Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing.

Objective: Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods: We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results: The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion: Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

Genomic Elucidation of a COVID-19 Resurgence and Local Transmission of SARS-CoV-2 in Guangzhou, China.

While China experienced a peak and decline in coronavirus disease 2019 (COVID-19) cases at the start of 2020, regional outbreaks continuously emerged in subsequent months. Resurgences of COVID-19 have also been observed in many other countries. In Guangzhou, China, a small outbreak, involving less than 100 residents, emerged in March and April 2020, and comprehensive and near-real-time genomic surveillance of SARS-CoV-2 was conducted. When the numbers of confirmed cases among overseas travelers increased, public health measures were enhanced by shifting from self-quarantine to central quarantine and SARS-CoV-2 testing for all overseas travelers. In an analysis of 109 imported cases, we found diverse viral variants distributed in the global viral phylogeny, which were frequently shared within households but not among passengers on the same flight. In contrast to the viral diversity of imported cases, local transmission was predominately attributed to two specific variants imported from Africa, including local cases that reported no direct or indirect contact with imported cases. The introduction events of the virus were identified or deduced before the enhanced measures were taken. These results show the interventions were effective in containing the spread of SARS-CoV-2, and they rule out the possibility of cryptic transmission of viral variants from the first wave in January and February 2020. Our study provides evidence and emphasizes the importance of controls for overseas travelers in the context of the pandemic and exemplifies how viral genomic data can facilitate COVID-19 surveillance and inform public health mitigation strategies.

Susceptibility of influenza A(H1N1)/pdm2009, seasonal A(H3N2) and B viruses to Oseltamivir in Guangdong, China between 2009 and 2014.

Nasopharyngeal swabs were collected from patients through the influenza surveillance network of the CDC of Guangdong. All specimens between 2009 and 2014 were checked for influenza virus using MDCK cells and further subtyped. Of those collected, 542 H1N1pdm09, 230 A(H3N2)and 448 B viruses selected at random were subjected to fluorescence-based NAI assays. Viral RNA was extracted from resistant isolates, and their NA genes were amplified by RT-PCR. Alignment of nucleotides and amino acids was performed. We performed structural modelling and simulations of mutants using Modeller 9.x and AutoDock and analyzed conformations and binding affinities. All tested seasonal type B and H3N2 viruses from 2009 to 2014 remained sensitive to oseltamivir. However, there were five strains (out of 198 tested isolates acquired between June and September 2013) that were resistant to oseltamivir. Another three resistant strains were identified among isolates from March to April 2014. We found that 2013/2014 oseltamivir-resistant strains and 2012/2013/2014 oseltamivir-sensitive strains had all or some of the following mutations: N44S, N200S,V241I, I321V,N369K, N386 K and K432E. MutationsV241I, N369K, N386K and K432E, alone or in conjunction with H275Y, had a significant impact on the binding pattern and affinity of oseltamivir for neuraminidase, rendering neuraminidase less susceptible.

Attitudinal changes toward control measures in live poultry markets among the general public and live poultry traders, Guangzhou, China, January-February, 2014.

Two sets of cross-sectional surveys were conducted among the general public and live poultry traders (LPTs) during January-February, 2014, to monitor attitudes toward human cases of avian influenza A(H7N9)-related control measures among these 2 parties in Guangzhou, China. We found generally high support for regular market rest days among the general public and LPTs, but only limited support for permanent central slaughtering of poultry. LPTs' support for relevant control measures declined after the citywide wet market closure.