Method For Detection Of Virus Research Articles

Graphene oxide based electrochemical immunosensor for rapid detection of groundnut bud necrosis orthotospovirus in agricultural crops

Groundnut bud necrosis orthotospovirus (GBNV) is one of the causative plant viruses responsible for the outbreak of many viral epidemics in food crops across India and other south-Asian countries. Its management is a major challenge due to fast vector transmission, and the non-availability of appropriate agrochemical treatment. The timely detection of GBNV becomes indispensable for the effective management of viral infection and the periodic monitoring of plant health. We report the fabrication of graphene oxide (GO) based electrochemical immunosensor for the rapid and sensitive detection of GBNV. The immunoelectrode is prepared by depositing GO onto indium-tin oxide (ITO) coated glass substrates and functionalized by anti-GBNV antibodies using N-ethyl-N′-(3- dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) conjugation chemistry. The response measurements of the immunoelectrodes revealed a sensitivity of 221 ± 1 μA μg−1 mL−1(n = 3) and limit of detection (LOD) of 5.7 ± 0.7 ng mL−1(n = 3) for the standard concentrations of GBNV antigen. Further, the GBNV detection was carried out in infected leaf extracts of three different host plants i.e., Tomato, Cowpea, and N. benthamiana, and the results have been compared with the conventionally used direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) technique. The comparable results obtained for the detection of GBNV in infected plants using electrochemical immunosensing and DAC-ELISA techniques advocated the immense potential of GO based immunosensor as a point-of-care sensing device that is poised to overcome the limitations of the traditional methods of virus detection in field conditions and may transform the diagnostics in agriculture.

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples.

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.

Methods of Respiratory Virus Detection: Advances towards Point-of-Care for Early Intervention.

Respiratory viral infections threaten human life and inflict an enormous healthcare burden worldwide. Frequent monitoring of viral antibodies and viral load can effectively help to control the spread of the virus and make timely interventions. However, current methods for detecting viral load require dedicated personnel and are time-consuming. Additionally, COVID-19 detection is generally relied on an automated PCR analyzer, which is highly instrument-dependent and expensive. As such, emerging technologies in the development of respiratory viral load assays for point-of-care (POC) testing are urgently needed for viral screening. Recent advances in loop-mediated isothermal amplification (LAMP), biosensors, nanotechnology-based paper strips and microfluidics offer new strategies to develop a rapid, low-cost, and user-friendly respiratory viral monitoring platform. In this review, we summarized the traditional methods in respiratory virus detection and present the state-of-art technologies in the monitoring of respiratory virus at POC.

Biosensor as an alternative diagnostic method for rabies virus detection: A literature review.

Rabies virus as a neurotropic agent causes rabies in humans and animals. Rabies virus transmission usually occurs through direct contact with saliva of rabid animals. However, serological and molecular tests commonly are used in diagnosing rabies but all the detection methods of rabies have some limitations. It is necessary to develop a rapid, effective, and low-cost biosensor as an alternative tool to detect rabies virus. In this review, we studied related biosensor researches to rabies virus detection for comparing it with other detection test including serological and molecular methods. Given that very limited studies have been conducted in this field, biosensors as quick, effective, and high sensitivity tools can be used in diagnostic of rabies as an alternative tool instead of other detection methods. According to the important role of rapid detection of rabies in the control of infection and public health measures, development of a biosensor as a quick tool can be very significant in the diagnosis of rabies.

Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.

SARS-CoV-2 RNA screening in routine pathology specimens.

SummaryVirus detection methods are important to cope with the SARS‐CoV‐2 pandemics. Apart from the lung, SARS‐CoV‐2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom‐indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT‐PCR protocol for the detection of SARS‐CoV‐2 RNA in patients’ routine diagnostic formalin‐fixed and paraffin‐embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID‐19‐positive patients, (ii) unrecognized cases in selected tissues from negative or not‐tested patients during a pandemic peak, and (iii) retrospectively, pre‐pandemic lung samples. We identified SARS‐CoV‐2 RNA in seven samples from confirmed COVID‐19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID‐19 case in tonsillectomy samples. All pre‐pandemic lung samples were negative. In conclusion, SARS‐CoV‐2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID‐19, allow retrospective studies, and advance our understanding of SARS‐CoV‐2 organ tropism and effects.

A series of COVID-19 autopsies with clinical and pathologic comparisons to both seasonal and pandemic influenza.

Autopsies of patients who have died from COVID‐19 have been crucial in delineating patterns of injury associated with SARS‐CoV‐2 infection. Despite their utility, comprehensive autopsy studies are somewhat lacking relative to the global burden of disease, and very few comprehensive studies contextualize the findings to other fatal viral infections. We developed a novel autopsy protocol in order to perform postmortem examinations on victims of COVID‐19 and herein describe detailed clinical information, gross findings, and histologic features observed in the first 16 complete COVID‐19 autopsies. We also critically evaluated the role of ancillary studies used to establish a diagnosis of COVID‐19 at autopsy, including immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy (EM). IHC and ISH targeting SARS‐CoV‐2 were comparable in terms of the location and number of infected cells in lung tissue; however, nonspecific staining of bacteria was seen occasionally with IHC. EM was unrevealing in blindly sampled tissues. We then compared the clinical and histologic features present in this series to six archival cases of fatal seasonal influenza and six archival cases of pandemic influenza from the fourth wave of the ‘Spanish Flu’ in the winter of 1920. In addition to routine histology, the inflammatory infiltrates in the lungs of COVID‐19 and seasonal influenza victims were compared using quantitative IHC. Our results demonstrate that the clinical and histologic features of COVID‐19 are similar to those seen in fatal cases of influenza, and the two diseases tend to overlap histologically. There was no significant difference in the composition of the inflammatory infiltrate in COVID‐19 and influenza at sites of acute lung injury at the time of autopsy. Our study underscores the relatively nonspecific clinical features and pathologic changes shared between severe cases of COVID‐19 and influenza, while also providing important caveats to ancillary methods of viral detection.

Analysis of effectivity of the tickborne encephalitis virus detection methods in ixodid ticks.

Tick-borne encephalitis (TBE) is transmissible viral disease widely common in temperate zone of Eurasia. ELISA and PCR are used for express identification of the vector's infection, but the results of the two methods often do not agree. Aim of the work is comparative analysis for TBE virus of Ixodid ticks from nature using complex of methods, including ELISA, PCR, and isolation of the virus in laboratory mice. 18608 Ixodid ticks were collected during 2013-2019 in TBE natural foci of the Baikal Region. The ticks suspensions were examined individually, using ELISA (n=17610) and PCR (n=2999). Suckling mice were inoculated with the suspensions positive in the both tests. The TBEV antigen was found in 1.2 % of ticks in average. All ticks positive in ELISA were examined in PCR (Group 1). Randomly selected part of negative-ELISA samples were examined in PCR too (Group 2). The PCR results were positive in 68.9±3.13 % of the Group 1, with average Ct index 24.6±0.38. Positive results of PCR in Group 2 accounted for just 2.7±0.31 % with average Ct index 31.0±0.70. The average Ct margin of the Groups 1 and 2 is statistically significant (p < 0.001; df = 118). Isolation of strains was significantly more successful in Group 1 (21.7±2.77 %), than in Group 2 (8.2±5.26 %; p < 0.05; df = 50). ELISA is more useful for examining large amounts of ticks. To get a more complex picture about epidemically dangerous part of the vectors in TBE natural foci, the results of the two express-methods is better to sum. The isolation of the virus is useful to carry out of the samples positive in ELISA and PCR concurrently.

Viral Antibody in Saliva Based Diagnostics: A Systematic Review

Background: Salivary viral antibodies originated either from viral salivary gland infection or systemic diseases. Virus related antibodies in saliva are essential for the virus diagnosis and the leveragial measurement of viral infection. Objectives: This review aims to evaluate the general characteristics of salivary protein; the available knowledge on viral antibodies in saliva, the current progress of proteomic studies on salivary viral antibodies, and salivary antibody-based diagnosis methods for viral infection. Method: Searching articles carried out with the help of PubMed dan Science Direct. Then, the relevant articles screened accordingly. Result: Protein is the most abundant biomolecules in saliva, more than nucleic acids. The salivary protein composition is quite complicated. Salivary proteins are originated from the mouth tissues, microbiota, secretes of salivary glands, and antibodies, especially virus-induced antibodies—these virus antibodies found in saliva. There is a relationship between the infectious virus species with the salivary antibodies. Proteomic studies are fundamental for viral detection method, but they are not yet much carried out. The conventional diagnosis for a viral infection is relay on PCR-based methods. New promising practices, besides the proteomic approach, are nano biosensor and SERS (Raman Spectrometry). Conclusions: As part of a salivary protein, salivary viral antibodies have a very specify interaction with viral antigens. Information on the antibody as protein needs to be enriched by the proteomic studies. Further accuracy and specificity of salivary virus diagnosis methods depend on the progress of proteomic studies of salivary antibodies.

&lt;i&gt;In vitro&lt;/i&gt; methods for rabies virus detection, and evaluation of their use in the production of rabies immunoglobulin

Current highly sensitive methods for rabies virus and rabies antibodies detection in biological material can be used not only for diagnosis and experimental research, but also for the production of antirabies medicines used for postexposure prophylaxis. The aim of the study was to analyse existing methods for rabies virus and rabies antibodies detection and to assess the potential for using these methods at the control stages during production of heterologous antirabies immunoglobulin obtained from equine serum. The search for cutting-edge highly sensitive in vitro control methods that could compete with the biological method, which is the main method used in antirabies immunoglobulin control, is an important prerequisite for improvement of the production technology and the quality of antirabies medicines. The study demonstrated that the following test methods can be used in the production of antirabies immunoglobulin: fluorescent antibody technique, enzyme-linked immunosorbent assay, cell culture methods, atomic force microscopy, and flow cytometry. These methods could be used alone or as an alternative to the biological method in white mice. These methods were chosen because of their high sensitivity, specificity, rapid and easy implementation, cost-effectiveness, and automatic recording of test results.

Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel.

BackgroundInfectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities.ObjectiveTo compare the recovery and detection of infectious and heat-inactivated (65 °C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel.MethodsViral stocks were diluted in viral transport medium and deposited onto stainless steel test areas at 2 x 103 and 2 x 104 genomic copies for low and high, respectively. Test areas were sampled, and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by Probability of Detection (POD) statistics.ResultsThe low level, where fractional positive results (25–75%) are expected, yielded PODI = 0.80 (0.58, 0.92) for the infectious virus and PODHI = 0.15 (0.05, 0.36) for the heat-inactivated virus. The bias, dPODHI = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and heat-inactivated virus detection. No difference was observed at the high inoculation level.ConclusionDespite the statistical difference observed, the use of the heat-inactivated virus is a viable alternative for matrix extension studies using a method comparison study design.HighlightsThe use of heat-inactivated SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.

The virome of German bats: comparing virus discovery approaches

Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies.

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct. MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations. ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R2 >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations). ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.

Development of General Methods for Detection of Virus by Engineering Fluorescent Silver Nanoclusters.

Viruses have caused significant damage to the world. Effective detection is required to relieve the impact of viral infections. A biomolecule can be used as a template such as deoxyribonucleic acid (DNA), peptide, or protein, for the growth of silver nanoclusters (AgNCs) and for recognizing a virus. Both the AgNCs and the recognition elements are tunable, which is promising for the analysis of new viruses. Considering that a new virus such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently requires a facile sensing strategy, various virus detection strategies based on AgNCs including fluorescence enhancement, color change, quenching, and recovery are summarized. Particular emphasis is placed on the molecular analysis of viruses using DNA stabilized AgNCs (DNA-AgNCs), which detect the virus's genetic material. The more widespread applications of AgNCs for general virus detection are also discussed. Further development of these technologies may address the challenge for facile detection of SARS-CoV-2.

Development of integrated swab chamber for drive thru Covid19 test system

WHO currently recommends molecular testing for all patients who are included in the suspect category. The molecular examination requires facilities with biosafety level 2 (BSL-2), meanwhile for culture testing at least BSL-3. Viral culture is not recommended for routine diagnosis. The recommended method for virus detection is nucleic acid amplification by real-time reverse-transcription polymerase chain reaction (rRTPCR) and by sequencing. The sample is said to be positive (confirm SARS-CoV-2) if the RRT-PCR is positive for at least two genomic targets (N, E, S, or RdRP) that are SARSCoV-2 specific; OR positive RRT-PCR for betacoronavirus, supported by sequencing results of part or all of the viral genome according to SARS-CoV-2. To reduce the spread of the virus, the government began to carry out massive swab tests. Molecular rapid tests are easier to perform and faster because the process is automated which greatly helps speed up detection. To support a safe swab process, in this study an integrated portable swab booth was built. The output of this research is expected to contribute to the prevention of the spread of disease caused by the 2019-nCoV virus.

Sensitive and high-throughput polyclonal antibody-based serological methods for rice stripe virus detection in both rice and small brown planthopper

Rice stripe virus (RSV) is a plant virus, and its infection leads to a devastating disease that often causes serious yield losses in rice-producing regions. In this study, the capsid protein (CP) gene of the RSV was cloned and expressed in E. coli (Rosetta), and the purified recombinant His-CPRSV protein was used to immunize New Zealand white rabbits. After six weeks, a polyclonal antibody against the His-CPRSV protein (PAb-CPRSV) was obtained from the serum of the immunized rabbits. Using the prepared PAb-CPRSV, three serology-based detection methods were developed, which could specifically test for RSV-infected rice plants or small brown planthoppers (SBPHs). Methods that combine dot-blot with enzyme-linked immunosorbent assay to high-throughput methods were also developed to detect RSV in rice seedlings and SBPHs simultaneously in wild fields. The field surveys indicated that the incidence of RSV in rice plants and SBPHs in the suburbs of Yangzhou City was much lower than the value ten years ago. This research presents multiple serology-based RSV detection methods, using the prepared PAb-CPRSV, which can simultaneously identify RSV-infected rice plants and SBPHs in wild field conditions. Our study will benefit RSV control in rice planting regions.

Swift and Reliable "Easy Lab" Methods for the Sensitive Molecular Detection of African Swine Fever Virus.

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this “easy lab” workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the “easy lab” strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the “easy lab” concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the “easy lab” strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.

Resource Optimization Through Group Testing in the Detection of Asymptomatic People with COVID-19

Background: The current coronavirus disease 2019 (COVID-19) pandemic is a serious global public health problem. Detection and isolation of people infected with the virus is crucial to public health measures. Hitherto, the real-time PCR (RT-PCR) test is the most useful method for viral detection, although high cost of performance and low supplies have limited testing. New ways to optimize RT-PCR testing performance must be achieved since many million tests have been performed almost exclusively on individual basis. We present the results of a pooling method for testing to optimize resources.Methods: Groups of two to 27 people with no COVID-19 symptoms were tested by RT-PCR. Positive groups were re-analyzed individually, and positive groups were compared to obtain the maximum number of persons per group to achieve optimal resource utilization.Results: Groups of four to six people were found optimal for resources optimization, up to 67%. Groups with higher or lower number of people had results from 37% and 47% that implied higher economic expenses.Conclusion: Group testing of 4 to 6 persons for COVID-19 by RT-PCR are practical and optimize time and economical resources for detection of asymptomatic people infected by the virus.Funding: None to declare. Conflict of Interest: No conflict of interest of any kind.Ethical Approval: The protocol was approved by the research department of the General Hospital of Mexico Eduardo Liceaga of Mexico City (Approval number DI / 20/405103150).

Metode single image-NDVI untuk deteksi dini gejala mosaik pada Capsicum annuum

Single image-NDVI method for early detection of mosaic symptoms in Capsicum annuum&#x0D; Mosaics are a symptom of a disease often found in red chilies (Capsicum annuum) and is generally caused by viral infections such as the Tobacco mosaic virus. Severe infection can cause stunting and significant yield loss. Serological and molecular detection is a common detection method for plant viruses although they are time-consuming, relatively inefficient for large samples, and are destructive to plants. On the other hand, direct symptoms observation is hampered by human visual abilities and latent symptoms in virus infection. Therefore, detection method based on the plant’s ability to absorb and reflect various spectrums of sunlight, such as the normalized difference vegetation index (NDVI), has the potential to be developed. This study aims to evaluate the potential of a single image-NDVI as an NDVI variant for the early detection of mosaic symptoms in red chilies. The main activity involved image recording of chili plants that were not inoculated (V0) and inoculated (V1) by the virus, and given minimal nutrients (M) using an unmodified RGB camera and lens filter to capture blue and Near-Infrared light reflection. Furthermore, image processing is carried out using the Photo Monitoring plugin on the Fiji-ImageJ application. The recording was done one day after inoculation (dai) until the symptoms were visible. The results showed that there was an increasing trend in the integrated NDVI value in all treatments. Howewer, the increasing trend in V1 was not significant compared to V0 and M. The difference in the mean value of integrated NDVI between V1 was very significant compared to V0 (at 5 dai) and M (at 1 dai). This method’s level of sensitivity, specificity, and accuracy ranges from 80–90% at 5 dai.

Respiratory syncytial virus B sequence analysis reveals a novel early genotype

Respiratory syncytial virus (RSV) is a major cause of respiratory infections and is classified in two main groups, RSV-A and RSV-B, with multiple genotypes within each of them. For RSV-B, more than 30 genotypes have been described, without consensus on their definition. The lack of genotype assignation criteria has a direct impact on viral evolution understanding, development of viral detection methods as well as vaccines design. Here we analyzed the totality of complete RSV-B G gene ectodomain sequences published in GenBank until September 2018 (n = 2190) including 478 complete genome sequences using maximum likelihood and Bayesian phylogenetic analyses, as well as intergenotypic and intragenotypic distance matrices, in order to generate a systematic genotype assignation. Individual RSV-B genes were also assessed using maximum likelihood phylogenetic analyses and multiple sequence alignments were used to identify molecular markers associated to specific genotypes. Analyses of the complete G gene ectodomain region, sequences clustering patterns, and the presence of molecular markers of each individual gene indicate that the 37 previously described genotypes can be classified into fifteen distinct genotypes: BA, BA-C, BA-CC, CB1-THB, GB1-GB4, GB6, JAB1-NZB2, SAB1, SAB2, SAB4, URU2 and a novel early circulating genotype characterized in the present study and designated GB0.