Date Published: February 20, 2019
Publisher: Public Library of Science
Author(s): Yuan Qiu, Suchun Wang, Baoxu Huang, Huanxiang Zhong, Zihao Pan, Qingye Zhuang, Cheng Peng, Guangyu Hou, Kaicheng Wang, Michelle Wille.
With rapidly increasing animal pathogen surveillance requirements, new technologies are needed for a comprehensive understanding of the roles of pathogens in the occurrence and development of animal diseases. We applied metagenomic technology to avian virus surveillance to study the main viruses infecting six poultry farms in two provinces in eastern China. Cloacal/throat double swabs were collected from 60 birds at each farm according to a random sampling method. The results showed that the method could simultaneously detect major viruses infecting farms, including avian influenza virus, infectious bronchitis virus, Newcastle disease virus, rotavirus G, duck hepatitis B virus, and avian leukemia virus subgroup J in several farms. The test results were consistent with the results from traditional polymerase chain reaction (PCR) or reverse transcription-PCR analyses. Five H9N2 and one H3N8 avian influenza viruses were detected at the farms and were identified as low pathogenic avian influenza viruses according to HA cleavage sites analysis. One detected Newcastle disease virus was classified as Class II genotype I and avirulent type according to F0 cleavage sites analysis. Three avian infectious bronchitis viruses were identified as 4/91, CK/CH/LSC/99I and TC07-2 genotypes by phylogenetic analysis of S1 genes. The viral infection surveillance method using metagenomics technology enables the monitoring of multiple viral infections, which allows the detection of main infectious viruses.
Animal disease surveillance is fundamental for animal disease prevention and control, and it is also a tool to get the information used to make decisions about control and eradication strategies. A variety of laboratory techniques have been used to survey the epidemiology of animal infectious diseases, among which animal pathogen surveillance and tracking the molecular epidemiology of pathogens are important strategies [1, 2]. These methods usually involve pathogen isolation and identification, reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and sequencing analyses. These methods often target a single known pathogen; however, the analysis of multiple pathogens in one or more hosts requires multiple detection methods, personnel, or even laboratories. Furthermore, these approaches cannot monitor and provide an early warning for pathogens of other animal diseases and viruses that have not yet been sequenced.
We used a metagenomics-based pathogen surveillance method to monitor viral infections in this study. The main infectious viruses in the six poultry farms were shown to be AIV and IBV, followed by NDV, ALV-J, DHBV, and rotavirus G, which is consistent with the current prevalence of poultry viral disease in China . The high prevalence of viruses in the poultry farms could indicate insufficient farm biosecurity. We also analyzed the subtype of AIV, genotypes of NDV and IBV, and pathogenicities of AIV and NDV in this study. All the detected AIVs and NDV were low pathogenic or avirulent viruses. These kinds of viruses can be usually found in Chinese poultry farms [41, 42]. The three avian IBVs detected in the three farms were identified as genotypes 4/91, CK/CH/LSC/99I, and TC07-2, by phylogenetic analysis of S1 genes compared with 33 reference viruses. The TC07-2-type was first detected in Guangdong province in 2007 , and classified as CH VI lineage by S1 gene phylogenetic analysis . In this study, the lineage was named as TC07-2-type, consistent with the principles of genotype nomenclature for molecular analysis of IBVs . Because the assembled contigs from the NGS reads was too short (about 250 bp) or located in the genes which was not suitable for phylogenetic analysis, the phylogenetic analysis of AIV and IBV was based on the sequence of PCR fragments. Few reads matched to rotavirus G, DHBV, and ALV-J, and these matched reads were not located on genes available for phylogenetic analysis, and it was therefore not possible to construct phylogenetic trees for these three detected viruses. Because of the low abundance of viruses in the clinical samples, the quantity and quality of the viral sequence in the NGS should be enhanced to analyze the genotype and phylogenetic relationship from the NGS data directly. Because animals are fed in flocks, animal disease surveillance focuses on the infecting pathogens of each flock. Therefore, the metagenomics-based pathogen surveillance method established in this study also focused on poultry groups. The poultry in the same field were considered as a single sampling pool. The high sensitivity of the NGS technology ensures that major kinds of viral pathogens in mixed samples can be detected. In this study, the results were validated by traditional PCR or RT-PCR techniques, which were consistent with the metagenomics-based pathogen surveillance method to certify whether the viruses were percent or absent in the clinical samples. However, this method had some limitations. The absence of pathogens on farms where none were detected by NGS was not confirmed by comparing the current metagenomics results with an independent set of test results. Furthermore, no sequences other than the virus sequences were analyzed.