Viral pathogen discovery

Abstract

Viral pathogen discovery is of critical importance to clinical microbiology, infectious diseases, and public health. Genomic approaches for pathogen discovery, including consensus polymerase chain reaction (PCR), microarrays, and unbiased next-generation sequencing (NGS), have the capacity to comprehensively identify novel microbes present in clinical samples. Although numerous challenges remain to be addressed, including the bioinformatics analysis and interpretation of large datasets, these technologies have been successful in rapidly identifying emerging outbreak threats, screening vaccines and other biological products for microbial contamination, and discovering novel viruses associated with both acute and chronic illnesses. Downstream studies such as genome assembly, epidemiologic screening, and a culture system or animal model of infection are necessary to establish an association of a candidate pathogen with disease.

Graphical abstract

Figure 1

Genomic approaches to pathogen discovery. Clinical samples are subjected to pathogen enrichment and host depletion methods, followed by genomic analysis using consensus PCR, pan-microbial microarrays, and/or NGS. After a novel agent is identified, downstream studies are needed to establish a causal association between the candidate pathogen and disease.

Figure 2

Microarray and NGS analyses of pandemic 2009 influenza A(H1N1) infection in humans. (a) Heat map of ViroChip microarray hybridization patterns obtained from nasal swab samples from patients with influenza-like illness and asymptomatic negative controls (‘neg’). The samples (x-axis) and microarray probes (y-axis) are clustered using a hierarchical clustering algorithm [45]. High-intensity probes derived from swine influenza A(H1N1) and human influenza A(H1N1) sequences are observed in samples from patients infected by pandemic 2009 influenza A(H1N1), with higher relative signal intensity in the swine influenza A(H1N1) probes. In contrast, the ViroChip signature in nasal swabs from patients infected with seasonal H3N2 influenza consists primarily of influenza A(H3N2) probes. No microarray cross-hybridization is observed in patients infected with other respiratory viruses or negative controls. Note that the influenza probes on the ViroChip microarray shown here were designed before onset of the pandemic 2009 influenza A(H1N1) outbreak. (b) Computational pipeline for analysis of NGS data. Preprocessing and computational subtraction of host (human) sequences are then followed by alignment to pathogen reference databases. The percentages show the remaining proportion of reads after each step, beginning with 100% of the preprocessed reads. Abbreviations: DBs, databases; rRNA, ribosomal RNA; mRNA, messenger RNA.

Figure 3

Discovery of Bas-Congo virus (BASV), a novel rhabdovirus associated with acute hemorrhagic fever in humans. (a) Map of Africa showing viral hemorrhagic fever outbreak regions. Hemorrhagic fever due to flaviviruses, such as dengue and yellow fever, is widespread throughout the continent. The location of the BASV hemorrhagic fever outbreak is designated by a red star. (b) Deep sequencing and de novo genome assembly of BASV. The BASV genome is highly divergent, sharing only 25% amino acid identity with rabies and <42% amino acid identity with any other rhabdovirus.

Figure 4

Baboon and human infections from a novel adenovirus species. (a) A 1997 acute respiratory outbreak in a baboon colony. A novel adenovirus, named simian adenovirus C (SAdV-C), was discovered in association with an outbreak at a primate research facility that sickened 4 of 9 baboons and resulted in two cases of fatal pneumonia. (b) Serological testing of staff personnel at the facility and controls (five epidemiologically unrelated young children) for exposure to simian adenoviruses SAdV-B and SAdV-C. Neutralizing antibodies to SAdV-C are absent before the outbreak but detected in 6 of 6 staff personnel after the outbreak, indicating recent or prior exposure to the virus. Abbreviations: BAdV, baboon adenovirus; SAdV, simian adenovirus; Pre, pre-outbreak; Post, post-outbreak; N/A, not applicable.