A systematic approach to novel virus discovery in emerging infectious disease outbreaks

Abstract

The discovery of novel viruses is of great importance to human health-both in the setting of emerging infectious disease outbreaks and in disease syndromes of unknown etiology. Despite the recent proliferation of many efficient virus discovery methods, careful selection of a combination of methods is important to demonstrate a novel virus, its clinical associations, and its relevance in a timely manner. The identification of a patient or an outbreak with distinctive clinical features and negative routine microbiological workup is often the starting point for virus hunting. This review appraises the roles of culture, electron microscopy, and nucleic acid detection-based methods in optimizing virus discovery. Cell culture is generally slow but may yield viable virus. Although the choice of cell line often involves trial and error, it may be guided by the clinical syndrome. Electron microscopy is insensitive but fast, and may provide morphological clues to choice of cell line or consensus primers for nucleic acid detection. Consensus primer PCR can be used to detect viruses that are closely related to known virus families. Random primer amplification and high-throughput sequencing can catch any virus genome but cannot yield an infectious virion for testing Koch postulates. A systematic approach that incorporates carefully chosen combinations of virus detection techniques is required for successful virus discovery.

Figure 1

Algorithm for novel virus discovery.

Figure 2

Methods of virus detection applied to novel coronavirus discovery. A: Vero kidney epithelial cells infected by Middle East respiratory syndrome–coronavirus (MERS-CoV) showing cytopathic effect in the form of cell shrinkage and syncytium formation. B: Electron micrograph of severe acute respiratory syndrome coronavirus (SARS-CoV) showing characteristic enveloped virus particles with protruding spikes. C: Gel electrophoresis image of DNA amplified by coronavirus pol gene-derived consensus primer RT-PCR. Lane 1, bacteriophage ΦX174 DNA HaeIII digest marker; 2, negative sample; 3, negative control; 4, positive control; 5, nasopharyngeal aspirate sample with human coronavirus HKU1. D: Phylogenetic analysis of selected human and animal coronaviruses on the basis of the pol gene by the neighbor joining method. Lineage A betacoronavirus (A), lineage B betacoronavirus (B), and lineage C betacoronavirus (C).