Viral Lysate Research Articles

Reactivation of a transplant recipient's inherited human herpesvirus 6 and implications to the graft.

The implications of inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) in solid organ transplantation remain uncertain. Although this trait has been linked to unfavorable clinical outcomes, an association between viral reactivation and complications has only been conclusively established in a few cases. In contrast to these studies, which followed donor-derived transmission, our investigation is the first to examine the pathogenicity of a recipient´s iciHHV-6B and its impact on the graft. We used hybrid capture sequencing for in-depth analysis of the viral sequences reconstructed from sequential liver biopsies. Moreover, we investigated viral replication through in situ hybridization (U38-U94 genes), real-time PCR (U89/U90 genes), immunohistochemistry, and immunofluorescence (against viral lysate). We also performed whole transcriptome sequencing of the liver biopsies to profile the host immune response. We report a case of reactivation of a recipient´s iciHHV-6B and subsequent infection of the graft. Using a novel approach integrating the analysis of viral and mitochondrial DNAs, we located the iciHHV-6B intra-graft. We demonstrated active replication via the emergence of viral minor variants across time points, in addition to positive viral mRNAs and antigen stainings in tissue sections. Furthermore, we detected significant upregulation of cell surface molecules, transcription factors, and cytokines associated with antiviral immune responses, arguing against immunotolerance. Our analysis underscores the potential pathological impact of iciHHV-6B, emphasizing the need for close monitoring of reactivation in transplant recipients. Most crucially, it highlights the critical role that the host's virome can play in shaping the outcome of transplantation, urging further investigations.

Immunogenic Material Vaccine for Cancer Immunotherapy by Structure-Dependent Immune Cell Trafficking and Modulation.

Inherently immunogenic materials offer enormous prospects in enhancing vaccine efficacy. However, the understanding and improving material adjuvanticity remain elusive. Herein how the structural presentation of immunopotentiators in a material governs the dynamic dialogue between innate and adaptive immunity for enhanced cancer vaccination is reported. The immunopotentiator manganese into six differing structures that resemble the architectures of two types of pathogens (spherical viruses or rod-like bacteria) is precisely manipulated. The results reveal that innate immune cells accurately sense and respond to the architectures, of which two outperformed material candidates (151nm hollow spheres and hollow microrods with an aspect ratio of 4.5) show higher competence in creating local proinflammatory environment with promoted innate immune cell influx and stimulation on dendritic cells (DCs). In combination with viral peptides, model proteins, or cell lysate antigens, the outperformed microrod material remarkably primes antigen-specific CD8 cytolytic T cells. In prophylactic and therapeutic regimens, the microrod adjuvanted vaccines display optimal aptitude in tumor suppression in four aggressive murine tumor models, by promoting the infiltration of heterogeneous cytolytic effector cells while decreasing suppressive immunoregulatory populations in tumors. This study demonstrates that a rationally selected architecture of immunogenic materials potentially advances the clinical reality of cancer vaccination.

A smartphone-controllable molecular diagnostic platform for SARS-CoV-2 detection by reverse-transcription recombinase polymerase amplification

We constructed a portable molecular diagnostic microsystem, enabling the whole processes of genetic analysis for SARS-CoV-2. The proposed platform was a miniaturized size and was comprised of three main parts: a microfluidic device with a 3D-printed solution loading cartridge, an operating system including a tailor-made heater board, a syringe pump and an optical fluorescence detector, and a smartphone with a customized app for the remote and automatic control. The microfluidic device was designed to control the complicated fluidic flow through only the passive valves, and could perform the RNA extraction from the viral lysate via glass membranes and the target gene amplification by reverse-transcription recombinase polymerase amplification (RT-RPA). The 3D-printed cartridge allowed the facile chemical solution storage and loaded a designated solution into the microfluidic device. A smartphone with a browser-based app could control the on-and-off status of solenoid valves, the workflow of a syringe pump to automatically introduce solutions on a chip, the heater board to supply the constant temperature for RT-RPA, and the periodic activation of the Pi camera and a 488 nm laser to measure the fluorescence signal in the reaction chambers that showed the amplification profiles on the smartphone display in real-time. Using the proposed smartphone-controllable diagnostic platform, the N gene and S gene of SARS-CoV-2 were successfully detected at a limit-of-detection (LOD) of 10 copies/μL within 1 hr in a sample-to-answer-out format. Ten out of 22 clinical samples were successfully analyzed for SARS-CoV-2, demonstrating the practical applications for the on-site diagnostics of patient samples.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was≤0.10 SD (<1.00S/C) with a≤10% CV (≥1.00S/C). Assay reagents were stable on board the instrument for 14days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0×106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30days) at-20°C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was≥98.5% and sensitivity was≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Diversity in Infection Specificity between the Bloom-forming Microalga &lt;i&gt;Heterosigma akashiwo&lt;/i&gt; and Its dsDNA Virus, Heterosigma akashiwo Virus

Heterosigma akashiwo virus (HaV) is a dsDNA virus that infects the bloom-forming raphidoflagellate Heterosigma akashiwo. Both the host and its virus are phenotypically diverse in terms of infection specificity. Their relationships have been examined based on the occurrence or absence of algal lysis following virus inoculation; however, variations in the strain-level host-virus relationship regarding infectivity and lysis rates remain unclear. Therefore, we performed a series of cross-infectivity tests using 60 H. akashiwo and 22 HaV strains isolated from the coastal waters of western Japan. The host strains were divided into 5 different groups and viruses into 4 groups. Using a representative strain from each group, algal lysis was observed in 14 of the (5×4=) 20 host-virus combinations; the concentration of infectious units in each HaV suspension was then assessed using the most probable number (MPN) assay on the five host strains. Virus titers ranged between 1.1×101 and 2.1×107 infectious units mL-1; the titer of each viral lysate was differently estimated using distinct H. akashiwo strains as hosts. These results suggest that (1) a clonal viral lysate comprises virions with different intraspecific infection specificities and/or (2) the efficiency and error rates of each intracellular replication process vary in each host-virus combination.

#40 An RSV live-attenuated vaccine candidate lacking G protein mucin domains is highly attenuated, immunogenic, and effective in RSV prevention in BALB/c mice

Abstract Background RSV is a leading viral respiratory pathogen in infants. Live attenuated vaccines (LAV) show promise to reduce the disease burden of RSV disease. The RSV attachment protein (G protein) is heavily glycosylated with mucin domains that promote viral attachment. They may also aid in immune evasion through steric shielding of immunogenic epitopes. The objective of this study was to create and rescue RSV LAV candidates lacking the RSV G protein mucin domains with attenuated LAV replication and increased immunogenicity. Method LAV vaccine candidates “G155” and “G155-S” were generated from recombinant A2-line19F strain RSV by deletion of the G-protein mucin domains. G155-S additionally underwent deletion of the transmembrane domain, such that G155-S only expressed the secreted form of the G protein, while G155 expressed both transmembrane and secreted forms. Western blotting was used to characterize F and G protein expression in both vaccine candidates in comparison to wild-type (wt) A2-line19F. Enzyme-linked immunosorbent assays (ELISAs) were used to measure monoclonal antibody binding to LAV virus lysate. Viral growth kinetics were measured in HEp2 cells and in BALB/c mouse lungs by a fluorescent focus unit (FFU) assay. Immunogenicity was determined by measuring serum neutralizing antibody titers in BALB/c mice following a prime/boost regimen on days 0 and 28. Mice were challenged with wt RSV on day 60, and LAV efficacy was determined by measuring RSV lung viral loads by FFU assay and RT-PCR on day 4 post-RSV challenge. Results The vaccine candidates showed increased monoclonal antibody binding to surface F protein by ELISA, despite similar levels of F protein expression to wt, consistent with F epitope de-shielding. Both vaccine candidates were significantly attenuated in vitro and were undetectable in mouse lungs via FFU assay. By RT-PCR, LAV viral RNA was reduced 3-fold for G155-S, and 22-fold for G155 compared to wt RSV in mouse lungs. Both vaccine candidates elicited similar neutralizing antibody titers compared to wt A2-line19F on day 28 (G155 GMT 13, 95% CI 3, 53; G155-S GMT 10, 95% CI 2, 51; A2-line19F GMT 13, 95% CI 12, 15), and significant increases in titers were observed on day 59 (G155 GMT 440, 95% CI 5, 36057; G155-S 436, 95% CI 354, 537; A2-line19F 275, 95% CI 134, 567). Following RSV challenge, vaccinated mice showed no detectable lung virus by FFU assay and a reduction in lung viral RNA by RT-PCR of 560-fold for G155 and 604-fold for G155-S compared to RSV-challenged unvaccinated mice. Conclusion G155 and G155-S are highly attenuated RSV LAV candidates that protect against RSV challenge in mice and produce high levels of neutralizing antibodies comparable to wt RSV.

Effect of Janus kinase inhibitors on T cell responses to herpes zoster in rheumatoid arthritis patients.

The incidence of herpes zoster (HZ) in rheumatoid arthritis (RA) patients is greater than that in healthy controls (HC), particularly in RA patients treated with Janus kinase inhibitors (JAKi). Here, we examined the effect of JAKi on CD4+/CD8+ T cells, cytokine production, and regulation of transcriptional factors in RA patients and HC. Peripheral blood mononuclear cells (PBMCs) obtained from RA patients (n=14) and HCs (n=7) were stimulated with varicella zoster virus lysates and exposed to three JAKi inhibitors (ruxolitinib [JAK1/2 inhibitor]; AG490 [JAK2 inhibitor]; and WHI-P154 [JAK3 inhibitor]) in the presence/absence of methotrexate. The CD4+ and CD8+ T cell populations were measured by flow cytometry. Cytokine levels in culture medium were measured by ELISA. Transcription factor expression was examined by RT-qPCR. There was a reduction in the CD4+IFN-γ+, CD4+CD69+IFN-γ+, CD8+IFN-γ+, and CD8+CD69+IFN-γ+ populations, and an increase in the CD4+CD25highFoxp3+ cell population, in PBMCs from RA patients and HCs after exposure to the three JAKi. ELISA revealed a reduction in IFN-γ and granzyme B levels in the presence of JAKi. JAKi reduced expression of mRNA encoding STAT1 and T-bet, but increased that of mRNA encoding STAT5 and Foxp3. Methotrexate plus the highest dose of each JAKi did not affect the Th1, cytotoxic T cell, or Treg populations, the levels of IFN-γ and granzyme B, or expression of transcription factors, significantly. JAKi reduce the Th1/cytotoxic T cell population and increase the Treg population in both RA patients and HC patients.

Heterogeneous viral contribution to dissolved organic matter processing in a long-term macrocosm experiment

Viruses saturate environments throughout the world and play key roles in microbial food webs, yet how viral activities affect dissolved organic matter (DOM) processing in natural environments remains elusive. We established a large-scale long-term macrocosm experiment to explore viral dynamics and their potential impacts on microbial mortality and DOM quantity and quality in starved and stratified ecosystems. High viral infection dynamics and the virus-induced cell lysis (6.23–64.68% d−1) was found in the starved seawater macrocosm, which contributed to a significant transformation of microbial biomass into DOM (0.72–5.32 μg L−1 d−1). In the stratified macrocosm, a substantial amount of viral lysate DOM (2.43–17.87 μg L−1 d−1) was released into the upper riverine water, and viral lysis and DOM release (0.35–5.75 μg L−1 d−1) were reduced in the mixed water layer between riverine water and seawater. Viral lysis was stimulated at the bottom of stratified macrocosm, potentially fueled by the sinking of particulate organic carbon. Significant positive and negative associations between lytic viral production and different fluorescent DOM components were found in the starved and stratified macrocosm, indicating the potentially complex viral impacts on the production and utilization of DOM. Results also revealed the significant viral contribution to pools of both relatively higher molecular weight labile DOM and lower molecular weight recalcitrant DOM. Our study suggests that viruses have heterogeneous impact on the cycling and fate of DOM in aquatic environments.

Antitumor immunity enhancement through Newcastle viral oncolysate in mice model: A promising method to treat tumors.

A Newcastle disease virus (NDV) oncolysate has been established as a unique and effective immune-stimulatory root for tumor treatment. Thus, the aim of the current study was to investigate the effects of intratumoral administration of NDV oncolysate on immune response and tumor regression of C57BL/6 mouse model of human papillomavirus (HPV) related transplanted with TC-1 syngeneic cancer cells. To further investigate the mechanism underlying the antitumor response, cytolytic and lymphocyte proliferation responses in splenocytes were measured using lactate dehydrogenase (LDH) release and MTT assays, respectively. In this regard, levels of IL-10, IFN-γ, and IL-4 were measured using ELISA after re-stimulation. The immune responses efficacy was evaluated by in vivo tumor regression assay. The results showed that immunization with the different titers of NDV lysate significantly reduced tumor volume in comparison with a combination of virus lysate and tumor cell lysate. Also, virus lysate could significantly enhance cytotoxic T lymphocyte production and lymphocyte proliferation rates versus tumor cell lysate. Also, our major findings are that the peritumorally injection of NDV oncolysate effectively induces antitumor immune responses through increased levels of IL-4, IFN-γ, and reduction of IL-10. These results indicate that this treatment is a specific, active immune mechanism stimulator, and may prove to be a useful therapeutic for a treatment against cervical cancers and merits further investigation.

Tergitol-15-S-9 inactivates SARS-CoV-2 and boosts immunoassay signals.

Inactivation of SARS-CoV-2 virus is necessary to mitigate risk but may interfere with diagnostic assay performance. We examined the effect of heat inactivation on a prototype SARS-CoV-2 antigen immunoassay run on the ARCHITECT automated analyzer. Recombinant full-length SARS-CoV-2 nucleocapsid protein and virus lysate detection was reduced by 66 and 31%, respectively. Several nonionic detergents were assessed as inactivation alternatives based on infectivity in cultured Vero CCL81 cells. Incubation of SARS-CoV-2 in 0.1% Tergitol 15-S-9 for 10 min significantly reduced infectivity and increased the immunoassay signal for cultured lysate and patient specimens. Tergitol 15-S-9 can inactivate SARS-CoV-2 while preserving epitopes on the nucleocapsid protein for enhanced detection by immunoassay antibodies.

Multi-modality detection of SARS-CoV-2 in faecal donor samples for transplantation and in asymptomatic emergency surgical admissions.

Background: Faecal transplantation is an evidence-based treatment for Clostridioides difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit. Methods: Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10 -1 to 10 -5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results: In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10 -2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10 -1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusion: RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.

693. Shaking Things Up: Direct-to-PCR Viral Detection off Swabs Using Shaker-Mill Homogenization

BackgroundAs the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. These assays often involve procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify it’s nucleotides, and then run the purified genetic material via PCR for detection of a gene product needed to confirm the patient’s suspected diagnosis. This process requires time to complete and is dependent on the availability of the reagents and plastics required to complete the lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off a swab using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR based assay, bypassing the reagent heavy and time consuming extraction and purification steps.MethodsUsing Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs with clinically relevant levels of the virus for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2e7 copies/mL to 1.2e1 copies/mL. The swabs were then placed in 2mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 uL of viral lysate was run in RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection from the sample.ResultsHCoV-229E spiked swabs were run through the two-step process of homogenization direct to RT-qPCR for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2e3 viral copies/mL with 96.30% sensitivity in vitro.ConclusionWe have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR based assays for the detection of virus. This finding allows for decreased run time in traditional PCR based diagnostics and reduces the reagents and plastics required for each sample, ultimately reducing the cost and time of each viral test when compared to traditional PCR based methods.DisclosuresZachary P. Morehouse, MS, OMS-III, Omni International Inc (Consultant) Caleb Proctor, BS, Omni International Inc (Employee) Gabriella Ryan, BS, Omni International Inc (Employee) Rodney J. Nash, PhD, Omni International Inc (Employee)

Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9.

Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3′,5,5′-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.

Specific and sensitive detection of Influenza A virus using a biotin-coated nanoparticle enhanced immunomagnetic assay.

This study reports the development of a sensitive magnetic bead-based enzyme-linked immunoassay (MELISA) for the pan-reactive detection of the Influenza A virus. The assay combines immunomagnetic beads and biotin-nanoparticle-based detection to quantify a highly conserved viral nucleoprotein in virus lysates. At the capture step, monoclonal antibody-coated magnetic microbeads were used to bind and concentrate the nucleoprotein in samples. The colorimetric detection signal was amplified using biotinylated silica nanoparticles (NP). These nanoparticles were functionalized on the surface with short DNA spacers bearing biotin groups by an automated supported synthesis method performed on nano-on-micro assemblies with a DNA/RNA synthesizer. A biotin-nanoparticle and immunomagnetic bead-based assay was developed. We succeeded in detecting Influenza A viruses directly in the lysis buffer supplemented with 10% saliva to simulate the clinical context. The biotin-nanoparticle amplification step enabled detection limits as low as 3 × 103 PFU mL−1 and 4 × 104 PFU mL−1 to be achieved for the H1N1 and H3N2 strains respectively. In contrast, a classical ELISA test based on the same antibody sandwich showed detection limit of 1.2 × 107 PFU mL−1 for H1N1. The new enhanced MELISA proved to be specific, as no cross-reactivity was found with a porcine respiratory virus (PRRSV). Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-020-03081-x.

A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E

BackgroundCurrently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps.MethodsUsing Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 × 106 to 1.2 × 101 copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 μL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection.ResultsHCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 103 viral copies/mL with 96.30% sensitivity.ConclusionWe have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Cesium Chloride Gradient Sedimentation.

Centrifugation to equilibrium in cesium chloride gradients has been used for more than 40 yr to purify viruses. The application of high G-forces for a long period of time to a solution of CsCl generates a density gradient that allows separation of empty, partially packaged, and fully packaged viral particles from cellular debris, proteins, and nucleic acids in the crude viral lysate on the basis of their buoyant densities. This protocol describes the use of CsCl gradients to purify AAV vectors from crude viral lysates.

Development and Investigation of Diagnostic Efficiency of a Test Kit for the Detection of IgM-antibodies to Individual Antigens of Rubella Virus by Immunoblotting (Western Blot)

Relevance . During serological diagnosis of rubella by enzyme immunoassay (detecting IgM), it is possible to obtain an indefinite or false-positive result. In this case, is needed an additional confirmatory test. It can be carried out by immune blotting, which allows detecting antibodies to specific rubella antigens. To date, in the Russian Federation, there is no such kit of reagents, which makes its development quite actual. Aim . The aim of this research was the development of a Russian test kit for detecting IgM-antibodies to individual rubella virus antigens by immune blotting (Western blot format). Materials and methods . Production of immunosorbent for a new test kit included electrophoresis of the native rubella virus lysate in a polyacrylamide gel to separate proteins by molecular weight and transfer (blotting) of separated proteins onto a nitrocellulose membrane. Investigation of sensitivity and specificity of the new test kit was carried with standard panels of serums containing and not containing IgM to the rubella virus, and with clinical samples characterized by the ELISA method. Results and its discussion . As a result of this work, was designed a Russian test kit that allowed detection IgM-antibodies to individual rubella virus antigens by immune blotting. This test kit has analytical sensitivity and specificity -100%, diagnostic sensitivity - at least 99.61% and diagnostic specificity - and at least 99-99%. The diagnostic efficiency is not less than 99.5%. Conclusion . The developed test kit has high rates of analytical and diagnostic sensitivity and specificity, as well as high diagnostic efficiency. It is intended for confirmatory research in the diagnosis of rubella infection.

A nanoparticle pseudo pathogen for rapid detection and diagnosis of virus infection

We herein report a new rapid blood test for virus infection detection and diagnosis. A citrate gold nanoparticle is first coated with a virus lysate to form a gold nanoparticle pseudo pathogen. The gold nanoparticle pseudo virus is then mixed with a blood plasma or serum samples. If the blood sample is from a positive patient, the activated immune molecules in the blood such as antibodies, complement proteins and others will react with the nanoparticle pseudo virus, leading to nanoparticle aggregate formation. The nanoparticle aggregate formation is detected and measured using a particle sizing technique called dynamic light scattering. In this study, we applied this test for Zika virus infection detection. We tested blood plasma samples from 85 Zika positive patients, 40 Dengue positive patients, 10 Chikungunya positive patients, and 78 non-patient control samples collected from both endemic and non-endemic locations. The study shows that the new test has a higher sensitivity compared to some existing commercial tests in the market, while maintaining a similar specificity. Within 7 days from the symptom onset, the new test can detect 43% of the infected patients while a commercial anti-Zika IgM test detects only 26% of the infected patients. Within 14 days from the symptom onset, our new test detects 73% of the infected patients while the same commercial anti-Zika IgM test detects 53% of the infected patients. The test is extremely simple, easy to develop, with test results obtained within minutes. This new test platform may be potentially adapted for the detection and diagnosis of a wide range of viral infectious diseases, for example, the currently ongoing COVID-19.

Microbial transformation of virus-induced dissolved organic matter from picocyanobacteria: coupling of bacterial diversity and DOM chemodiversity

Picocyanobacteria make up half of theocean's primary production, and they are subjected to frequent viral infection. Viral lysis of picocyanobacteria is a major driving force converting biologically fixed carbon into dissolved organic carbon(DOC). Viral-induced dissolved organic matter (vDOM) released from picocyanobacteria provides complex organic matter to bacterioplankton in the marine ecosystem. In order to understand how picocyanobacterial vDOM are transformed by bacteria and the impact of this process on bacterial community structure, viral lysate of picocyanobacteria was incubated with coastal seawater for 90 days. The transformation of vDOM was analyzed by ultrahigh-resolution mass spectrometry and the shift of bacterial populations analyzed using high-throughput sequencing technology. Addition of picocyanobacterial vDOM introduced abundant nitrogen components into the coastal water, which were largely degraded during the 90 days' incubation period. However, some DOM signatures were accumulated and the total assigned formulae number increased over time. In contrast to the control (no addition of vDOM), bacterial community enriched with vDOM changed markedly with increased biodiversity indices. The network analysis showed that key bacterial species formed complex relationship with vDOM components, suggesting the potential correspondence between bacterial populations and DOM molecules. We demonstrate that coastal bacterioplankton are able to quickly utilize and transform lysis products of picocyanobacteria, meanwhile, bacterial community varies with changing chemodiverisity of DOM. vDOM released from picocyanobacteria generated a complex labile DOM pool, which was converted to a rather stable DOM pool after microbial processing in the time frame of days to weeks.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.

Preliminary evaluation of Thermo Fisher TaqMan® Triplex q-PCR kit for simultaneous detection of chikungunya, dengue, and Zika viruses in mosquitoes.