Screening And Validation Of Unknown Pathogens With High-throughput Sequencing

Background and ObjectivesEmerging infectious disease outbreaks pose important global threats. Infection rates are increased by many factors, including global warming, population increases, antibiotic overuse, economic integration, rapid urbanization, ecological environmental changes, frequent contact between humans and wildlife, and high transnational tourism rates. According to a World Health Organization report, at least 48 new pathogens have been found since 1973, including some important viruses, such as Human immunodeficiency virus, SARS virus, Ebola virus, Marburg virus, and avian and swine influenza viruses. New diseases are appearing at an unprecedented rate(one per year on average) and spreading across the world. Therefore, the prompt screening for and identification of emerging infectious pathogens and the rapid acquisition of their genomic data have significant implications for epidemic and biohazard control.Traditional pathogen identification techniques have become increasingly inadequate. With the emergence of PCR technologies, pathogen identification based on nucleic acid detection has significantly improved the clinical success rate. The most commonly used detection method based on PCR requires genome sequence information for each pathogen. However, in some cases, the genomic sequence of the pathogen is unavailable. For new, previously unreported types of human pathogens, the lack of genomic sequence data limits the detection of the pathogen causing an outbreak. In the clinical context, infections with unknown pathogens account for a high proportion of disease diagnoses.In recent years, increasingly more researchers have identified new pathogens with important significance for human health using high-throughput sequencing(HTS), even though these pathogens have not been detectable with traditional biotechnological methods. This suggests that HTS technology has great utility in the identification of pathogenic microorganisms. The development of HTS has made it possible to study complex microbial specimens and to determine the various sources from among diverse microbial communities, including those in the ocean, soil, and the human body, thus providing new perspectives. These studies use both 16 S rDNA gene sequencing to determine the phylogenetic relationships and more comprehensive shotgun sequencing to predict detailed species and gene compositions.The 16S rDNA molecule contains not only highly conserved regions, but also moderately conserved and highly variable regions, so it is suitable for studying the relationship of bacteria in complex samples. Despite the moderate size of the 16 S rDNA molecule(its sequence length is about 1 540 bp), it contains sufficient information for this type of research. Whole-genome sequencing can also facilitate the study of genetic diversity, the genetic laws of evolution, pathogenesis, and the propagation of resistance genes. The new-generation HTS technology requires far less performance time than the earlier technology, and the cost is steadily declining. It circumvents the limitations of traditional biological research methods and has greatly advanced research in comparative genomics, epidemiology, and microbial evolution. Methods and ResultsIn recent years, the number of zoonoses has increased, and infectious diseases such as SARS, rabies, and avian influenza pose a huge threat to public health and human health security. Simian varicella(SV) disease caused by simian varicella virus(SVV) is similar in its clinical presentation to human varicella–zoster virus(VZV) infection, and an antigenic correlation between SVV and VZV has been demonstrated. Therefore, an in-depth study of the zoonotic potential of SVV is required. In 2014, an outbreak of an unknown disease in an animal breeding center caused the death of many African green monkeys. We used HTS to explore the cause of death in this outbreak. Liver and lung tissue samples were taken from the dead green monkeys, total viral DNA was extracted, HTS was performed using the Life Ion Torrent Personal Genome Machine(PGM) platform based on a viral metagenomic technique, and the sequence data were analyzed with bioinformatic tools to identify the virus involved. Based on the HTS results, quantitative PCR(qPCR) primers were designed for the quantitative detection of the virus, and to confirm the presence of the virus in the tissue samples. In total, 674 Mb of raw sequence data was generated from the green monkey tissue samples, with 4.76 million reads. The whole genome sequence of the virus(approximately 124 kb) was obtained with sequence splicing. Sequence alignments showed that a large number of sequences shared a high degree of homology with the monkey varicella–zoster virus identified, which was more than 98% identical to a known virus designated Cercopithecine herepesvirus 9. Based on our sequencing results, herpes-virus-specific primers were designed, and the virus was quantitatively detected with qPCR in both tissue samples and pure viral cultures. The cycle threshold values for the liver, lung, and viral culture were 23, 20, and 16, respectively. These indicate that large amounts of the virus were present in the two organs of liver and lung, and that the monkey herpes virus titers were higher in the lung tissues than in the liver tissues. This study used HTS as a rapid screening technique to identify an infectious disease and confirmed that the lethal pathogen responsible for the green monkey deaths was simian varicella virus.Emerging infectious diseases can be caused by either viruses or bacteria, and the structural differences of these agents demand that different procedures be followed. A cynomolgus monkey was found dead at an experimental animal center and the cause of death was unknown. The fatal pathogens were identified with HTS technology. In this experiment, bacterial DNA was extracted directly from tissue samples of the brain lesions, and the V1–V2 hypervariable regions of 16 S rDNA was amplified. Metagenomic sequencing and a bioinformatic analysis rapidly identified the microflora in the brain lesions without the need for culture. In order to identify the possible pathogens, the brain tissue homogenate was then cultured. Two bacteria, which were then identified as Streptococcus lutetiensis and Macrococcus sp., were separated from the cultured brain tissue homogenate. Shotgun sequencing and sequencing a large-fragment mate-pair library were used to analyze the separated bacteria. The whole genomes were annotated with the RAST program, and the possible resistance and virulence genes present in the genomes were analyzed. Finally, the virulence of the two bacterial strains was confirmed in animals. The combination of metagenomic sequencing and a histopathological examination suggested that the death of the cynomolgus monkey was caused by a pathogenic bacterial infection. After shotgun genomic sequencing for Streptococcus lutetiensis and Macrococcus sp., 27 contigs from the S. lutetiensis sequence data showed 98% homology between our sequence and the reference sequence S. lutetiensis 033(GenBank: CP003025.1). The 28 contigs from the Macrococcus sp. sequence data showed only 74% homology between our sequence and the reference sequence M. caseolyticus(Gen Bank: AP009484.1). Therefore, the isolated Macrococcus may be a novel species. Mate-pair sequencing was performed to determine the whole genome sequence of Macrococcus sp.(2 391 704 bp). RAST annotation showed that the genome contains 2 430 coding sequences, 73 transcribed RNAs, nine virulence genes, including ClpP, ClpX, and ResD, and six resistance genes to tetracycline, aminoglycoside, fluoroquinolone, rifampicin, and so on. When we injected BALB/c mice intracranially with the bacteria, using an engineered strain of Escherichia coli as the control, the results suggested that the S. lutetiensis and Macrococcus sp. strains were relatively highly pathogenic, and caused the death of the mice. These results suggest that a mixed infection of S. lutetiensis and Macrococcus sp. caused the death of the cynomolgus monkey. However, further research is required to clarify the specific disease mechanism.