Pathogenic viruses: Molecular detection and characterization

Abstract

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Keywords: DNA sequencing; Pathogenic viruses; Polymerase chain reaction; Unidirectional workflow; Virus detection.

Fig. 1

A). Schematic diagram of influenza A virus, an example of RNA virus. The influenza A virus is an enveloped virus. The envelope is a lipid bilayer membrane obtained from the host cell in which the virus multiplies. Beneath the lipid membrane is the viral matrix protein forming a shell that gives strength and rigidity to the lipid envelope. Within the interior of the virion are 8 capsidated segments of RNA, the genetic material of the virus. The haemaggutinin protein (HA) is shown to have a rod-shaped structure, while the neuraminidase protein (NA) is depicted to have a mushroom-shaped structure. Influenza A virus envelope also contains M2 ion channels. B). Schematic diagram of hepatitis B virus, an example of DNA virus. The hepatitis B virus particle has a spherical structure consists of a lipid envelope containing HBsAg that surrounds an inner icosahedral nucleocapsid composed of hepatitis B core antigen (HBcAg) complexed with polymerase and the viral partially double stranded DNA genome. HBeAg, an indicator of active viral replication, can be found between the icosahedral nucleocapsid core and the lipid envelope. The surface antigen (HBsAg) has large (L), medium (M) and small (S) variants.

Fig. 2

Diagram of the one-step and two-step reverse transcription polymerase chain reaction (RT-PCR). In one-step RT-PCR, the reverse transcription step is coupled with PCR. The viral RNA undergoes reverse transcription to cDNA followed by PCR amplification in a single reaction. In the two-step RT-PCR, the process is carried out in two steps. The first step is a reverse transcription reaction to form cDNA. The second step is the amplification of cDNA using PCR (Wacker and Godard, 2005; Singh et al., 2014).

Fig. 3

Recommended layout and workflow of molecular diagnostic laboratory. The workflow direction is shown by the arrow. Workflow between these rooms/areas must be unidirectional in that from clean areas to contaminated areas, but not from contaminated areas to clean areas.