Indian Flying Foxes, Sri Lanka

A novel lyssavirus was isolated from brains of Indian flying foxes (Pteropus medius) in Sri Lanka. Phylogenetic analysis of complete virus genome sequences, and geographic location and host species, provides strong evidence that this virus is a putative new lyssavirus species, designated as Gannoruwa bat lyssavirus.