Development of a real-time RT-PCR assay for detection of Hendra and Nipah viruses

The article is devoted to the development of a method for detection of viral RNA of two highly pathogenic zoonotic viruses from the genus Henipavirus — Hendra and Nipah using real-time reverse transcription polymerase chain reaction. In the natural environment, these viruses are carried by flying foxes in the family Pteropodidae. Horses and pigs, respectively, are susceptible to infection. The diseases are also transmitted to humans through contact with sick animals, their biological excreta and from person to person. In infected humans and animals, clinical signs of infection may be asymptomatic, or may present with flu-like symptoms at the onset of the disease and progress to neurologic disease and acute respiratory infection, followed by death. In Australia, the subunit vaccine HeV-sG is used against Hendra virus in horses. There is no treatment or vaccine for Hendra or Nipah viruses for humans. The need to develop new detection methods and search for new viral targets remains an urgent task due to the large area of distribution of the described viruses, high contagiousness and mortality of animals and humans. The study describes the original designed primers and probes for conserved regions of the genomes of two viruses: the gene encoding the nucleocapsid protein of Hendra virus and the gene encoding the glycoprotein of Nipah virus. Synthetic controls for the extraction and reverse transcription PCR stages have been created, confirming the quality of the developed method. Biological samples from healthy people (blood plasma, swabs from oral and nasopharyngeal mucous membranes, cerebrospinal fluid) with the addition of artificial controls passed the stages of sample extraction and real-time reverse transcription PCR, thus confirming the quality of control samples. The detection limit of the described viral RNA identification methods was determined as 100 copies/mL for Hendra virus and 1000 copies/mL for Nipah virus. The amplification transit time is less than 90 minutes. The developed method will help in epidemiologic control of the spread of these infections, can be used in the diagnosis of Hendra and Nipah viruses and for solving research tasks to study the properties of these pathogens.