Development and evaluation of a RT-qPCR assay for fast and sensitive rabies diagnosis

Highlights

• Reliable qRT-PCR assay for RABV detection was developed and evaluated

• Analytical sensitivity of the assay was 10³ copies/ml, and the sensitivity measured using characterized strains was between 0.1 LD50/1 ml and 1.0 LD50/1 ml.

• Diagnostic sensitivity and specificity were both 100%

Abstract

Rabies virus is endemic to Russia, among other countries. It is therefore critical to develop a high-quality and high-precision diagnostic procedure for the control and prevention of infection.

The main objective of the research presented here was to develop a reliable RT-qPCR assay for rabies diagnostics.

For this purpose, a RABV strains from various biological and geographical origins were used. In addition, rabies-positive and rabies-negative samples, as well as nucleic acids from other viruses and DNA extracted from the brain tissues of mice, dogs, cats, bats and humans, were studied using the developed assay.

The analytical sensitivity of the assay, as assessed using armored recombinant positive control dilutions, was 10³ copies/ml, and the sensitivity measured using characterized strains was between 0.1 LD50/ml and 1.0 LD50/ml. A broad range of RNA from RABV strains circulating in different regions of Russia, as well as RNA from RABV-positive primary brain samples from 81 animals and two humans, was detected using the developed assay. No false-positive or false-negative results were obtained.

Given that high analytical and diagnostic sensitivities and a high specificity were verified for this assay, it has high potential as a screening test that may be suitable for the epizootiological monitoring of animals and for the fast postmortem diagnosis of rabies.

Introduction

Classical rabies virus (RABV) is a neurotropic negative-sense single-stranded RNA virus that belongs to the genus Lyssavirus within family Rhabdoviridae. RABV causes one of the deadliest zoonotic diseases, namely, rabies, and is estimated to be responsible for up to 70,000 human deaths worldwide each year (WHO Expert Consultation on Rabies, 2013). Apart from RABV, the genus Lyssavirus includes 15 species, some of which are circulating in the territory of the former Soviet Union, including European bat lyssaviruses 1, Khujand lyssavirus, Aravan lyssavirus, Irkut lyssavirus, and West Caucasian bat lyssavirus (https://talk.ictvonline.org/ictv-reports/ictv_online_report/, 2016). All of these viruses are neurotropic infectious agents that cause irreversible brain lesions in humans and other warm-blooded animals, resulting in rabies-like diseases (Delmas et al., 2008). However, the greatest epidemiological and epizootiological significance is attributed to RABV because it is widespread and causes the vast majority of the known disease cases related to Lyssavirus species in both humans and animals. In contrast, other members of the genus Lyssavirus are known to have restricted areas of distribution and a limited number of host species, with bats being the main host (WHO Expert Consultation on Rabies, 2013).

All currently known RABV can be divided into seven major genetic groups. Two of these groups, the Cosmopolitan and the Arctic/Arctic-like groups, circulate in the Russian Federation. Within these two major groups, members of six subgroups have been reported in Russia: A. Arctic rabies (northern parts of Siberia), B. Arctic-like rabies (Khabarovsk Krai, Transbaikal region), C. steppe rabies (Eurasian Steppe), D. Central European Russian rabies, E. Northeast European Rabies, and F. Caucasian rabies (Deviatkin et al., 2017).

Rabies is a zoonotic disease transmitted by direct contact with infected domestic and wild animals through bites or scratches, usually via saliva (http://www.who.int/mediacentre/factsheets/fs099/en/). Globally in up to 99% of human cases, RABV is transmitted by domestic dogs. Despite significant advances in research within the fields of epidemiological surveillance and prophylaxis (Dietzschold et al., 2003, Simonova and Khadarcev, 2014), cases of infection are still being registered on all continents except for Antarctica (http://www.who.int/mediacentre/factsheets/fs099/en/).

In Russia alone, 39,895 disease cases were registered within the period of 2000–2016 (39,783 in animals and 114 in humans) (http://www.who-rabies-bulletin.org/Queries/Surveillance.aspx). Approximately 400,000 people come into contact with potentially rabid animals and receive post-exposure prophylaxis each year (Deviatkin et al., 2017). Rabies is enzootic in Russia from the western borders to the Far East. According to surveillance data, this disease is maintained in Russia by wild canids, including the arctic fox, raccoon dog, steppe wolf and wolf. However, the majority of rabies cases are associated with the red fox, which is the main RABV reservoir (Kuzmin et al., 2004). Therefore this is a peculiarity of enzootic process in Russia.

Currently, the most applicable rabies diagnostic method in Russia is the fluorescent antibody test (FAT), which is applied to autopsy material. In cases of negative or ambiguous results, additional diagnostic procedures are applied, including virus isolation from animals (mouse inoculation test (MIT)) or cell cultures and an enzyme-linked immunosorbent assay (ELISA) (Anonymous, 2013, Methodological guideline for laboratory diagnostics of rabies in animals, 1997, WHO Expert Consultation on Rabies, 2013). For now, antemortem diagnosis is not performed, and all of the utilized methodology is based on World Health Organization (WHO) recommendations. Within these guidelines, molecular diagnostic techniques are considered as auxiliary or additional for rabies diagnosis. However, molecular techniques have high potential and could be faster confirmatory methods due to their prevailing procedural simplicity and the ease of interpreting their results. The reported diagnosis of two rabies cases in humans from the Astrakhan region supports this opinion (Dedkov et al., 2016).

The use of molecular methods for RABV diagnosis or for the diagnosis of all members of the Lyssavirus genus is not new. The efficiency of PCR techniques for the routine diagnosis of rabies has been convincingly demonstrated by many researchers (Ermine et al., 1990, Heaton et al., 1997, Kamolvarin et al., 1993, Kulonen et al., 1999, Limaa et al., 2005, McColl et al., 1993, Sacramento et al., 1991). In the last decade, a great number of molecular assays for RABV and pan-lyssavirus diagnostics have been developed and evaluated (Wadhwa et al., 2017). These assays are based mainly on real-time reverse transcription-PCR (RT-qPCR) via intercalating dye or TaqMan probe methods (Coertse et al., 2010, Dupuis et al., 2015, Hayman et al., 2011, Mani et al., 2014, Nadin-Davis et al., 2009, Suin et al., 2014, Wakeley et al., 2006). However, no reliable real-time RT-qPCR assay has been developed for RABV diagnostics in Russia and neighboring countries. Such a method is necessary for the diagnosis of rabies and surveillance of the virus in wildlife.

The main objective of the research presented here was to develop a reliable RT-qPCR assay for postmortem rabies diagnosis in both humans and animals. We deliberately focused on the diagnosis of RABV only because other members of the Lyssavirus genus, although prevalent in Russia, are extremely rare, and the diseases associated with them are unique (Leonova et al., 2010).