Overcoming taxonomic challenges in DNA barcoding for improvement of identification and preservation of clariid catfish species

DNA barcoding without assessing reliability and validity causes taxonomic errors of species identification, which is responsible for disruptions of their conservation and aquaculture industry. Although DNA barcoding facilitates molecular identification and phylogenetic analysis of species, its availability in clariid catfish lineage remains uncertain. In this study, DNA barcoding was developed and validated for clariid catfish. 2,970 barcode sequences from mitochondrial cytochrome c oxidase I (COI) and cytochrome b (Cytb) genes and D-loop sequences were analyzed for 37 clariid catfish species. The highest intraspecific nearest neighbor distances were 85.47%, 98.03%, and 89.10% for COI, Cytb, and D-loop sequences, respectively. This suggests that the Cytb gene is the most appropriate for identifying clariid catfish and can serve as a standard region for DNA barcoding. A positive barcoding gap between interspecific and intraspecific sequence divergence was observed in the Cytb dataset but not in the COI and D-loop datasets. Intraspecific variation was typically less than 4.4%, whereas interspecific variation was generally more than 66.9%. However, a species complex was detected in walking catfish and significant intraspecific sequence divergence was observed in North African catfish. These findings suggest the need to focus on developing a DNA barcoding system for classifying clariid catfish properly and to validate its efficacy for a wider range of clariid catfish. With an enriched database of multiple sequences from a target species and its genus, species identification can be more accurate and biodiversity assessment of the species can be facilitated.

Genome characterization and mutation analysis of human influenza A virus in Thailand

The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/ H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in hemagglutinin (HA) and neuraminidase (NA) genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, HA, and NA genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.

Metagenomic analysis of viral genes integrated in whole genome sequencing data of Thai patients with Brugada syndrome

Brugada syndrome (BS) is an autosomal dominant inheritance cardiac arrhythmia disorder associated with sudden death in young adults. Thailand has the highest prevalence of BS worldwide, and over 60% of patients with BS still have unclear disease etiology. Here, we performeda new viral metagenome analysis pipeline called VIRIN and validated it with whole genome sequencing (WGS) data of HeLa cell lines and hepatocellular carcinoma. Then the VIRIN pipelinewas applied to identify viral integration positions from unmapped WGS data of Thai males, including 100 BS patients (case) and 100 controls. Even though the sample preparation had noviral enrichment step, we can identify several virus genes from our analysis pipeline. The predominance of human endogenous retrovirus K (HERV-K) viruses was found in both cases andcontrols by blastn and blastx analysis. This study is the first report on the full-length HERV-Kassembled genomes in the Thai population. Furthermore, the HERV-K integration breakpointpositions were validated and compared between the case and control datasets. Interestingly,Brugada cases contained HERV-K integration breakpoints at promoters five times more oftenthan controls. Overall, the highlight of this study is the BS-specific HERV-K breakpoint positionsthat were found at the gene coding region "NBPF11" (n = 9), "NBPF12" (n = 8) and longnon-coding RNA (lncRNA) "PCAT14" (n = 4) region. The genes and the lncRNA have been reported to be associated with congenital heart and arterial diseases. These findings provide another aspect of the BS etiology associated with viral genome integrations within the humangenome.

Comparative genome characterization of Leptospira interrogans from mild and severe leptospirosis patients

Leptospirosis is a zoonotic disease caused by spirochetes from the genus Leptospira. In Thailand, Leptospira interrogans is a major cause of leptospirosis. Leptospirosis patients present with a wide range of clinical manifestations from asymptomatic, mild infections to severe illness involving organ failure. For better understanding the difference between Leptospira isolates causing mild and severe leptospirosis, illumina sequencing was used to sequence genomic DNA in both serotypes. DNA of Leptospira isolated from two patients, one with mild and another with severe symptoms, were included in this study. The paired-end reads were removed adapters and trimmed with Q30 score using Trimmomatic. Trimmed reads were constructed to contigs and scaffolds using SPAdes. Cross-contamination of scaffolds was evaluated by ContEst16s. Prokka tool for bacterial annotation was used to annotate sequences from both Leptospira isolates. Predicted amino acid sequences from Prokka were searched in EggNOG and David gene ontology database to characterize gene ontology. In addition, Leptospira from mild and severe patients, that passed the criteria e-value < 10e-5 from blastP against virulence factor database, were used to analyze with Venn diagram. From this study, we found 13 and 12 genes that were unique in the isolates from mild and severe patients, respectively. The 12 genes in the severe isolate might be virulence factor genes that affect disease severity. However, these genes should be validated in further study.

Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR

BACKGROUND: Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. METHODS: Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). RESULTS: Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. CONCLUSIONS: Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations.

Erythrocyte Binding Preference of Human Pandemic Influenza Virus A and Its Effect on Antibody Response Detection

BACKGROUND: Validation of hemagglutination inhibition (HI) assays is important for evaluating antibody responses to influenza virus, and selection of erythrocytes for use in these assays is important. This study aimed to determine the correlation between receptor binding specificity and effectiveness of the HI assay for detecting antibody response to pandemic influenza H1N1 (pH1N1) virus. METHODS: Hemagglutination (HA) tests were performed using erythrocytes from 6 species. Subsequently, 8 hemagglutinating units of pH1N1 from each species were titrated by real-time reverse transcription-PCR. To investigate the effect of erythrocyte binding preference on HI antibody titers, comparisons of HI with microneutralization (MN) assays were performed. RESULTS: Goose erythrocytes showed most specific binding with pH1N1, while HA titers using human erythrocytes were comparable to those using turkey erythrocytes. The erythrocyte binding efficiency was shown to have an impact on antibody detection. Comparing MN titers, HI titers using turkey erythrocytes yielded the most accurate results, while those using goose erythrocytes produced the highest geometric mean titer. Human blood group O erythrocytes lacking a specific antibody yielded results most comparable to those obtained using turkey erythrocytes. Further, pre-existing antibody to pH1N1 and different erythrocyte species can distort HI assay results. CONCLUSIONS: HI assay, using turkey and human erythrocytes, yielded the most comparable and applicable results for pH1N1 than those by MN assay, and using goose erythrocytes may lead to overestimated titers. Selection of appropriate erythrocyte species for HI assay allows construction of a more reliable database, which is essential for further investigations and control of virus epidemics.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

OBJECTIVE@#To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV) infection.@*METHODS@#We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.@*RESULTS@#The assay's sensitivity was 97.65%, specificity was 92.59% and accuracy was 95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction between CHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.@*CONCLUSIONS@#This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Novel H1N1 2009 influenza virus infection in Bangkok, Thailand:effects of school closures

Background: Influenza virus is the major cause of respiratory illness, especially in young and older age groups. Since 1918, many subtypes, defined by hemagglutinine (HA) and neuraminidase (NA), have caused global infections or pandemics. The recently isolated swine origin influenza virus (S-OIV) subtype H1N1 has been defined by the World Health Organization (WHO) as the cause of the present influenza pandemic. Objective: To describe and attempt to predict the epidemiology of the novel H1N1 2009 in Bangkok and to evaluate the effects of school closures during the outbreaks. Materials and methods: The first two human cases infected by this S-OIV subtype H1N1 or H1N1 2009 in Thailand have been reported in May 12, 2009 by the Ministry of Public Health. Between May 12 and July 30, 2009, 1212 nasopharyngeal (NP) swabs from four private hospitals and Chulalongkorn Hospital, Bangkok have been sent to a laboratory for Influenza virus diagnosis. The diagnosis was based on real time RT-PCR for seasonal influenza (H1, H3) and H1N1 2009. Results: One thousand two hundreds and twelve specimens of patients with influenza like illness were tested using real time RT-PCR detection. Between mid June and early July, the number of H1N1 2009 increased rapidly with a high prevalence among the 6- to 20-year olds. School closure policy, long public holiday, and additional preventive measures have led to a rapid reduction in the number of H1N1 2009 positive patients. Conclusion: Preventive measures including school closures are important to slow down the outbreak and thus enable health care centers to cope with the large number of patients. Everyone should play a role in delaying the spread of this pandemic.