Identification Of Viral Pathogens Research Articles

Age characteristics and possibilities of laboratory diagnosis of infectious diarrhea in children in the tashkent city

Objective: to study the age characteristics of pathogens of infectious diarrhea in children and evaluate the possibilities of laboratory diagnosis of intestinal infections in the Tashkent city. Material and methods. The study included 360 children aged 6 months to 18 years, hospitalized in Tashkent with a diagnosis of “Acute diarrhea”. Diagnosis of intestinal infection was carried out by bacteriological, immunochromatographic and PCR methods using RIDA®QUICK tests (R - BIOPHARM AG, Germany) and kits from Inter Lab Service “AmpliSens® OKI screen-FL”. Results. Analysis showed that the majority (56.7%) of children with diarrhea belonged to the age group of 3-7 and 7-14 years. In the etiological structure of diarrhea, the main share was occupied by viral and bacterial agents (30% and 45.8%, respectively). In 3.6% of cases parasitic diarrhea was observed and in 20.6% of patients the etiology of diarrhea remained unclear. A comparative evaluation of laboratory methods demonstrated the high efficiency of immunochromatographic and PCR methods compared with bacteriological examination in terms of early diagnosis of the etiological agent of diarrhea and the identification of viral pathogens. Conclusions. In children in the younger age group, diarrhea of viral etiology is more often observed, in contrast to older age groups, where bacterial ones are more common. Immunochromatographic and PCR methods enable earlier diagnosis of the causative agent of diarrhea and, consequently, the start of etiotropic treatment.

The Identification of Viral Pathogens in a Physostegia virginiana Plant Using High-Throughput RNA Sequencing.

Physostegia virginiana is an important ornamental and cut-flower plant in China. Its commonly used method of clonal propagation leads to virus accumulation in this plant. However, which viruses can infect the Physostegia virginiana plant remains to be illuminated. In this work, five viral pathogens in a Physostegia virginiana plant with virus-like symptoms of yellow, shriveled, and curled leaves were identified using RNA-seq, bioinformatics, and molecular biological techniques. These techniques allowed us to identify five viruses comprising one known alfalfa mosaic virus (AMV) and four novel viruses. The novel viruses include a virus belonging to the genus Fabavirus, temporarily named Physostegia virginiana crinkle-associated virus 1 (PVCaV1); two viruses belonging to the genus Caulimovirus, temporarily named Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a virus belonging to the genus Fijivirus, temporarily named Physostegia virginiana fijivirus (PVFV). The genome sequences of PVCaV1, PVCV1, and PVCV2, and the partial genome sequence of PVFV were identified. Genome organizations and genetic evolutionary relationships of all four novel viruses were analyzed. PVCaV1 has a relatively close evolutionary relationship with five analyzed fabiviruses. PVCV1 and PVCV2 have separately a closest evolutionary relationship with lamium leaf distortion-associated virus (LLDAV) and figwort mosaic virus (FMV), and PVFV has a close evolutionary relationship with the five analyzed fijiviruses. Additionally, PVCaV1 can infect Nicotiana benthamiana plants via friction inoculation. The findings enrich our understanding of Physostegia virginiana viruses and contribute to the prevention and control of Physostegia virginiana viral diseases.

Rapid screening and identification of viral pathogens in metagenomic data

BackgroundVirus screening and viral genome reconstruction are urgent and crucial for the rapid identification of viral pathogens, i.e., tracing the source and understanding the pathogenesis when a viral outbreak occurs. Next-generation sequencing (NGS) provides an efficient and unbiased way to identify viral pathogens in host-associated and environmental samples without prior knowledge. Despite the availability of software, data analysis still requires human operations. A mature pipeline is urgently needed when thousands of viral pathogen and viral genome reconstruction samples need to be rapidly identified.ResultsIn this paper, we present a rapid and accurate workflow to screen metagenomics sequencing data for viral pathogens and other compositions, as well as enable a reference-based assembler to reconstruct viral genomes. Moreover, we tested our workflow on several metagenomics datasets, including a SARS-CoV-2 patient sample with NGS data, pangolins tissues with NGS data, Middle East Respiratory Syndrome (MERS)-infected cells with NGS data, etc. Our workflow demonstrated high accuracy and efficiency when identifying target viruses from large scale NGS metagenomics data. Our workflow was flexible when working with a broad range of NGS datasets from small (kb) to large (100 Gb). This took from a few minutes to a few hours to complete each task. At the same time, our workflow automatically generates reports that incorporate visualized feedback (e.g., metagenomics data quality statistics, host and viral sequence compositions, details about each of the identified viral pathogens and their coverages, and reassembled viral pathogen sequences based on their closest references).ConclusionsOverall, our system enabled the rapid screening and identification of viral pathogens from metagenomics data, providing an important piece to support viral pathogen research during a pandemic. The visualized report contains information from raw sequence quality to a reconstructed viral sequence, which allows non-professional people to screen their samples for viruses by themselves (Additional file 1).

Testing for Common Respiratory Viruses in Children Admitted to Pediatric Intensive Care: Epidemiology and Outcomes.

Viral infections are common in children, but there is a lack of data on severe viral infections in critically ill children. We investigated testing for viral infections in children requiring PICU admission and describe the epidemiology and outcomes. Multicenter retrospective study. Results of viral testing for nine respiratory viruses using polymerase chain reaction were collected. Children less than 16 years old nonelectively admitted to PICU over a 6-year period. Two tertiary PICUs in Queensland, Australia. None. Primary outcome was PICU length of stay. Secondary outcomes included need for and duration of intubation and mortality in PICU. Univariate and multivariate regression analyses were performed, adjusting for age, indigenous status, comorbidities, and severity of illness. Of 6,426 nonelective admissions, 2,956 (46%) were polymerase chain reaction tested for a virus of which 1,353 (46%) were virus positive. Respiratory syncytial virus was the most common pathogen identified (n = 518, 33%), followed by rhinovirus/enterovirus and adenovirus. Across all patients who underwent polymerase chain reaction testing, identification of a respiratory virus was not significantly associated with longer overall length of stay (multivariate odds ratio, 1.08; 95% CI, 0.99-1.17; p = 0.068) or longer intubation (p = 0.181), whereas the adjusted odds for intubation and mortality were significantly lower (p < 0.01). Subgroup analyses restricted to patients with acute respiratory infections (n = 1,241), bronchiolitis (n = 761), pneumonia (n = 311), confirmed bacterial infection (n = 345), and malignancy (n = 95) showed that patients positive for a virus on testing had significantly longer PICU length of stay (multivariate p < 0.05). In children with pneumonia, identification of a respiratory virus was associated with significantly increased duration of ventilation (p = 0.003). No association between positive test results for multiple viruses and outcomes was observed. Viral infections are common in critically ill children. Viral infections were associated with lower intubation and mortality rates compared with all children testing negative for viral infections. In several subgroups studied, identification of viral pathogens was associated with longer PICU length of stay while mortality was comparable. Prospective studies are required to determine the benefit of routine testing for respiratory viruses at the time of PICU admission.

Establishment and characterization of a new cell line derived from half-smooth tongue sole Cynoglossus semilaevis kidney

In this study, we established a new cell line (Cynoglossus semilaevis kidney cells, CSK) from kidney of the half-smooth tongue sole (Cynoglossus semilaevis). The cells were subcultured over 1 000 days and passaged for more than 100 times. Additionally, CSK cells were optimally maintained in Dulbecco’s modified Eagle medium nutrient mixture F-12 supplemented with HEPES, antibiotics, fetal bovine serum, 2-mercaptoethanol, and basic fibroblast growth factor. The optimum growth temperature for CSK cells was 25°C, and the cells showed a fibroblast-like phenotype. Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2n=42. CSK cells were susceptible to Grouper nervous necrosis virus (NNV), and cytopathic effects were observed at 3–5 days postinfection. The NNV sensitivity of CSK cells was related to the high abundance of virions in the cytoplasm, as observed by electron microscopy. Additionally, CSK cells could be successfully transfected with a green fluorescent protein reporter plasmid, and fluorescent signals were easily observed. Finally, immunocytochemistry analysis showed that CSK cells were supporting cells. Overall, we established this new cell line, which may have potential applications in the identification of viral pathogens affecting the half-smooth tongue sole.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data

BackgroundNext-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge.MethodsIn order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells.ResultsThe program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes.ConclusionsSearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface.Availability and implementationSearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

In-situ hybridization 법을 사용한 양식 넙치, Paralichthys olivaceus의 바이러스 감염 질병 특성 고찰

PCR (polymerase chain reaction) 법은 신속하고 정확하여 바이러스성 질병진단을 위해 널리 사용되지만 조직병리학적인 정보를 제공하지 못한다. 반면에 in-situ hybridization (ISH) 법을 사용하면 바이러스를 빠르게 검출할 수 있을 뿐 아니라 조직에서의 분포도 알 수 있다. 본 연구에서는 RSIV, VHSV, 그리고 VNN 바이러스들의 조직내 분포 및 조직 병리학적인 특성을 확인하기 위하여 이 바이러스들에 감염된 에 감염된 양식 넙치의 어류의 다양한 조직들을 대상으로 ISH 법을 적용하였다. 그 결과 이들 세 종류의 바이러스가 각각 다른 조직 및 세포들에 감염함을 확인 할 수 있었다. 본 연구 결과는 ISH법이 어류 병원성 바이러스의 신속 검출 뿐 아니라 조직 병리학적인 특성 확인에도 유용함을 제시한다. Polymerase chain reaction (PCR) is the most rapid and widely used method to detect viral pathogens. However, this method does not provide histopathologic nature of the virus. In situ hybridization (ISH) with oligonucleotide probes is attractive because it is a rapid method for detection and identification of viral pathogens at sites of tissue infection. In order to understand the histopathologic characterictics of Red sea bream iridovirus (RSIV), viral-hemorrhagic septicemia (VHS) virus and viral nervous necrosis (VNN) virus to cultured olive flounder, we her applied ISH method to various kinds of olive flounder tissues with PCR-positive for these three viruses. We found that these viruses showed different tissue tropism and were detected from different cell types. Our results suggest that ISH is useful not only in rapid detection of viral pathogens but also in understanding the histopathologic characters of specific viral pathogens.

Detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus PCR, reverse-transcription PCR and electrospray ionization mass spectrometry

BackgroundRapid detection and identification of viruses are important for early diagnosis and effective surveillance of hand, foot, and mouth disease (HFMD). We described a novel assay using multilocus PCR and reverse transcription-PCR coupled with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) to simultaneously detect and identify human enterovirus A–D, adenovirus A–F, human herpesvirus 1–8, parvovirus B19 and polyomavirus. ObjectivesTo evaluate the accuracy and efficacy of the RT-PCR/ESI-MS method, to detect and type enterovirus from specimens of clinical diagnosed HFMD patients. Study designIn this study, 152 specimens of clinically diagnosed HFMD patients were studied by the assay using RT-PCR/ESI-MS method. The detection and typing of enterovirus by RT-PCR/ESI-MS were compared with results from reference molecular methods. ResultsThe assay detected enteroviruses in 97 (63.8%) specimens, resulting in a sensitivity of 93.8% (95% CI: 91.8–95.7%) and a specificity of 87.5% (95% CI: 84.8–90.2%) compared with a reference clinical diagnostic test. Most enterovirus genotypes (65/84; 77%) determined by the platform were consistent with 5′ UTR sequence analysis, and most misidentifications resulted from the virus library, which could be further improved by updating the enterovirus database. In addition to enteroviruses, herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses were detected and identified in 55 (36%) HFMD specimens by RT-PCR/ESI-MS. ConclusionWith the capability of high throughput and detection and typing of multiple clinically relevant viruses simultaneously, RT-PCR/ESI-MS can be a rapid and robust laboratory tool for identifying viral pathogens.

Issues and Updates in Emerging Neurologic Viral Infections

This review discusses the recent advances in the identification of viral pathogens and other etiologies responsible for cases of suspected viral encephalitis. The authors describe new molecular diagnostic strategies for identifying novel causes of viral encephalitis, including MassTag PCR, DNA microarrays, and high-throughput DNA pyrosequencing. They also highlight the increasing recognition of immune-mediated causes of encephalitis among those cases previously thought to be viral encephalitis of unknown etiology. Lastly, they review some of the most recent updates in the field of emerging neurologic viral infections impacting the United States, including the neurologic complications of H1N1 virus and the reemergence of dengue virus in the Florida Keys.

A sample-in-answer-out instrument for the detection of multiple respiratory pathogens in unprepared nasopharyngeal swab samples

Multiplex RT-PCR suspension array assays provide a powerful tool for identifying the causative agent(s) of respiratory infections. These assays are time consuming and laborious on a time-per-sample basis if only a few samples require processing. To address this shortcoming and provide an automated solution for fast detection and identification of viral pathogens, we developed the first automated multiplex RT-PCR suspension array instrument capable of handling unprepared clinical samples. The instrument requires less than 3 minutes of hands-on time for a result generated in approximately 2.5 hours. In analytical studies, the instrument performed as well as manually performed assays. The performance of the instrument and loaded multiplex viral detection assay was then tested using unprepared nasopharyngeal samples. The instrument-performed assay detected 61 of 71 RSV positive samples, for a sensitivity of 85.9%. Adenovirus (n = 5) and influenza B (n = 3) were less prevalent in the sample set, but detected to similar levels, 80% and 75%, respectively. The same sample set was also tested using FDA approved immuno-assay rapid tests, and the instrument was found to be more sensitive than the rapid tests with the sole exception being influenza A (n = 16), which was poorly detected due to significant sequence mismatches between the influenza A primer/probe set included in the multiplex mixture and the circulating influenza A strains. Overall, these data demonstrate the developed prototype platform performs multiplex array assays as well as hand-performed assays, and that the instrument's sensitivity and specificity are dictated by the quality of the loaded multiplex assay.

Viral illness, applied molecular virology and antivirals

The advent of molecular techniques for diagnosis has arguably affected the identification of viral pathogens in illness more than any other segment of pathology, addressing existing problems with timely direct diagnosis of viral pathogens, e.g., viral culture was often lengthy, highly specialised, and insensitive, and there was a relatively low clinical need as antiviral agents were often toxic, infrequently used, and their use was not informed by laboratory assessments. The world-wide human immunodeficiency virus (HIV) and hepatitis C virus (HCV) epidemics and increasing use of antiviral therapy, has driven laboratory surrogate markers of clinical response, particularly quantitation of viral RNA genotype determination, drug-resistance testing particularly for HIV. The techniques now routinely in use for HIV and HCV quantitation have flowed on to laboratory diagnosis of other viruses, particularly cytomegalovirus (CMV), human papillomavirus (HPV) and others. Molecular techniques are typically more sensitive, specific, semi-automatable, and sometimes more rapid. Viral quantitation is increasingly available for more viral pathogens, routinely for HIV, HCV, CMV, and Epstein-Barr virus (EBV). Multiplexing used to detect multiple agents of similar clinical relevance (respiratory viruses such as respiratory syncytial virus [RSV], influenza, adenovirus, and parainfluenza virus [PIV] 1,2,3) is now routinely in use in several laboratories, and commercial assays are available. Commercial assays available for quantitating HIV and HCV include RTPCR (Roche, in-house assays), bDNA (Siemens-Bayer), NASBA (Biomerieux), real-time RTPCR (Roche Taqman, Biomerieux EasyQ, Abbott Celerus) and many more are under development. Techniques improving throughput include automation of nucleic acid preparation and cycling, pooled targets (e.g., ARCBS HIV/HCV), use of new targets for diagnosis, and determination of resistance genotypes to direct therapy. Effective HIV and HCV treatment, with emergence of resistance to antivirals, has driven development of NAT targeting mutation sites associated with resistance, and software using sequence information to derive virtual phenotypes, processes now used for determining resistance of viruses, including influenza (H1N1 strains in the 2008 season), herpes simplex virus (HSV;immunocompromised patients on long term aciclovir), CMV (foscarnet and ganciclovir), hepatitis B virus (HBV) and less frequently varicella zoster virus (VZV). The era of assays that are highly specific (such as PCR), brought together in a multiplex format to answer specific questions is upon us, and design of such assays for individual patients (personalised medicine) is likely becoming a reality. Driving the use of such assays will be reductions in cost that are already occurring, and most importantly, clinical need.

Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5′ biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain

The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), Aujeszky’s disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease.

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5′ ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.

Molecular Methods for the Detection and Identification of Viral Pathogens

Abstract This article addresses the use of nucleic acid amplification technology, specifically polymerase chain reaction (PCR), for the detection of viral nucleic acids in tissue specimens. Two different approaches are being used for the detection of viral DNA in tissue by PCR, solution PCR and in situ PCR (ISPCR). Solution PCR is the classic form of the procedure that uses DNA isolated from cells or tissue as target. ISPCR is where target DNA amplification occurs in the cell or tissue and the amplified product is visualized within the context of the intact specimen.Solution PCR is a very powerful technique for detection of viruses in tissue. While solution PCR does not afford target localization capability, it is a well researched and highly utilized procedure that can be readily adapted to routine use in a clinical laboratory. Unfortunately, the inherent difficulties associated with ISPCR obviates its use in the clinical laboratory. At present, ISPCR is in the developmental stages and its use should be ...

The Diagnosis of Viral Disease by Electron Microscopy

Recent years have brought a reversal in the attitude of the medical profession toward the diagnosis of viral infections. Identification of bacterial pathogens was formerly thought to be faster than identification of viral pathogens. Viral identification was dismissed as being of academic interest or for confirming the presence of an epidemic, because the patient would recover or die before this could be accomplished. In the past 10 years, the goal of virologists has been to present the clinician with a viral identification in a matter of hours. This fast diagnosis has the potential for shortening the patient's hospital stay and preventing the administering of toxic and/or expensive antibiotics of no benefit to the patient.