OBJECTIVE: Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease. METHODS: A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay. RESULTS: The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay. CONCLUSION A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.
Encephalitis Viruses, Tick-Borne ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; RNA, Viral ; analysis

OBJECTIVEUnbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.

METHODSA viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.

RESULTSA minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.

CONCLUSIONThe VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

Base Sequence ; Genome, Viral ; High-Throughput Nucleotide Sequencing ; Humans ; Nucleic Acid Amplification Techniques ; methods ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; Virus Diseases ; diagnosis ; virology ; Viruses ; isolation & purification

One unusual human G3P[3] group A rotavirus (RVA) strain M2-102 was identified in stool sample collected from a child with diarrhea in Guangxi Province, China in 2014. It is well known that G3P[3] is a genotype commonly identified in feline and canine RVAs. However, the preliminary phylogenetic analyses of the VP7 and VP4 genes of strain M2-102 indicated that these two genes were closely related to bat RVA strain MYAS33 and simian strain RRV, respectively, whereas both clustered distantly to feline/canine-like RVA strains. In this study, full genome sequencing and molecular analyses were conducted to obtain the true origin of strain M2-102. It was revealed that strain RVA/Human-wt/CHN/M2-102/2014/G3P[3] exhibited a G3-P[3]-I3-R3-C3-M3-A9-N3-T3-E3-H6 genotype constellation for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes. Phylogenetic analyses revealed that 5 genes (VP7, VP1, VP2, NSP2 and NSP3) from strain M2-102 were closely related to those of bat strain MYAS33 from Yunnan Province which was thought a true bat RVA strain rather than a virus transmitted between species, while another 5 genes (VP4, VP3, NSP1, NSP4 and NSP5) clustered closely with those of simian strain RRV, yet the VP6 gene was closely related to that of human G3P[9] strain AU-1 and AU-1-like RVAs. The epidemiological data indicated that the child infected with M2-102 came from a countryside village, located in Dong Autonomous County of Sanjiang (subtropical hilly wooded area), Liuzhou city in Guangxi Province which might provide natural environment for reassortment events occurring among animal and human RVAs. Therefore, the data suggest that human strain M2-102 might originate from multiple reassortment events among bat, simian and human AU-1-like RVAs, yet it is not clear whether the genomic backbone based on bat MYAS33 (5 genes) and simian RRV (5 genes) like rotaviruses had been obtained through reassortment before being transmitted to the human. This is the first report on whole genome analysis of human G3P[3] RVA from China.
Child, Preschool ; China ; Genome, Viral ; Genomics ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Reassortant Viruses ; classification ; genetics ; isolation & purification ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Infections ; virology ; Viral Proteins ; genetics

Until the recent emergence/re-emergence of human-pathogenic viruses in ticks, tick-borne viruses have been neglected as causative agents of human disease (particularly in China). To gain insight into the diversity of tick-borne viruses in Xinjiang Uygur Autonomous Region (northwestern China), we conducted illumina deep sequencing-based screening for virus-derived small RNAs in field-collected Ixodes persulcatus ticks. We found 32, 631 unique virus-matched reads. In particular, 77 reads mapped to the tick-borne group within the genus of Flavivirus, and covered 3.8%-2.4% viral genomes. In addition, 32 unique reads were specific to the Siberian subtype of tick-borne encephalitis viruses (TBEV-Sib) which have never been reported in Chinese TBE loci. We confirmed the potential existence of TBEV-Sib by amplification (using reverse transcription-polymerase chain reaction) of genomic fragments from the envelope gene or 3' genomic terminus from the pools of examined ticks. Both sequences demonstrated high homology to TBEV-Sib strains attached geographically to southern Siberia with nucleotide identity of 97.2%-95.5% and aminoacid identity of 99.4%-98.3%, respectively. In conclusion, we report, for the first time, detection of TBEV-Sib in the natural TBE loci of China. These novel data may provide genetic information for further isolation and epidemiologic investigation of TBEV-Sib.
Animals ; Arachnid Vectors ; virology ; China ; Encephalitis Viruses, Tick-Borne ; classification ; genetics ; isolation & purification ; Encephalitis, Tick-Borne ; transmission ; virology ; Genome, Viral ; Humans ; Ixodes ; virology ; Molecular Sequence Data ; Phylogeny

To understand the potential causes of laboratory-acquired infections and to provide possible solutions that would protect laboratory personnel, samples from a viral laboratory were screened to determine the main sources of contamination with six subtypes of Rhinovirus. Rhinovirus contamination was found in the gloves, cuffs of protective wear, inner surface of biological safety cabinet (BSC) windows, and trash handles. Remarkably, high contamination was found on the inner walls of the centrifuge and the inner surface of centrifuge tube casing in the rotor. Spilling infectious medium on the surface of centrifuge tubes was found to contribute to contamination of centrifuge surfaces. Exposure to sodium hypochlorite containing no less than 0.2 g/L available chlorine decontaminated the surface of the centrifuge tubes from Rhinovirus after 2 min.
Equipment Contamination ; statistics & numerical data ; Humans ; Laboratories, Hospital ; manpower ; standards ; statistics & numerical data ; Occupational Exposure ; analysis ; statistics & numerical data ; Virus Diseases ; virology ; Viruses ; genetics ; growth & development ; isolation & purification

OBJECTIVES: An outbreak of acute febrile illness occurred in the Republic of Korea Air Force boot camp from May to July 2011. An epidemiological investigation of the causative agent, which was of a highly infective nature, was conducted. METHODS: Throat swabs were carried out and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed to identify possible causative factors. RESULTS: The mean age of patients who had febrile illness during the study period was 20.24 years. The multiplex RT-PCR assay identified respiratory syncytial virus (RSV) as the causative agent. The main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%), and rhinorrhea (55.9%). The mean temperature was 38.75degreesC and the attack rate among the recruits was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. The prognosis was generally favorable with supportive care but recurrent fever occurred in 10.1% of the patients within a month. CONCLUSIONS: This is the first epidemiological study of an RSV outbreak that developed in a healthy young adult group. In the event of an outbreak of an acute febrile illness of a highly infective nature in facilities used by a young adult group, RSV should be considered among the possible causative agents.
Adolescent ; Adult ; Antiviral Agents/therapeutic use ; Body Temperature ; Disease Outbreaks ; Humans ; Male ; Military Personnel ; Multiplex Polymerase Chain Reaction ; Oseltamivir/therapeutic use ; Pharynx/virology ; RNA, Viral/chemistry/genetics/metabolism ; Republic of Korea/epidemiology ; Respiratory Syncytial Virus Infections/drug therapy/*epidemiology/virology ; Respiratory Syncytial Viruses/*genetics/isolation & purification ; Sputum/virology ; Young Adult

Small RNAs (sRNA) are produced abundantly in either plants or animals and function in regulating gene expression or in defense against virus infection. Deep sequencing of small RNAs is an emerging technology in virus identification and de novo assembly of virus genomes and is demonstrated to be an effective method to discover new viruses and monitor virus variation. A significant number of viruses from plants, invertebrates and human cells has been successfully identified using this technology. In this paper, we summarized the principle, operation process and latest advances of sRNA deep sequencing We also showed the feasibility of sRNA deep sequencing by bioinformatic analysis using sRNA deep sequencing dataset public available for the detection of viruses.
Genomics ; High-Throughput Nucleotide Sequencing ; methods ; RNA, Small Untranslated ; genetics ; RNA, Viral ; genetics ; Sequence Analysis, RNA ; methods ; Viruses ; genetics ; isolation & purification

In nature, honeybees are the most important pollinators. They play a vital role in both protecting the diversity of natural ecosystems, and maintaining the yield-improving effects of agroecosystems. But in recent years, epidemic disease in bees has caused huge losses. Black Queen Cell Virus (BQCV) is a bee pathogen that was first reported in 1955. It mainly infects bee larvae and pupae, making their bodies turn dark and black, and causing a massive decrease in the bee population. More specifically, the virus makes the exterior of the cell walls in the larvae and pupae turn black. BQCV is a seasonal epidemic, spread by means horizontal and vertical transmission, and is often unapparent. BQCV not only infects a variety of bee species, but also spiders, centipedes and other arthropods. It can also be coinfected with other honeybee viruses. In recent years, research has shown that the Nosema intestinal parasite plays an important role in BQCV transmission and bees carrying Nosema that become infected with BQCV have increased mortality. Here we summarize current research on the incidence, prevalence, geographical distribution and transmission of BQCV.
Animals ; Bees ; virology ; Dicistroviridae ; classification ; genetics ; isolation & purification ; physiology ; Insect Viruses ; classification ; genetics ; isolation & purification ; physiology

Respiratory virus poses a serious threat to human life and health. Airway epithelial cells are the body's first line of defense from a wide variety of foreign pathogens, such as viruses and bacteria. Therefore, successful airway epithelial cell culture can provide a model for investigating the mechanisms underlying respiratory pathogenic diseases following airway virus infection. This respiratory disease model can also be used for the potential development of novel therapeutics. Here we provide a brief review of recent developments on the culture of cells derived from human trachea-bronchial airway epithelium, and the application of this model for studying respiratory virus and disease.
Animals ; Cell Culture Techniques ; Epithelial Cells ; virology ; Humans ; Respiratory Tract Diseases ; virology ; Virus Diseases ; virology ; Virus Physiological Phenomena ; Viruses ; genetics ; isolation & purification

Honeybee pupae were collected from Jilin apiaries and RNA was extracted for use as a tefnplate for amplification. Based on the complete genome sequences of black queen cell virus (BQCV) published on GenBank, we designed 10 pairs of primers to amplify genes by reverse transcription-polymerase chain reaction (RT-PCR). Using this approach, we have obtained the first complete genome sequence of a BQCV isolate in China. The genome of the isolated strain, named BQCV-JL1, is composed of 8358 nucleotides and shares between 86% and 93% homology with the complete genome sequences of the other six BQCV strains published on GenBank. ORF 1 of BQCV-JL1 is positioned between nucleot ides (nt) 546 and 4676 (4131 nt), while ORF 2 is located between nt 5750 and 8203. Between the two ORFs of BQCV-JL1 there is a short ORF, called ORF 3, between nt 4891 and 5433 (543 nt). The first functional gene ex- pression domain of the BQCV-JL1 strain is positioned between nt 546 and 5 429, encompassing both ORF 1 and ORF 3. There is an internal ribosome entry site (IRES) located before ORF 2, the last three bases of which are CCU (nt 5642-5644). These bases act as an initiation.codon facilitating the translation of ORF 2. The second functional gene expression domain of the BQCV-JL1 strain is located between nt positions 5642 and 8203. The BQCV-JL1 strain was found to share high sequence identity (93%) with the Hungary 10 genotype at the whole-genome level and analysis of the nucleotide and amino acid sequences revealed that the BQCV-JL1 strain also shows close genetic relationships with the South Korea strain, suggesting that both the BQCV-JL1 and South Korea strains may have migrated from European countries. BQCV-JL1 strain was different from the other 6 strains in dividing the nucleotides positions of QRF, which vqs because of the gene mutation.
Amino Acid Sequence ; Animals ; Bees ; China ; Genome, Viral ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; RNA Viruses ; classification ; genetics ; isolation & purification ; Viral Proteins ; genetics