Highly sensitive Taqman PCR detection of Puumala hantavirus

Abstract

An increasing number of clinical cases of Hantavirus infections have been reported from various regions in Asia, Europe and North America. Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped and possess a single-stranded trisegmented RNA genome of negative polarity. Rodents or insectivores are natural hosts of hantaviruses and transmit the virus to humans chiefly by aerosolisation. These viruses are the causative agents of haemorrhagic fever with renal and pulmonary syndromes. In the northeast of France, Puumala hantavirus causes, every year, more than 150 mild forms of haemorrhagic fever with a renal syndrome known as nephropathia epidemica. Serological tests may lack sensitivity for diagnosing early stages of infection and virus isolation is limited because it grows poorly in cell culture. Since reverse transcription (RT)-PCR amplification is an efficient method for detecting viral genomes in patient specimens, we developed an assay using a Taqman probe and compared it with the classical RT-PCR amplification. To achieve this goal, a Puumala strain was grown in Vero E6 cells and RNA extracted from the culture supernatant. We found that the semi-nested RT-PCR detected a minimal amount of 300 TCID(50) mL(-1), while the Taqman PCR allowed detection of less than 10 TCID(50) mL(-1 )and provided a quantitative analysis.