NanoSTR: A method for detection of target short tandem repeats based on nanopore sequencing data.

Short tandem repeats (STRs) are widely present in the human genome. Studies have confirmed that STRs are associated with more than 30 diseases, and they have also been used in forensic identification and paternity testing. However, there are few methods for STR detection based on nanopore sequencing due to the challenges posed by the sequencing principles and the data characteristics of nanopore sequencing. We developed NanoSTR for detection of target STR loci based on the length-number-rank (LNR) information of reads. NanoSTR can be used for STR detection and genotyping based on long-read data from nanopore sequencing with improved accuracy and efficiency compared with other existing methods, such as Tandem-Genotypes and TRiCoLOR. NanoSTR showed 100% concordance with the expected genotypes using error-free simulated data, and also achieved >85% concordance using the standard samples (containing autosomal and Y-chromosomal loci) with MinION sequencing platform, respectively. NanoSTR showed high performance for detection of target STR markers. Although NanoSTR needs further optimization and development, it is useful as an analytical method for the detection of STR loci by nanopore sequencing. This method adds to the toolbox for nanopore-based STR analysis and expands the applications of nanopore sequencing in scientific research and clinical scenarios. The main code and the data are available at https://github.com/langjidong/NanoSTR.

Nano2NGS-Muta: a framework for converting nanopore sequencing data to NGS-liked sequencing data for hotspot mutation detection.

Nanopore sequencing, also known as single-molecule real-time sequencing, is a third/fourth generation sequencing technology that enables deciphering single DNA/RNA molecules without the polymerase chain reaction. Although nanopore sequencing has made significant progress in scientific research and clinical practice, its application has been limited compared with next-generation sequencing (NGS) due to specific design principle and data characteristics, especially in hotspot mutation detection. Therefore, we developed Nano2NGS-Muta as a data analysis framework for hotspot mutation detection based on long reads from nanopore sequencing. Nano2NGS-Muta is characterized by applying nanopore sequencing data to NGS-liked data analysis pipelines. Long reads can be converted into short reads and then processed through existing NGS analysis pipelines in combination with statistical methods for hotspot mutation detection. Nano2NGS-Muta not only effectively avoids false positive/negative results caused by non-random errors and unexpected insertions-deletions (indels) of nanopore sequencing data, improves the detection accuracy of hotspot mutations compared to conventional nanopore sequencing data analysis algorithms but also breaks the barriers of data analysis methods between short-read sequencing and long-read sequencing. We hope Nano2NGS-Muta can serves as a reference method for nanopore sequencing data and promotes higher application scope of nanopore sequencing technology in scientific research and clinical practice.

Pathogenicity and molecular characterization of a fowl adenovirus 4 isolated from chicken associated with IBH and HPS in China.

BACKGROUND: Since July in 2015, an emerging infectious disease, Fowl adenovirus (FAdV) species C infection with Hepatitis-Hydropericardium syndrome was prevalent in chicken flocks in China. In our study, one FAdV strain was isolated from commercial broiler chickens and was designated as SDSX1.The phylogenetic information, genetic mutations and pathogenicity of SDSX1 were evaluated. RESULTS: The phylogenetic analysis indicated that SDSX1 is a strain of serotype 4, FAdV-C. The amino acid analysis of fiber-2 showed that there were more than 20 mutations compared with the non-virulent FAdV-C strains. The pathogenic evaluation of SDSX1 showed that the mortality of one-day-old chickens inoculated SDSX1 was 100%. The typical histopathological changes of SDSX1 were characterized by the presence of basophilic intranuclear inclusion bodies in hepatocytes. The virus copies in different tissues varied from107 to 1011 per 100 mg tissue and liver had the highest virus genome copies. CONCLUSION: In conclusion, the isolate SDSX1, identified as FAdV-4, could cause one-day-old chicks' typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were the highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

Development and application of an indirect ELISA for the detection of antibodies against encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. It has been recognized worldwide as a pathogen infecting many species and causes substantial economic losses. In the present study, an indirect ELISA was developed for the detection of antibodies to EMCV. The VP1 gene of EMCV was amplified by reverse transcription-quantitative polymerase chain reaction and expressed in Escherichia coli with 49.3 kDa under the condition of isopropyl-ß-d-thiogalactoside. Following this, the authors obtained the recombinant protein VP1 as a coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. Compared with viral neutralization tests, the sensitivity and specificity of the indirect ELISA was 95.7% and 92.9%, respectively. A total of 265 clinical swine serum samples from different pig farms in China were used to a serological survey. The seropositive rate of the serum samples was 81.9%. In conclusion, the developed indirect ELISA assay is sensitive and specific, which will be useful for large-scale serological survey in EMCV infection and monitoring antibodies titers against EMCV.

Complete Genomic Characterization of Porcine Reproductive and Respiratory Syndrome Virus Strain HB-XL.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causal agent of a serious disease of swine. Here, we report the genome sequence of PRRSV strain HB-XL isolated from a pig farm with a clinical outbreak of porcine reproductive and respiratory syndrome. The genome is 15,323 bp long and has nine open reading frames (GenBank: KP162169). Comparative and phylogenetic analysis showed that HB-XL belongs to the highly pathogenic PRRSV (HP-PRRSV) subfamily in the family PRRSV. The viral nonstructural protein 2 (Nsp2) of the HB-XL strain contained 30 discontinuous amino acid (AA) deletions relative to that of the Nsp2 of the VR2332 strain. The AA substitutions R13 and R151 suggested high virulence of the HB-XL strain. The unique mutations in glycoprotein 5 (GP5) and Nsp2 revealed that HB-XL might be a novel variant PRRSV strain recombined with vaccine strains. However, the low morbidity and mortality in the pig herd from which HB-XL was isolated indicate that the virulence of the virus was weak, so it has potential as a future vaccine strain.

Development of a nanoparticle-assisted PCR assay for detection of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is an important pig pathogen that can cause vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. In this study, a nanoparticle-assisted polymerase chain reaction (nanoPCR) assay targeting the N gene of PEDV was developed and the sensitivity and specificity were investigated. Under the optimized conditions for detection of PEDV RNA, the nanoPCR assay was 100-fold more sensitive than a conventional RT-PCR assay. The lower detection limit of the nanoPCR assay was 2.7 × 10(-6) ng/µL of PEDV RNA and no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nanoPCR assay for the detection of PEDV. The sensitive and specific nanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of PEDV-infection.

Complete Genome Sequence of Porcine Circovirus Type 2 Strain HB-MC1.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Here, we report the complete genome sequence of PCV2 strain HB-MC1, which belongs to PCV2d.

Development of a TaqMan-based real-time reverse transcription polymerase chain reaction assay for the detection of encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) is one of the major zoonosis pathogens and can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, a TaqMan-based real-time reverse transcription polymerase chain reaction (RT-PCR) assay targeting 3D gene of EMCV was developed and their sensitivities and specificities were investigated. The results indicated that the standard curve had a wide dynamic range (10(1)-10(6) copies/µL) with a linear correlation (R(2)) of 0.996 between the cycle threshold (Ct) value and template concentration. The real-time RT-PCR assay is highly sensitive and able to detect 1.4×10(2) copies/µL of EMCV RNA, as no cross-reaction was observed with other viruses. These data suggested that the real-time RT-PCR assay developed in this study will be suitable for future surveillance and specific diagnosis of EMCV-infection.

Distinct promoters affect pyrroloquinoline quinone production in recombinant Escherichia coli and Klebsiella pneumoniae.

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters-lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)-were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.

Rapid detection of encephalomyocarditis virus by one-step reverse transcription loop-mediated isothermal amplification method.

The encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect EMCV RNA. The RT-LAMP assay was highly sensitive and able to detect 2.2 × 10(-5)ng of EMCV RNA, as no cross-reaction was observed with other viruses. The RT-LAMP assay was conducted in isothermal (62 °C) conditions within 50 min. The amplified products of EMCV could be detected as ladder-like bands using agarose gel electrophoresis. This is the first report to demonstrate the application of a one-step RT-LAMP assay for the detection of EMCV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in clinical diagnosis and field surveillance of EMCV.

Isolation and molecular analysis of porcine encephalomyocarditis virus strain BD2 from northern China.

Encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, an EMCV strain (BD2) was isolated from the suspected piglets with EMCV in China. It was identified by an indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. The virus could reproduce on BHK-21 cells and reach a peak titer by 16 hpi with maximum titers of 10(8.22)TCID50/0.1 ml. Phylogenetic analyses of open reading frame and the VP3/VP1 genes using neighbor-joining method revealed that EMCV isolates fell into two clusters: groups I and II. The BD2 isolate belonged to group I, along with strains NJ08, HB1, BJC3, CBNU, K3, K11, BEL-2887A, GX0601, GXLC, pEC9, and PV21, whereas four strains (D variant, EMCV-B, EMCV-D, and PV2) belonged to group II.

[Construction and characterization of EGFP reporter gene labeled Sindbis virus].

OBJECTIVE: To construct and characterize EGFP reporter gene labeled Sindbis virus (SINV). METHODS: The reporter gene EGFP was inserted into the genome of infectious clone pBR-XJ160 by using multi-fusion long fragment PCR method. Then apply reverse genetic manipulation technique to rescue and obtain EGFP labeled SINV. RESULTS: We successively obtained labeled SINV, which has good fluorescent expression characteristics and genetic stability. CONCLUSION: The labeled virus can be seen in living cells and living body, and this serves as a good tool for cell and tissue tropism and biological function study of viruses. This study laid a foundation for further studying the cell tropism, biological functions and infection mechanism of SINV.

Complete Genome Sequence of Porcine Encephalomyocarditis Virus Strain BD2.

Encephalomyocarditis virus (EMCV) causes acute myocarditis in young pigs or reproductive failure in sows, and it is divided into two main groups. Here, we report the complete genome sequence of EMCV strain BD2, which belongs to group I.