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Viral Infection - Science topic

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Working on viral infectious disease to evaluate different microRNAs profile using RT-PCR. I used mostly SV40 miRNA that's commonly used as an internal control, but sometimes in literature other internal controls used as U6, RNU44, U1, B-actin ,GAPDH etc.
Which type of internal control has the best accuracy for normalization instead of SV40?
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Hello, I am currently establishing a virus infection model in my lab. The cell I use is a vero cell and the virus is porcine epidemic diarrhea virus (PDEV). I plan to use the virus stock without concentration titration to make the new virus stocks, and I will conduct TCID50 analysis of the virus stocks in the future.
First, 0.5 ug/ml to 2 ug/ml TPCK-trypsin test was conducted in 96 well for the condition of virus infection culture (including 0.3% BSA). The virus infection culture was treated after washing twice with plain DMEM using 80-90% vero cells (Figure 1). Then, the appropriate concentrations (0.5 ug/ml, 0.75 ug/ml, 1 ug/ml) were selected and PEDV infection was performed.
Similar to the above process, after washing twice with plain DMEM, the virus stock of unknown concentration was diluted by virus infection culture (by TPCK-trpysin concentration) at 1:10. Finally 5 ml was dispensed into 100 dishes for 1 hour incubation. Next, I washed it twice with plain DMEM, added 10ml of the virus-infected culture medium, and incubated it for 3 days (Figure 2).
What I am curious about is the concentration (0.5 ug/ml or 0.75 ug/ml?) of TPCK-trypsin to establish a virus infection model and the timepoint of harvesting cell supernatant for virus stock manufacturing. What state should the vero cell be in to manufacture the virus stock? Please advise us to establish a virus infection model. Thank you!
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Vero cells either primary or continuous cell line prefered for porcine virus diarrhea because minimize mixed infection transmission and protective titer
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Hello,
I would like to ask which is the best terminology to use for describing a negative control in an experiment (drug testing in-vitro, viral infection): NC (negative control) or NTC?
Thank you for being so helpful.
Also, I recently saw several articles use the term naive to describe the negative control for non-infected animals.
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I appreciate your answer. As far as I know, NTC stands for non-template control in PCR. However, seevral papers used the term NTC for negative control, which is why I got a bit confused. As you said, using NC is the way.
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Hello every one
Can any one suggest me trends subjects in viral infection in cancer patients for PHD thesis?
Thanks in advance.
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Dominic Worku
Many thanks, doctor.
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I am having difficulty trying to increase the concentration of both low molecular and high molecular weight Poly I:C without increasing the volume. Can anyone help?
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Titrating the concentration of poly I:C (polyinosinic:polycytidylic acid) involves testing a range of concentrations to determine the optimal concentration for stimulating the desired immune response or cellular effects while minimizing potential cytotoxicity or off-target effects. Here's how you can titrate the concentration of poly I:C:
  1. Prepare Stock Solution: Start by preparing a stock solution of poly I:C at a known concentration. Dissolve poly I:C powder in a suitable sterile buffer, such as phosphate-buffered saline (PBS) or cell culture medium, to prepare a concentrated stock solution.
  2. Dilution Series: Prepare a series of dilutions of the poly I:C stock solution to create a range of concentrations for testing. Use serial dilution to create two-fold or ten-fold dilutions, depending on the desired concentration range and the sensitivity of the assay or experimental system.
  3. Experimental Setup:Determine the cell type, tissue, or experimental model system that will be exposed to poly I:C. Plate cells or prepare tissues in suitable culture vessels or experimental formats according to the specific experimental design.
  4. Treatment:Apply each concentration of poly I:C to separate wells, samples, or experimental units. Ensure that appropriate controls are included, such as untreated cells or samples and cells treated with vehicle (buffer) alone.
  5. Incubation:Incubate the cells, tissues, or experimental samples with poly I:C for an appropriate duration. This may vary depending on the experimental objectives and the kinetics of the immune response or cellular effects induced by poly I:C.
  6. Assessment of Response: After the incubation period, assess the response to poly I:C treatment using relevant assays, techniques, or readouts. This may include:Measurement of cytokine or chemokine secretion using ELISA or multiplex assays. Analysis of gene expression profiles using quantitative PCR (qPCR) or RNA sequencing. Evaluation of cell viability, proliferation, or apoptosis using viability assays, flow cytometry, or microscopy. Assessment of immune cell activation or differentiation using flow cytometry or immunofluorescence staining.
  7. Data Analysis:Analyze the data to determine the concentration of poly I:C that elicits the desired immune response or cellular effects while minimizing cytotoxicity or off-target effects. Calculate dose-response curves, IC50 values, or other relevant parameters to quantitatively assess the concentration-dependent effects of poly I:C.
  8. Optimization:Based on the results of the titration experiment, select the optimal concentration of poly I:C for your specific experimental goals. Consider factors such as the magnitude and specificity of the response, as well as practical considerations such as cost, availability, and reproducibility.
By systematically titrating the concentration of poly I:C and evaluating its effects in a range of experimental conditions, you can identify the optimal concentration for stimulating immune responses or cellular effects in your specific research context.
l Reviewing the protocols listed here may offer further guidance in addressing this issue
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Hi,
I have Illumina RNA sequencing data for viral infection only or viral treated. I would quantify the viral gene expression, but the total reads counts is various between library. Please see fig. I am wondering do I need to downsample the total reads to 5 M or is it ok to quantify using the total viral mapped reads ?? Thanks in advance
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Great, thanks for your answer. But the viral genes have different lengths. I am wondering if I use the EDgR package. It will not take into consideration these differences. Thanks in advance
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Hello
How can i get my article :
Mly Ismail El Karimi, Khalid Hattaf, Noura Yousfi, Dynamics of an immunological viral infection model with lytic and non-lytic immune response in presence of cell-to-cell transmission and cure of infected cells, Commun. Math. Biol. Neurosci., 2022 (2022), Article ID 119
in ResearchGate
Thank you
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My dear,
Send me your mail and I will send invitation to you.
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I have two DEG sets for 2 disease conditions (from mild to severe condition) of the same viral infection. When I look at the common gene from these two sets of DEGs, I found that some genes show opposite expression among these two conditions ( Like a gene downregulated in mild but up-regulated in severe or vice versa). So what I want to know is that,
1) If this phenomenon is normal in viral infection??
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The shift between two opposite direction of change is the rule for regulatory genes that work as 'toggle.switches' in which the biphasic alternation of two conditions is the basis for a sort of digital control of biological regulation. It is not by chance that a great part of toggle-switches are retroviral origin sequences that mirror the lytic-lysogenic phases of viruses and phagi, see:
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Which viral infection might be more severe?a transported virus from an animal to a human or a virus directly invade human?
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The severity of virus depends on : Viruence ,immunological status ,concurrent infrction and infected area (vargin or exposed previously).
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Hi everyone, i'm having problems with the MTT assay i'm performing, since two weeks.
I'm performing viral infection on Beas2B cells and a dose response curve of compounds with citotoxicity in parallel.
When i add MTT on the infected cells the wells get completely purple, while on the non-infected (citotoxicity) I see normal metabolism inside the cells.
What could be the reason? Do any of you have experimented something like this?
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Zuzanna Koziara Thank you for your answer.
1. the citotoxicity and the viral infection were performed in parallel, i thought about a possible contamination but i should have seen the very same result in the non infected cells (unless the tube where i made the viral dilution was contaminated).
2. the same compounds' dilution were use for both the experiments.
The experiment was performed in one plate, half was infected, the other half wasn't.
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Hi all
I've been struggling with this problem for a few months now. I am transfecting a BMI1 overexpression plasmid with GFP (https://www.addgene.org/21577/) into Phoenix cells for retroviral production. The Phoenix cells are GFP+ 24 hours post transfection so I know that step is working.
However, when I try to collect the media (which presumably contains virus) and infect target cells using polybrene, GFP is not expressed up to 4 days post transduction. I cannot figure out what's going on. A test infection of 293T cells also did not express GFP so I suspect the problem is with viral production?
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Thanks Yannick Frey I'll give that a try. Would I also have to cotransfect a different envelope plasmid? I believe Phoenix express VSV.G
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What is the best way to isolate extracellular vesicles from cell culture media if they are infected with viruses?
I am talking about an experiment in which we want to study the components inside the exosome after viral infection. I want to make sure that the isolation is clean of viruses.
If you mention the kit and the solutions, I will be grateful to you.
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Hey Faisal,
Maybe you could try an approach that targets surface markers of extracellular vesicles. I attached a protocol for an affinity chromatography method. For more details regarding the unlerlying technology, you can have a look here: https://www.iba-lifesciences.com/strep-tag/exosome-isolation/
Best,
Alex
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Crimean Congo hemorrhagic fever is an acute febrile viral disease with high mortality rate, the detection of this disease is through PCR for detection viral nucleic acid and ELISA to detect antibodies specific to the virus, carriers among human are uncommon and there is no many researches about human carriers.  
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During these epidemics, the infection of humans with Crimean-Congo hemorrhagic fever virus (CCHFV), the causative agent of CCHF, was diagnosed using qRT-PCR. We have identified 88 cases of CCHF, including 13 fatalities reported during five epidemics that occurred in 2010, 2011, 2015, 2019, and 2020.Apr 28, 2022
Epidemics of Crimean-Congo Hemorrhagic Fever (CCHF) in Sudan ...
Feedback
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My PI has given me a protocol for viral infection with HAZARA virus using Opti-mem media. The protocol calls for replacing the growth media with Opti-mem and adding the virus stock directly, incubating this for 90 minutes at 37°C with intermittent swirling, then removing this media and replacing with 2% supplemented DMEM. We have been mainly using HEK and Vero cells and getting no results. Could Opti-mem be interfering with the viral infection?
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Please can you give us a reference about the use of Optic_Men with best regards
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ADAM17 activity is rapid and reversible. Want to check if ADAM17 activated after viral infection in my cell line (Huh 7.5, A549) and if I can detect that by flow cytometry.
Can anyone share any detail protocol how I can detect activated ADAM17 on cell surface by flow cytometry?
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Hi
Prepare an antibody for TACE/ADAM17.
The below is an example. Please confirm its applications including flow cytometry. And, you can select some fluorescence conjugation.
Protocol
1. cell collection -Cell count 5x10^5cells / tube
2. 5min 1500rpm RT
3. 1%FCS/PBS 5mL wash
5min 1500rpm RT 5.
5. removal of supernatant
6. addition of the antibody
Example: Detailed conditions are different for each antibody. 1µL
or Isotype control 1uL
7. 45 min 4℃
8. 1% FCS/PBS 5mL
9. 1500rpm 5min 4℃ RT
10. 1%FCS/PBS 500~1000uL suspension on ice
11. Put the cell suspension through the filter in a round tube with 35 micro meter filter.
12. FACS analysis
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We are having trouble measuring viral genome copy numbers using qPCR. The CT values for dengue virus are quite varied; between technical replicates (same sample), we may see 10 CT differences and sometimes no detection. Has anyone had a similar experience and found a way to correct the issue?
We speculate it may be generating cDNA using Random Primers. We usually use oligo dT for cDNA synthesis but switched with viral infection to random primers. Previously, we have had no issues with qPCR and have gotten consistent CT values.
The Random primers we use are a stock concentration of 10uM, added in 1ul volume to a 20 ul cDNA reaction. This is the concentration recommended by the kit (https://www.abmgood.com/onescript-plus-cdna-synthesis-kit.html).
Any tips would be greatly appreciated!
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agree with Jack Greenhouse here. You can also do it as two step and use the reverse PCR primer for the cDNA synthesis.
Best Sven
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Hello
I am working on viral infection in PK-15 cells and then IFA in 96 well plates. After fixing the cells with 50%methanol and acetone for overnight at 40C and IFA, the wells turn completely white. Can anyone suggest what can be done for IFA in 96 well plates? I have also attached pictures herewith
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It could be, the overnight fixing is too long and damages the plastic. I would try shorter fixing, 10-30 minutes at room temperature should be long enough.
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Egypt has the highest prevalence of HCV. What is the major risk factor or history behind it?
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IFN detection is hard in vivo and I would like to use a reporter to quantify IFN during a viral infection in vivo using flow. The Locksley lab has been reported to have a week signal so I would love to get suggestions on what other reporter mice to use.
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Hi Lisa, Thank you for the suggestion!! Have you had any experience with it/heard of someone that has used it? Lisa Miorin
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I'm looking for published or unpublished papers.
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We are looking for some viral disease on Tuberose, what do you think of this symptom?
The main goal is detection of Tuberose mild mosaic virus, please check this paper to compare symptoms:
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This symptom is likely to be a viral disease. How is the disease incidence or distribution on the field? This question helps in proper diagnosis because one is not present in your field to know exactly what is happening. Do us great help by providing us with some conditions of cultivation. Thank you
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I made an cytokines expression curve, in 7dpi and 14 dpi Interferon gamma have a expression peaks, but at 28 dpi I observed an expression below the mock / control. Could this result be only related to the resolution of the viral infection, or a possible way of evading the immune response?
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Just assuming- It may happen due to immune cell exhaustion. The producers of IFNg like Helper T cells, NK cells may be in a dampened functional state after a massive host-pathogen battle.
Does the virus directly affect these IFNg producing cells? Then the virus can remain in a latent state and downregulate immune function.
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I'm working with primary human cell lines and I'm having difficulty with a viral infection using lentivirus with a plasmid backbone ~13kb. I use a positive control, lentiGFP-NEO, which infects well but my experimental plasmid does not.
Other technical things I've tried:
- I use 4ug/ml of polybrene at the time of the infection.
- I've tried just applying the viral media directly to the cells and also have tried a spinfection
- I've also tried adding PEG-IT when harvesting the virus.
The positive control that I use for infection is the same positive control that I use for transfection. I've been carrying it through the extent of the experiment, which makes me think that my viral production (I make virus in HEKT 293Ts) is not the source of the problem.
Any and all suggestions would be appreciated!
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Hi, yes the VSVG envelope is compatible with psPAX2, which is very similar to delta8.2. 13kb is a big plasmid! What's the sequence length between the viral LTRs? LV titers will go down fairly predictably as viral RNA size increases so if the viral RNA is longer than ~5kb you'll see a decrease in titer. You can easily get a 10-fold decrease in titer if you have a long sequence
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We found many cases of covid-19 infection who suffer second time within 2-3 months after the first infection. It indicates the status of immunity of the infection which in most cases of other viral infection that imparts long time immunity even life long immunity. It is same type of immunity like influenza infection. Effectivity of influenza vaccine usually last for 6 months due to changing its antigenicity. So this vaccine lost its popularity in most of the countries particularly in developing countries and manufacturers failed to make business from this vaccine. In the same way, I am afraid that Covid-19 vaccine will fail to make much money of the manufacturer in few months and no more than 6 months.
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COVID-19 vaccines are not going to provide life long immunity against the SARS-CoV-2 infection.
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I am performing an experiment analyzing the cytokine production of macrophages in response to viral infection.
I recently harvested and cultured peritoneal macrophages from mice. I plated these cells on 12-well plates and have been changing the media enriched with 1:1000 mCSF every two days. Once the cells reached 80-90% confluent I washed the cells two times with PBS and infected these cells with an inoculum of virus at an MOI of 3. After 1hr incubation, I washed the cells again two times with PBS and put 750uL of media back on cells. After 24hrs, I collected this 750uL of media to be used for 36-spot Luminex.
I'm concerned that I placed too much media on the cells after infection - do you think I have diluted the supernatant too much and my cytokines will all be below the limit of detection when I run a Luminex assay?
Thank you for your help.
Olivia
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You have not told us the problem you encountered, only your fear. I do not know what the problem is and so cannot tell where it lies.
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Hi,
I'm trying to stain a viral infection of the pancreas in paraffin sections of adult (3 month old) BALB/c mice.
Prior to starting my histology analysis, I have titrated the organ to confirm that I actually have a viral load in the pancreas of my infected animals.
I have also used a organ (liver) were I know that I have abundancy of viral positive cells as a control for my work.
In my protocol I did Citrate Buffer retrieval (I have also tried Tris-EDTA) on 97C, used a commercial block of endogenous HRP, and 3%BSA to block nonspecific binding. My primary antibody is a nuclear marker, and is biotinylated (I have also tried un-biotinylated). My secondary antibody S-POD (also tried Envision for un-biotinylated Ab) and I use DAB as my chromogen.
When developing my chromogen I observed little no-specific staining, mostly in the areas that have dried. However, I did observed a lot of small brown DAB spots that mostly fade once I counterstain. And the biggest problem is that I didn't observe a single positive nuclear staining in 3 separated experiments. In each experiment I used pancreases from 3 different mice.
I observed a sufficient viral titer, which meant that the virus is present in the pancreas and specific positive cells with no background staining in my positive control which meant that the protocol and my work was ok?!
I know that the maybe I should think about changing my viral marker, however, this marker is mostly abundantly present in other tested organs.
I was wondering if the pancreas as a organ has some specific guidelines that I must follow, or if someone has some tips or tricks for pancreas staining in IHC!
Thank you for your help and suggestions,
Fran
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10.1007/978-1-62703-287-2_3
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We're suddenly unable to get a good protein content after cell lysis preparation.  I mean, almost no protein by coomasie blue staining of the gel, yet our protein standards/ladders show up fine. Previously, I had no problems with overall protein content or detecting desired bands with my protocols.  We've purchased new RIPA buffer and protease inhibitors; varied lysis time, sonication, boiling, loading buffer...everything we can think of and none of it has made a difference. Does anyone have any ideas? Something I'm missing? Coinciding with the onset of this problem are other cellular changes that suggest our mice may be harboring a retrovirus. Is it possible that a viral protease is untouched by our inhibitor cocktail and be chewing up the proteins in our samples?  I did try lysing with boiling laemmli buffer to overcome that possibility; it didn't help, but I may not have performed that correctly.  
Any ideas or thoughts?
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I suggest using 10x Laemmli Sample Buffer from Ecotech Biotechnology. You do not need to dilute your protein too much if you use this buffer.
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Hello,
I am trying to measure the cytokine storm markers following SARS-Cov-2 viral infection of lung cells and macrophages under different conditions. However, finding a safe way to deactivate the virus while preserving the markers is not straightforward to find in literature, any suggestions?
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Although infection with coronavirus regarded as viral infection, however, its effect on immunity may lead to decreased immunity of the human body and lead to opportunistic infection, so maybe an introduction of potent antibiotics like 4th generation cephalosporin to be followed by other common usual antibiotics which currently used could have a potent effect in increase the improvement rate, especially for those with abdominal symptoms.
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Hello, I am looking for a computational simulator of in vitro infection of cells by virus, to be used for teaching purposes. Do you know any tools/resources ? I would like students to set infection conditions, such as MOI, time, temperature and could visualize outcomes of infections as cytophatic effect, for example.
We usually do actual infections in the lab, but we are looking for a tool to perform this activities also in the virtual platform of the course.
Thank you very much in advanced
Kind regards.
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If NAT(nucleic acid test) shows negative and CT shows positive, which should we believe?
Can we calculate their degrees of confirmation to make decision?
There are many confirmation measures. Are they practical?
Are they helpful for diagnosing viral infection?
The attached figure shows that NAT has lower sensitivity (0.5) and higher specificity (0.95); CT has higher sensitivity (0.80) and lower specificity (0.75).
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Welcome to discuss!
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The adaptive, or acquired, immune response takes days or even weeks to become established—much longer than the innate response; however, adaptive immunity is more specific to pathogens and has memory. Adaptive immunity is an immunity that occurs after exposure to an antigen either from a pathogen or a vaccination. This part of the immune system is activated when the innate immune response is insufficient to control an infection. In fact, without information from the innate immune system, the adaptive response could not be mobilized. There are two types of adaptive responses: the cell-mediated immune response, which is carried out by T cells, and the humoral immune response, which is controlled by activated B cells and antibodies. Activated T cells and B cells that are specific to molecular structures on the pathogen proliferate and attack the invading pathogen. Their attack can kill pathogens directly or secrete antibodies that enhance the phagocytosis of pathogens and disrupt the infection. Adaptive immunity also involves a memory to provide the host with long-term protection from reinfection with the same type of pathogen; on re-exposure, this memory will facilitate an efficient and quick response.
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Please see the following PDF attachment.
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How much favourable is -6.78 delta G value for docking a ligand for an enzyme, please help me with this as it will help me intern to create in-silico evidence for a viral infection.
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Ibelieveare belive there is no criteria for delta G.
it depends on your active site and your ligand.
I worked on a project and I analyzed the known inhibitor delta G was -3.7 which computationally can be considered poor an unfavourable but this inhibitor is better than two drugs performed -4.6 and -4.5.
again it depends on the acive site and your ligand if your ligand fit inside the active site with no clashes or bump don't care about the delta G.
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I am not fully aware of all investigators.
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Lymphocytes are a type of white blood cell that play several roles in the immune system, including protection against bacteria, viruses, fungi, and parasites. Lymphocyte increased in response to infections or cancer. However, in covid-19 infection the lymphocyte decreased.
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The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. Some of COVID-19 patients enrolled, severe cases showed significant and sustained decreases in lymphocyte counts but increases in neutrophil counts than mild cases. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8 + T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-CD8+ T cell ratio (N8R) were identified as the most powerful prognostic factor affecting the prognosis for severe COVID-19.  
 The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R may serve as a useful prognostic factor for early identification of severe COVID-19 cases.  
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Immunoglobulin is used to treat viral infection. There are different immunoglobulin already used for the treatment of different viral diseases. Whether immunoglobulin is available or not against the corona virus.
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Please take a look at this useful RG link.
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I have two groups of viral sequences classified by year of isolation. How do I compare between these groups to determine any genetic differences that may have contributed to an increase in the infection rate of the virus?
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Make Phylogenetic tree with the orthologues of the viral genomes. Use partition and concatenation technique.
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I have used the statics to define the diseased from Non diseased. however this comes by improving the process of laboratory diagnosing viral infection. These to best of knowledge , independently were done which cause redundancy and noise in the final rests , are they ever possible factors to include to have the true decision rather than cut-off point inclusion and exclusion since it is precedent by type I and type II errors any suggestions!
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Start reading this paper on Research Gate
Or simply google Researchgate & 'Diagnostic accuracy" and find plenty of literature.
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I am using IgM Mac ELISA for diagnosis of viral infection in our laboratory. I am strictly following the protocol as per the instruction from the manufacturer, but unfairly getting the high absorbance value in negative control. Have tried with all trouble shooting and even changed the kit batch number too. So can anyone help me out in rectifying the issue and solve it.
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Interesting, but how does your positive controls behaves? I suggest you re-validate your ELISA apparatus and if possible, proceed on inter-laboratory testings as a form QA; compare outcomes.
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So, I can't wrap my head around the logic behind these calculations. I've figured out 2 methods, but they give me different answers. So I have 40 wells (80, 000 cells each), and I need 300 uL of virus (MOI 1) in each well.
Stock Concentration=1.81 x 10^9 PFU/mL; 5 uL of this is kept in each eppendorf
Method 1:
1.81 x 10^9 PFU/mL = 1.81 x 10^6 PFU/uL = 9.02 x 10^6 PFU/ 5 uL
So, we have 9.02 x 10^6 virions in each eppendorf.
And so, if we take the 5 uL of stock and add it to 2257.5 uL, we essentially end up with 9.02 x 10^6 virions in 2262.5 uL. This means that the concentration has become 4000 PFU/ uL. And so, 20 uL of this diluted virus contains 80 000 virions.
So, then I added 20 uL of this diluted virus and topped it off with 280 uL of media. This step is what confuses me. I understand that this may dilute the virus more, but don't I still have 80 000 virions in each well regardless? So the MOI is still 1, and shouldn't the cells still be infected?
Method 2:
I need 80 000 virions per well, with 40 wells. So, in total I need 3.2 x 10^6 virions. I have 1.81x10^6 PFU/uL stock. So, 3.2/1.81= 1.768 uL. So, this means i need 1.768 uL of stock. I have 40 wells which need 300 uL media each = 12 000uL media total.
Hence, I need 12 000-1.768= 11,998.232 uL of media added with 1.768 uL of stock concentration virus, to give me 12 000 uL of diluted virus with 80 000 virions for each 300 uL.
I already performed my infection with method 1, but I feel that it is wrong. Method 2 seems to make sense. Can anyone confirm and explain the correct logic behind it? Thanks.
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" don't I still have 80 000 virions in each well regardless? So the MOI is still 1" Yes your are right the ratio number of virus/number of cells is still 1. The volume of infection is not (very) important; it should cover the cells to avoid them to dry. (of course if you put viruses in a very large volume there is little chance that they could contact the cell) . remember that with a moi of 1 only 2third of the cells will get at least one virus (so 1/3 will not be infected at the first round of infection)(Poisoon's law)
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What are the data needed and exclusion criteria for the researches studying the prevalence of infectious diseases or viral infection incidence in epidemiological researches?
How to design the study and what are the suggestions to get a good questionnaire form?
Regards;
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I am trying to generate a stable cell line using lentiCRISPRv2 infection with my desired gRNA. This plasmid has the Puromycin selection marker. I used 2ug/mL Puromycin for selecting cells, but some cells in control group (No viral infection) are still present even after 3days of Puromycin containing media. I changed the media every 24 hrs. Can anyone guide in this how these cells are becoming resistant?
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I would recommend to make the kill curve first , However i did several CRISPR/Cas9 knockouts in A549 cells and i can suggest you to go up to 2.5µg Puromycin with no problems.
I don't think the cells can antibiotic develop resistance on their own !
Good luck!
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Hi, I am doing my macrophages culture from Porcine PBMCs of 3 months old piglets, So is it necessary that checking of maternally derived antibodies of a particular virus in that species? Again we have to give viral infection to macrophages after its complete growth and then harvest the cells. So please suggest me checking of MDA in piglets serum by cELISA is necessary?
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Since you are isolating macrophages, culturing them and then infecting them in vitro, I do not think you need to assess the presence of MDA. Your macrophage cultures would be free of serum/circulating antibodies, thereby minimizing any impact. If you were doing in vivo infections, you would have to give some consideration to MDA (depending on how prevalent the virus is in your pig colonies and how long maternally derived antibodies stick around in weanlings).
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I have read many papers when the author refer to a MOI value, I know what MOI means but I dont know what is a good MOI
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Thanks. Your answer help me a lot
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the potential risk for Reye’s syndrome among children and teenagers when Infection with viral diseases and take Aspirin is taken as a hypothermia
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Reye’s syndrome is a rare, but extremely serious condition that affects children and teenagers recovering from viral illness such as the flu or chickenpox. It most commonly causes swelling of the brain and a large build up of fat in the liver. Rapid breathing, diarrhoea, continuous vomiting, irritable or aggressive behaviour excessive lethargy and decreased level of consciousness are some of the symptoms of Reye’s syndrome. The exact cause of Reye’s syndrome is not known, however links have been made between patients with an underlying fatty acid oxidation disorder and taking aspirin during viral infections. Please take a look at the following links for more details.
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I have 4 X 10 E10 PFU of my viral stock and I have to follow a protocol for the viral infection which suggests a MOI of 0.01, diluting the virus in 40 mL of BHI for infection per O157:H7 (containing 1.5 X 10 E8 cells). I tried to make calculations but I'm not sure they are correct. How many microliters do I have to use from my viral stock?
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Virus stock titers:
4X10^10 pfu/ml ->40 folder dilution ->1X10^9 pfu/ml
Set the volume as X,
X ml times 1X10^9pfu/ml divided by 1.5x10^8 cells=0.01 pfu/cell (MOI)
X=0.01 times 1.5 x10^8 divided by 1X10^9=1.5X10^-3ml =1.5ul
Based on your description, you will need 1.5 ul 40-folder diluted virus stock.
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Hi,
Currently i am performing QC on new RNA-seq data. My fastq files were generated by Paired-end sequencing with a read length of 151 bases. Total rna was extracted and depleted of rRNA. RNA was isolated from a viral infection experiment. Upon looking at the fastqc output of the un-trimmed fastq files, most of the parameters were satisfied except for those mentioned above. I did get a warning for over represented sequences but these were found to be sequences related to the virus genome due to cellular replication.
My question is, that is this issue due to adapters of more likely a subsequence of the experiement conditions. I have attached pictures of output for failed modules.
Thanks.
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Hi!
You can trim your adapter sequences and run fastQC again.
About GC content - in one organism the are sequences with different GC content. It could be contamination but you can separate your reads based on GC content and analyse all peaks individually.
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Hello,
I would like to detect viral infection in may plant tissue cultures. I know there is Elisa tests and RT-PCR methods but all of them are refered to one specific virus. Is there any methods to screen my plants and after positive outcome make decision about virus identification or to throw out same cultures. I would be grateful for any sugestions.
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Dear Iwona
Virus isolation in cell cultures, immunofluorescence based assay and molecular techniques to determine nucleic acid, have all been used successfully to detect and identify the viruses. Since each of these established methods has several limitations, improved techniques of virus identification and quantification, such as mass spectrometry and next-generation sequencing/metagenomics are explored to overcome their limitations.
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 It seems that viral infections have been described for virtually every human organ except for Bone. Certainly there are bony sequelae of viral infections, such as osteomyelitis variolans seen in smallpox,  but I am not aware of a viral osteomyelitis per se. Is it possible that some pathologic or radiographic findings, may be explained by an as yet undescribed viral osteomyelitis?
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I find a recent study of association between Rhinoviruses and Kingella kingae osteoarticular infections in children, but there is not direct damage of bone by virus. Certainly, certain viruses may be the aetiological agents as written above.
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My question refers to how to prevent viral infection by the pulverization of potential mice fecal material / urine on the leaf litter that is been examined.
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I Follow the answers
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Our lab has a frozen stock solution of polyinosinic:polycytidylic acid (poly IC). It's been in our -20C freezer since 2004. We've used it on and off since then. The concentration is 2.5mg/mL
I recently defrosted it for a cell culture. But once it became a solution, I saw a transparent mass in the Eppendorf. It looks like a small piece of plastic wrap/Ziploc bag. I tried to disperse it using a P1000 pipette, but the clump would not dissolve
I looked at the literature, and no paper mentions a precipitate.
Does anyone know if this clump is normal? Or should I get a new source of poly IC?
Thanks very much.
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Incubation at 37 degree C for 15-30 min should help.
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what amount of viral titre is being used for infecting c.elegans?
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Try to check the viral titre used in other animal models
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Can we use the processing waste of shrimp affected with viral infection, in particular WSSV, in aquafeed formulation?
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The short answer is No. However, Since the virus is an enveloped virus, you may deactivate it with high heat during the processing. You need to use the food only for fishes in which WSSV does not have any effects and only in the areas where WSSV is already prevalent. I strongly suggest not to use this food in non-WSSV affected areas. Goodluck
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Following the Zika virus epidemic in the Americas, a large proportion of the population has likely been immunized to Zika virus. Kawiecki et al reported DENV-2 enhancement by Zika virus-induced antibody response in vitro. Are there arguments that can make us expect more severe dengue outbreak in the Americas in the coming years?
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Hi Harold, I was at the American Society of Virology conference last month, and Dr Eva Harris presented some results on that subject on a Nicaragua population if I recall correctly. You might want to check her publications or contact her directly. She mentioned this Review you might have seen already: https://www.ncbi.nlm.nih.gov/pubmed/28655548 (Andrade et al, Virus Research June 2017).
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Explanation:
After storing viral stocks for too long at -80oc are used to infect (MOI) the suitable cell line  show's less infection as compared to the freshly prepared viral stock. What does it indicate? some of the virus particles in the stock get either dead, disappear or weak, as we are aware virus are called as biological puzzles outside the host they behave as dead and can stay too long even for years and while in inside their host they get activated and start replicating? Why is this decrease in infectivity when stored for too long?  
Thanks.                                                                                             
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A virus is a complex microscopic structure. There are three major types: filament (like Ebola), icosohedral (like cold viruses), and enveloped, like influenza and HIV. Of the set, the most sensitive to freeze damage are the enveloped viruses for the same reason cells are sensitive. The envelope is a cell membrane from the host and it isn't very strong.
Water expands when it freezes. It doesn't expand by much, but it's a very strong force. There's an old demo of a cast iron ball filled with distilled water. The ball was completely filled, with no air pocket. A tight fitting screw was used as a stopper. The ball was placed in dry ice inside an explosion containment chaimber. The ball exploded with enough force to embed fragments of iron into the steel of the door.
So that's a major factor. When ice forms, it pushes all viruses apart somewhat. When they thaw, the question is, will they go back together well enough or not to function. Even the tougher icosahedral, or filament viruses won't all be ok after thawing.
When you add glycerin, that acts as a glassifying agent. It prevents water from forming crystals, so it doesn't expand.
Another factor is local desiccation. As water freezes, it forms crystals, and those crystals force salts and non-water elements out. You can experience this in sea-ice in the arctic. Fresh ice you can't use for water because as the sea-ice forms, it creates tiny pockets of concentrated brine. But as the brine drains down through the ice, it becomes fresh water. The same process happens inside cells and viruses that get frozen, but at a tiny scale.
Those dry pockets with high salt concentrations damage proteins and DNA. Molecules can also be compressed and sheared into pieces by the ice crystals. One technique for dealing with that is to add sugars. Even sucrose will help in high concentration. It helps protect DNA and protein molecules during freezing.
Most of the damage is done by either the freezing process or by thawing. The more times you cycle it, the more damage you will do. There is some slower degradation that happens at very low temperature, but it's pretty slow. It also depends on your storage temp. Storing in liquid nitrogen or at -80 C will do better than storage at -20.
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I am looking for virus inactivation to discern the effect caused by virus replication itself and the effect caused by the already induced IFN by the virus infection (prsent in the vehicle of the virus). 
I irradiated with 240nm UV to vanish any replicative form from my supernatant. Now I am wondering if the irradiation also inactivated the IFN present in the supernatant. I checked, with the available lamp in our lab, that cells just die when cultured with UV-irradiated media. 
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Thanks you guys! I'm on it. 
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I want to use influenza A virus rescued from plasmids to infect MDCK cells. Could polybrene can improve the efficiency of the infection of influenza A virus?
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Could anyone recommend protocol for production of infectious Varicella zoster virus (VZV) on MRC-5 cells? 
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You most welcome
Houda
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I am using RetroNectin to coat plates for viral infection. The protocol (see the link below) says to block with 2% BSA for 30 min and wash with PBS after removing the RetroNectin coating. Does anyone know why these steps are needed, and would it affect the viral infection efficiency if not block/wash?
Also, the protocol says to spin down the virus first, and then add cells. Would it make a big difference if I spin the virus and the cells together? I am afraid that I would lose some virus if spin down the virus first and remove the supernatant.
Thanks for your help! 
RetroNectin protocol link:
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Typically, the BSA is added to reduce non-specific protein-protein interactions, the result is a decrease in noise levels and an increase in specificity, sensitivity, or in this case transduction efficacy. I would recommend to do it, and I have never tried to skip this step. 
You have two "classical" way to perform your transduction : the first one is to add the virus spin the plate, remove the supernatant and then add the cells, and second is to first add the cells and then put the virus and spin your plate. 
It will depend on what type on virus you're working with and what type of cells also. In my case I used primary CD4 T cells from mice. As I had to make them proliferate first to transduce them, here was my protocol : 
 Day 0: P24 Coating.
- Coat p24 wells with 3ug/ml of anti-CD3 at least 3H at 37°C.
-Wash with PBS
- Add 40ug/ml (200ul) of Retronectin and incubate O/N at 4°C
 Day 1: Stimulation of CD4 T Cells.
- Add 1ml of PBS 2% BSA and incubate 30 mins at room temperature.
- Harvest CD4 T cells.
- Plate cells at 500 000 cells in 1ml of OptiMEM medium containing 2ug/ml of anti-CD28 and 1 UI/ml of IL-2 (It also works with complete RPMI).  
Day 2: Transduction.
- 18H after stimulation (Doesn't work well after 24H) replace media by virus-containing  media (My virus was in OptiMEM) and centrifuge plates at 3000 RPM 32°C for 1,5 hour (The less volume you put the more you'll increase the transduction efficiency).
- After transduction add 500uL of OptiMEM containing 2X cytokines and CD28 mAbs.
Day 3: Same as Day 2 (Replace medium and put a second wave of virus, spin and add medium containing 2X cytokines
Day 4 : Put cells in complete RPMI medium 
 - Put cells in Complete RPMI media (+IL-2 + aCD28).
Depending also on what is your readout, you can add anti-IFNg it improves a lot the transduction efficiency in my case but I don't know whether it is because my cells survived/proliferate more or whether it was due to a better transduction efficiency.
I don't know if it helps. 
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Varicella zoster virus is the cause of chickenpox and zoster often produce a highly contagious and mild disease in children but if an adult acquires a primary VZV infection, it may lead to more severe disease.
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This may be related to immune response to VZV infection and various virus-host cell interactions. There are many evidences in favor to the crucial role of cell-mediated immunity in VZV infection. It was observed that an impaired cellular immune response favors severe forms of varicella and is associated with higher rate of complication and lethality. Likewise, the spread of the VZV in the body, after secondary viremia, is mostly by the intracellular route, rather than by release of cell-free virus in vesicular fluid. It is established that cell-mediated immune responses  enables virus transport via T cells to the skin but also lysis of target cells by cytotoxic T cells stimulated with VZV antigens. Natural killer cell and antibody-dependent cellular toxicity to VZV have been reported The strongest cell-mediated immune responses to VZV appear to be in early adulthood with declining with advancing age, starting at age 50. Hypothetically, it is possible, that appearance of the excessively strong and robust cell mediated immune response to VZV in adults may be sometimes harmful for the host leading to excessive damage of VZV infected cells which consequently more severe disease and higher percentage of complications.
  On the other hand, it is possible that adults have an enhanced primary VZV viremia, which predisposes severe forms of disease and does not leave much time to the immune system to react.  However, this requires further study for better understanding of differences in age-specific antiviral immunity in children and adults.
 
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I am trying to understand the expression of Fc receptor on microglia post viral infection. Hence, I am not adding Fc Block in the cells. However, as a control should I use Fc Block in the isotype? I assume that I should add Fc Block in the isotype controls otherwise my sample and isotype may show the same signal? Any suggestion?
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you most welcome
Houda
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Many scientists talked about first report of VNN in marine fish In many of scientific reports such as:
1) The first report of viral infection in Asian sea bass was made by Glazebrook et al. (1990), who described a picorna-like virus associated with mortalities of 15 to 20 d old larvae. This disease was also investigated by Munday et al. (1992) in Asian sea bass and is now recognized as being caused by a piscine nodavirus (Azad et.al., 2005).
2) Also, according to first record of VNN in North America by (Curtis et al., 2001), the viral aetiology of mass mortalities of white seabass, Atractoscion nobilis, cultured in southern California, USA was examined. Disease outbreaks occurred in juvenile fish reared at two culture facilities from June to December 1999, with clinical signs such as anorexia and erratic swimming motion. Microscopic lesions observed in moribund fish included marked vacuolation of brain, spinal cord and retina. The piscine nodavirus (Betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues by electron microscopy, indirect fluorescent antibody test (IFAT), reverse transcription–polymerase chain reaction (RT–PCR), and isolation in cell culture. The agent was identified as one of the four known genotypes of piscine nodavirus. In addition, a similar nodavirus was also detected in fish samples from disease outbreaks at the same facility in 1992. In the last decade, VNN has been reported among cultured populations of marine fish worldwide and this paper is the first record of the agent in North America (Curtis et al., 2001).
Meanwhile, we succeed to isolate and identify of VNN virus as first time in Golden grey mullet (Liza auratus) in the Caspian Sea in 2004 (as attached article). 
Furthermore, according to knowledge of my dear friend, Prof. G. Bovo from OIE reference Lab. of VNN, he showed me in EAFP 2013 Conference in Split, Croatia a document about first report of VNN in Senegal in 1996 (In second attached file).
What do you think about original and correct time about first outbreak or occurrence of VNN in the world?
Thanks again
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Yes but its not just my opinion. I have found many oyster herpesvirus by TEM before 1990 but the scientific community and the OIE will not accept that they are OSHV1. Likewise I spoke to T. Miyazaki about iridovirus and he agrees that today molecular confirmation of identity is essential.
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The trypsin can change the VERO receptors used by dengue virus infection.
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A large number of proteins are reported to act as entry receptors for dengue.  It has been argued that to efficiently spread, dengue has to be able to infect both mammalian and insect hosts, and also infect a variety of different cell types within those hosts.  The non-specific nature of dengue infection makes me skeptical that trypsin would be able to block virus entry completely.  Still, if you try, think about how you will sort out any drop in infectivity (due of receptor loss) from a drop infectivity (due of trypsin toxicity).  You might have to tag your virus so that you can compare cell viability against virus entry. Good luck.
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I need isolate the CDV in cell culture. 
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I think Pierre is right- the success of isolation is very dependent on the viraemic phase. Previous day -you could prepare some Vero cells in a small flask with 50-60% confluency. Then inoculate the monolayer with blood/plasma with sufficient amount of serum free media/sterile PBS to cover the monolayer- incubate at 37 degree incubator with 5% CO2.  Shake the flask every 10 minutes - after 2-3 hrs wash the monolayer at least twice with PBS followed by addition of fresh media with 2-5% serum. You'd probably start seeing some CPE 4-5 days later if there is live virus in your inoculum.
Let me know if you require any other information. Good luck with the isolation
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Is shrimp white spot virus can also infect fish
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As far as I know it is not possible but molluscs (e.g. blue mussels, different oysters) can bear WSV and distribute it to crustaceans back. I don't know if those animals replicate the WSV.
Best wsihes
Sven
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The 8ug/ml polybrene works well for the viral infection of multiple lung cancer cells in my hands. But recently I got a cell line isolated from mouse lung. 8ug/ml polybrene (with or without virus) can kill all the cells in 24 hours. Has anyone try to infect cells without polybrene? How low will the efficiency be?
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Hi Yongming,
It really all depends on your cell lines. I sometimes forget to add polybrene to my lentiviral transductions but get very high infection rates. So if your cell line is very sensitive you may either use a drastically lower concentration or don't you any at all, it may just work very well.
Good luck!
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viral infection (HIV-HBV-HCV) in hemodialysis patients are common now the question is : is there any viral particles passage possibility from the pores of dialyser ?
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Pleas read this article:
The pores in dialysis tubing are too small to allow the passage of virus, unless there were a defect. However, that is not the only way a virus could be passed between dialysis patients.
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During my preparation for state boards in Germany, I have come across a trend particularly for viral infections. In complete blood count analysis during an acute infection, one often finds an increase in Lymphocytes as well as a decrease in Thrombocyte counts. Is the thrombocytopenia a reaction due to this lymphocytopenia (due to bone marrow proliferation and cytokine release), or are the thrombocytes being degraded as a result of the infection (increase in spleen size due to sequestration or increased turnover of thrombocytes)? Any insight on this would be valuable. 
Thanx
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Dear Michael,
maybe this rather new review will help to answer your question!
Best,
Mathias
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I have been trying for quite sometimes now, however haven't been able to successfully get the transgene into the cells.
I have tried to use Polybrene 8ug/ml with various MOI starting from 50 up to 1000. I have also used LentiMag (magnetic particle) which suppose to enhance efficiency but still no luck.
I wanted the cells to overexpress a particular transcription factor in one cell, and shRNA specific to the same TF for Knockdown.
I produced the lentivirus based on Didier Trono protocol with PMD2G and PSPAX2 as the packaging and envelope. My transfer vector is purchased from system bio, Cumate inducible lentivirus backbone and the TRIPZ from GE Healthcare.
I used DMEM with 10% FBS as culture media, then collect supernatant over 72 hours and concentrate with the 10kDA CENTRICON.
I then centrifuge it at 10,000g for 4 hours with a sucrose cushion. Pellet is then resuspended with 200ul of PBS. I tested the titre of my virus by infecting 293T with different virus quantity. 
I always get approximately 1x10^8 - 1x10^9 IU/ml.
So I don't really know why my human iPSC are not infected properly.
Following are brief protocol on how I carried out the infection:
- I seeded approximately 75,000 cells into 24 well plate a day prior to infection
- On the day of infection I add 8ug/ml of Polybrene to E8 culture media and incubate for 15min
- I then add the appropriate ammount of virus from MOI 50 -1000, incubate at 37oC for 24 hour
- I have also tried double infection the next day however it doesnt seem to improve.
I was wandering if serum from the FBS cause any issue with the low infection rate in my human iPSC??
I would highly appreciate it if anyone could help me :)
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FBS might interfere with transduction. We performed infection with  highly concentrated vectors (with ultracentrifugation) in serum-free medium. Also, we perforemed the transduction step in a feeder-free system - without MEFs - on matrigel. Hope this is helpful
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  • Bacterial and Viral lung infections are a major cause of morbidity and mortality in hospitals as well as community. With neutropenic patients on a raise ..some fungal infections are also increasingly observed.
  • If we can have a/or some biomarkers to differentiate bacterial compared to viral and or fungal infections it will be useful in addressing the problem on early stage itself.
  • Are there any biomarkers available to differentiate bacterial infections from viral as well as from fungal and or parasitic infections that are available and in use or in research??
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Can someone please tell how many " cells" is to be seeded per well for HaCaT cells in a 6 well plate.
How much should be the ideal confluency for lentivirus infection?
Thanks
Apoorva 
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Hey thanks Gaurav
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Is there any relation established between viral infection and pain modulation?
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I think your question needs a little clarification. Viral infection is something that we can characterize precisely like we can know the quantity of viral particles, the interaction with other viruses...however the pain modulation is something really hard to characterize if not impossible. Seeing the previous answers, I think that the inflammation axe can be considered as promising but for me it is not sufficient alone.     
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correction to my last entry Should we be testing tissues from GBS patients for Zika?
(Last entry - From what tissues in the body can you isolate Zika virus? - I would like to know whether a real try has been made to isolate the virus from the affected babies, or from infecting mothers and a,Lao if it can be isolated those with GBS should be tested). 
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I think more data is needed for sure, and sending samples from a variety of sources is important. CDC recommends the following:
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