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Influenza - Science topic

This group is for discussion about any influenza research related topics
Questions related to Influenza
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Hello
1. Why this system does not recognise copies of same publication and remove them?
2. Also: My article on influenza and Pneumococcal vaccination was published in December and is my latest publication, yet your system decided from its head to make it published in January 24 and put it down in the list. Why is that?
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I don't know. It could be related to the system rules.
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Would the removal of methionine and alanine from the N-terminus of the Influenza A H3N8 nucleoprotein affect its function or structure, considering the goal of leaving a serine residue to create an optimal TEV protease cleavage site? My objective is to purify the nucleoprotein for use as an antigen in developing ELISA kits for diagnostic purposes, and I want to ensure that this modification does not compromise the protein's integrity or its suitability for diagnostic applications.
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My guess is no, for the following reasons:
Structure:
The N-terminal few residues don't seem to fold into any specific structure as they don't resolve in the various crystal structures of NP.
Fusing a large tag to the N-terminus (e.g. maltose binding protein and a factor Xa cleavage site; Elton et al 1999 JVI) worked just fine for purification and biochemical studies
We also got function in a minireplicon assay with an NP that we'd lopped the first 11 residues off (Elton et al 1999, virology). Though that was using a very old fashioned minireplicon system (it was last century!) and I suspect we mght not get quite the same result with a modern pol I-launched system.
Antigenicity:
The only time we've ever noticed any strain/sequence-dependent difference in NP antigenicity is with the commercial anti-NP clone AA5H, which doesn't react well with pdm09 NPs, or at least not all of them. We've never formally mapped the epitope using this, but I don't think it's N-terminal. This doesn't say that animals won't produce abs against the region you're interested in, but NP is very immunogenic so I doubt it would be a major confounder even if they did.
Only one way to find out for sure though :)
Cheers
Paul
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I have done 3 blind passages to concentrate the viral stock in the supernatant without adding TPCK Trypsin. After passage 1 my Ct value for INF A was 36.96. After P2 the value decreased to 33.36. However, I was expecting a much lower value in the mid-20s. I am freeze-thawing the flasks at -20 degrees to lyse the cells and release the virus into the medium. Is it the correct approach? and can I get a much higher titre without adding TPCK Trypsin?
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Dear Colleague,
I hope this message finds you well. Culturing Influenza A virus in MDCK cells typically involves the addition of TPCK trypsin to facilitate viral replication and ensure high viral titers. TPCK trypsin cleaves the hemagglutinin (HA) protein, enabling the virus to become infectious. However, you are inquiring about the possibility of obtaining high viral titers without the addition of TPCK trypsin. Below is a detailed and logical discussion on this topic.
Culturing Influenza A Virus Without TPCK Trypsin
Role of TPCK Trypsin
  1. Cleavage of Hemagglutinin (HA):TPCK trypsin cleaves the HA protein of the Influenza A virus, which is essential for viral entry into host cells and subsequent replication. Without this cleavage, the virus remains non-infectious, and the replication cycle is interrupted, leading to lower viral titers.
Alternative Approaches
  1. Trypsin-Free Culture Media:Using trypsin-free culture media generally results in significantly lower viral titers because the HA protein remains uncleaved. Some labs have experimented with alternative proteases or conditions that might support HA cleavage, but TPCK trypsin remains the most reliable and widely used method.
  2. Genetically Modified Virus:One potential approach is to use a genetically modified strain of Influenza A that can replicate without the need for HA cleavage by trypsin. However, such strains are not typically used for standard virology studies.
  3. Endogenous Proteases:Some cell lines express endogenous proteases that can partially cleave HA. However, MDCK cells typically require exogenous TPCK trypsin for optimal viral replication.
Experimental Considerations
  1. Optimization of Conditions:pH and Temperature: Ensure that the culture conditions (pH, temperature) are optimal for both MDCK cell growth and viral replication. Infection Dose: Use a higher multiplicity of infection (MOI) to compensate for the lower efficiency of viral spread without trypsin.
  2. Protease Activity Monitoring:Monitor the culture for signs of cytopathic effect (CPE) and viral replication using assays such as plaque assays, hemagglutination assays, or qPCR. Assess the presence of cleaved HA using Western blotting or similar protein analysis techniques.
  3. Alternative Proteases:Experiment with other proteases that might be able to substitute for TPCK trypsin. However, this requires careful validation to ensure they effectively cleave HA and do not adversely affect cell viability or viral integrity.
Practical Recommendations
While it is theoretically possible to culture Influenza A in MDCK cells without TPCK trypsin, achieving high viral titers under these conditions is challenging and often impractical. TPCK trypsin remains the gold standard for this purpose due to its reliability and effectiveness. If you decide to experiment with trypsin-free conditions, I recommend conducting parallel cultures with and without TPCK trypsin to directly compare the effects on viral titers.
Example Protocol with TPCK Trypsin
  1. Cell Preparation:Seed MDCK cells in culture flasks or plates and grow to 80-90% confluency.
  2. Virus Infection:Infect cells with Influenza A virus at the desired MOI. Allow the virus to adsorb for 1 hour at 37°C in a CO2 incubator.
  3. Media Change:Replace the inoculum with serum-free DMEM containing 1-2 µg/mL TPCK trypsin. Incubate the cells at 37°C in a CO2 incubator.
  4. Monitoring and Harvest:Monitor cells daily for CPE. Harvest the supernatant when CPE is observed (typically 2-4 days post-infection) and determine viral titers using a plaque assay or other appropriate method.
Conclusion
While culturing Influenza A virus in MDCK cells without TPCK trypsin is not impossible, it poses significant challenges and typically results in lower viral titers. TPCK trypsin facilitates efficient viral replication by cleaving the HA protein, making it a critical component for high-yield virus production. If you proceed with trypsin-free cultures, careful optimization and parallel comparison with TPCK trypsin-containing cultures are recommended.
With this protocol list, we might find more ways to solve this problem.
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Hi :)
How should I design primers for (-)ssRNA samples from Influenza A and hRSV viruses?
I need to select the protein, locate the gene sequence, and then use it to design primers. Should I retrieve the cDNA sequence from NCBI to work with?
The term 'cds' in FASTA means that it is the sequence without introns. Is it okay to use this type?
if you have any guides, videos or documents you would like to share with me, I would be very grateful.
Thanks
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Designing specific primers for reverse transcription (RT) involves several key considerations to ensure efficient and specific amplification of the target RNA. These considerations include target selection, primer length, GC content, Tm calculation, avoidance of self-complementarity, specificity, positioning, optional incorporation of a T7 promoter for IVT, quality control, and optional modifications. Following these guidelines can help optimize primer design and ensure successful RT-PCR amplification of the target RNA.
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Dear Researchers:
Could you please share some simple cures or prevention for COVID-19, Cold, Flu or Influenza, and possibly Other Viruses, and Cancers?
Updates on Oct. 10, 2023: First, many thanks to all contributors to this discussion. Here are some Natural Approaches found from surveying literature in medicine to Boost our Immune Systems against viruses such as COVID-19, Cold, Flu or Influenza infections and to avoid/minimize developing further inflammations in the lungs and hearts caused by some of those viruses:
Give it a try, please! Especially if you increase your Vitamin D level to a required level and consume Vitamin C sources, e.g., oranges, on a daily basis, you can check how rarely you would catch the virus. Or, even after catching the virus, the virus will likely develop very mild symptoms in your body.
1- Daily uptake of Vitamin D pills up to 100 IU per 1 kg weight is safe and very important, recommended by Afshar et al. (2000) and Dr. Hamid Sajjadi in an interview, to RAISE the Vitamin D level in our body to the POINT which is REQUIRED to BOOST our IMMUNE SYSTEMS against Viruses and Diseases including Cancers.
Vitamin D daily use needs to be adjusted based on our body weight.
Please read the following article by Afshar et al. (2000) about the importance of vitamin D and the required daily dose of it (Up to 100 IU per 1 kg weight) to boost our Immune Systems.
Please also read the following Review article by Jordan et al. (2022) about the importance of Vitamin D on the level of infection & disease progression for COVID-19. You may find in the article the importance of our Forgotten SUN.
Vitamin D is rarely available in food sources, except in fatty fish which needs to be eaten high enough to get the required amount of Vitamin D for a body.
Another good natural source is daily sunbathing with naked skin; however, in cloudy regions such as Europe, sunbathing doesn't work well.
Vitamin D helps to absorb Calcium in our intestines and thus, in order to avoid excessive absorption of Calcium by our body, it would be better to use Vitamin D pills with Calcium sources such as warmed-up milk and Magnesium sources such as bananas on a daily basis. Because magnesium competes with calcium in our intestines to get absorbed.
Here is a text from A Review article by Kulie et al. (2009) about some of the importance of Vitamin D on our health:
"Vitamin D is a fat-soluble vitamin that plays an important role in Bone Metabolism and seems to have some Anti-Inflammatory and Immune-Modulating properties. In addition, recent epidemiologic studies have observed relationships between low vitamin D levels and multiple disease states.
Low vitamin D levels are associated with increased overall and Cardiovascular mortality, Cancer incidence and mortality, and Autoimmune Diseases such as Multiple Sclerosis. Although it is well known that the combination of vitamin D and calcium is necessary to maintain Bone Density as people age, vitamin D may also be an independent risk factor for falls among the Elderly."
2- Having Good Nutrients including Protein sources, Minerals, and Other Vitamins, e.g., C, A, and E, sources from fresh fruits, vegetables, and nuts. For example, the good sources of fruits and vegetables for these vitamins could be a daily use of 1-2 Oranges for Vitamin C, Carrots for Vitamin A, and Almonds or Sunflower Seeds for Vitamin E.
As Vitamin C is a water-soluble vitamin, the excess of it will be excreted from the body, it needs to be consumed every day to provide everyday vitamin C requirements for the body, as it is the 2nd most important vitamin after Vitamin D to boost our Immune Systems against viruses and diseases.
And, Vitamin B family from grains, poultry, and meat sources.
3- After the infection by those viruses, gargling salty water to disinfect the throat to avoid further movement of the virus into the lungs as the virus may stay in the throat for a few days
4- Inhaling Steamed Fresh Leaves, if not available, the Oil, of Eucalyptus 4-5 times a day for several continuous days to kill the virus in the lungs.
Here is A Review article by Mieres-Castro et al. (2021) about the "Antiviral Activities of Eucalyptus Essential Oils: Their Effectiveness as Therapeutic Targets against Human Viruses"
Australian Aboriginals are very much using Eucalyptus to Treat Infections.
5- Having plenty of Warm Drinks to wash out the virus from our body and dilute the blood to avoid blood clotting.
6- Having enough sleep and daily activities/exercises
7- Kids are proven to have High Immunity Against COVID-19, likely due to having a high amount of Melatonin, the Sleep Hormone, in their blood. So, that is why kids sleep very much as you know.
Melatonin production in our body usually decreases with increasing age. Thus, we may use daily melatonin pills after the infection based on what physicians may prescribe for us.
Here is A Review article by Carrillo-Vico et al. (2013) about the Importance of Melatonin on the Functionality of Our Immune Systems:
8- Avoid Fear/Panic as it Substantially Deteriorates the Functionality of Immune Systems against viruses and diseases.
Here is an interview by Dr. Lauren Deville about How Fear Affects Our Immune System:
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Many thanks, Rohan RANJAN Waliya, for contributing to this discussion!
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This RG open question is linked to the previous about the dramatic evolution (partly unexplainable) of COVID19 in Northern Italy during wave 1.
The previous RG open question is reported below🔴 and resulted in a completely alternative model for the evolution🟨 of SARS-CoV/2 from pre-pandemic phase to pandemic phase.
In this specific RG question, the intention is to create an open discussion on the possible emergence of a violent outbreak of avian flu or similar in central Europe.
This concern arises from a qualitative model that links three events which in the past have always characterized the violent explosion of a bird flu or similar.
---Coronavirus Epidemic/Pandemic;
---Conflict/War partly out of control;
---Pandemic avian flu or similar.
The ABSTRACT of the model can be consulted directly here.. https://www.researchgate.net/figure/46_fig2_367046404
This RG open question will serve to accumulate data both for and against this dire possibility.
Thanks to all the participants.
|--sv--|
🔴The novel Coronavirus in N. Italy, Lombardia 【 COVID19 / 2019nCoV / SARSCoV2 】 shows a fatality rate compatible with SARS-MERS. Why?? MAR.2020. -- https://www.researchgate.net/post/The-novel-Coronavirus-in-N-Italy-Lombardia-COVID19-2019nCoV-SARSCoV2-shows-a-fatality-rate-compatible-with-SARS-MERS-Why
🟨Link between the start of pandemic SARS-CoV/2 (COVID19) and the Huanan Seafood Wholesale Market in Wuhan (Hubei: China): the furin cleavage site of spike protein. FEB.2022. -- https://www.researchgate.net/publication/358443761
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Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants. December 2022. Cell 186(2).
DOI:10.1016/j.cell.2022.12.018.
PMID:36580913.
PMCID:PMC9747694.
RG:366251425.
Abstract.--- The BQ and XBB subvariants of SARS-CoV-2 Omicron are now rapidly expanding, possibly due to altered antibody evasion properties deriving from their additional spike mutations. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals boosted with a WA1/BA.5 bivalent mRNA vaccine. Titers against BQ and XBB subvariants were lower by 13-81-fold and 66-155-fold, respectively, far beyond what had been observed to date. Monoclonal antibodies capable of neutralizing the original Omicron variant were largely inactive against these new subvariants, and the responsible individual spike mutations were identified. These subvariants were found to have similar ACE2-binding affinities as their predecessors. Together, our findings indicate that BQ and XBB subvariants present serious threats to current COVID-19 vaccines, render inactive all authorized antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.
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It is common during the season to increase the incidence of severe influenza, the symptoms of which are similar to Corona 19. In your opinion, is the weather the main cause, or is it the results of infection with Corona, or is it possible that a new variant is currently developing? Are there recent studies in Iraq on this subject?
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Uh oh! , COVID-19 is a "One Thousand and One Nights" story. It seems that we are at the beginning!
Has this epidemic become a point to chronicle date like the One Thousand and One Nights stories? هل أصبح هذا الوباء نقطة تأريخ مثل حكايات ألف ليلة وليلة؟
Let me ask the following three questions:
  • Is there a lack of interpretability and transparency related to this virus?
  • Did we reach a state with this pandemic that is hard to control and monitor?
  • Is COVID-19 one of nature's secretions or it has been fabricated in the laboratories of one or more countries?
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Covid news – live: Cases soar again in India as doctors warn of ‘new symptom’
A new coronavirus strain dubbed Arcturus appears to be driving a surge in Covid-19 cases in India, prompting the country to resume vaccine production and sparking fears it could lead to a rise in cases in the UK and elsewhere.
India on Friday recorded 11,109 new Covid infections, the biggest jump in almost a year. The country’s active case count is now up to 49,662.
The XBB.1.16 strain, a sub-variant of Omicron, has been found in 22 countries, including Singapore, Australia, the UK and the US. Research indicates Arcturus could be one 1.2 times more infectious than the last major sub-variant, making it likely to become the dominant strain.
The spread of the strain, first detected in late January in India, is worrying experts, as it seems to exhibit unique symptoms in children, one of which is conjunctivitis.
The symptoms of the variant include high fever, cough, and “itchy” conjunctivitis or pinkeye, according to Vipin Vashishtha, a paediatrician and former head of the Indian Academy of Pediatrics Committee on Immunisation.
COVID-19 and Influenza Activity
April 2, 2023 to April 8, 2023
These images provide a high-level assessment of respiratory virus activity in Ontario. Provincial percent positivity can be used to provide an estimate of the intensity of circulating viruses in the province. Percent positivity for the most recent week is used to assign influenza and COVID-19 to either a low, moderate, high or very high category. Weekly indicator change was determined by considering a combination of indicators (see Technical Notes). For further details, please refer to the Respiratory Virus Overview in Ontario report.
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I think, AI could advance medical science enormously. Deep Blue lost to Kasparov in the beginning. However, after being taught by chess grand masters it won.
AI of today should the same way be taught by "grand masters" of each and every scientific field and sub-field. It should be taught to write references. And it should be taught to ask questions (suggest research), and not only supply answers.
AI can read the entire literature of medicine. Human professors can hardly reach to read the new literature in his or hers specific sub-sub-field.
And human experts of different sub-sub-fields some times do not understand the expert-language used by the other expert.
Therefore the knowledge that can be obtained, by combining knowledge from different sub-fields is not produced.
Here and now, I do not think that AI can help us against the corona virus.
This might:
I do not think there is any reason to be afraid of AI. It is just a sophisticated search engine. It is humans, and how they use AI, we should be afraid of.
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According to most U. S. state health department records the levels of influenza recorded during the 2020-2021 flu season and the 2021-2022 flu season, when Covid 19 surges occurred, the levels of infection by influenza was below the baseline. Even the national data from the CDC shows the same thing, but no explanation has come forward to explain this phenomenon.
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According to the CDC and many state public health department websites, during the entire 2020-2021 flu season influenza remained below the baseline level in the U. S. In the 2021 - 2022 flu season influenza appeared from December until mid-January and suddenly dropped below the baseline when the omicron surge was declared. It is just too contrived to suggest that the Covid 19 virus could prevent infection by influenza. I suspect it is more likely that the rt-PCR test cannot discriminate between them.
John
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I have a question. Do prior immunity to a virus-like particle vaccine platform affects its immunogenicity for the next vaccination? For example, if we vaccinate an individual using a chimeric VLP consist of an antigen of interest fused into influenza M1 protein as the core, will it have a lower immune response if the individual were vaccinated with other VLP-based vaccine containing the same M1 protein? Will it happen as with the viral vector vaccine?
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The relationship betwween the efficacy of prior immunity and vaccine depends on titer of prior immuinty due to previous vaccination or infection ,if protective titer no need for vaccination ,if not protctive vaccine needed
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Hi Dear researcher
Some Influenza subtypes are recommended for vaccination by WHO. Unfortunately the current database of influenza have not update sequences about influenza. could you help me to find the protein sequences of following subtypes?
H1N1 A/Sydney/5/2021 (H1N1)pdm09-like virus
H3N2 A/Darwin/6/2021 (MDCK-SIAT derived)
B/Austria/1359417/2021 (MDCK derived)
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The isolation of substational numbers of viral sequence variants at highly variable viral protein domains remain a major challanges ,several trials depends on the following......increase sucess of gene cloning and streamline the proces of futural engenineering of novel viral variants ....Clustred randsmization in a full length gene......Stsbilizating of thr clones and reduction of stress on host cells......The use of poission diztribution is proposed to approxametly sequencing out put to achivd coast effectivess
For more detailed see the attached ref.https://www.Sciencedirect.com/sci.
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I am wondering if the use of collagen as a matrix on the plate, or other matrix, with the consequent cell polarization, is necessary to get good virus yields. Many studies does not report culture on collagen or other matrix.
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Thank you !!
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I plan to use BALB/c mice for studying pneumonia caused by the mouse-adapted influenza virus (A/PR8/H1N1).
I want to know, what is the requirement to determine the sex of an animal? ​It seems that most of them choose female mice, but there are also researchers who use half male and female mice. What is the difference between them? Does the sex of the mouse have a significant impact on the final result?
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One of the main issues using male mice (and housing several in the same cage) is that they tend to fight with each other. If this occurs, they need to be separated and placed into individual cages. This could certainly impact the results of your study.
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Information provided by (Responsible Party):
ZhiYong Peng, Zhongnan Hospital
Brief Summary:
2019 new coronavirus (2019-nCoV) infected pneumonia, namely severe acute respiratory infection (SARI) has caused global concern and emergency. There is a lack of effective targeted antiviral drugs, and symptomatic supportive treatment is still the current main treatment for SARI.
Vitamin C is significant to human body and plays a role in reducing inflammatory response and preventing common cold. In addition, a few studies have shown that vitamin C deficiency is related to the increased risk and severity of influenza infections.
We hypothesize that Vitamin C infusion can help improve the prognosis of patients with SARI. Therefore, it is necessary to study the clinical efficacy and safety of vitamin C for the clinical management of SARI through randomized controlled trials during the current epidemic of SARI.
Vitamin C, also known as ascorbic acid, has antioxidant properties. When sepsis happens, the cytokine surge caused by sepsis is activated, and neutrophils in the lungs accumulate in the lungs, destroying alveolar capillaries. Early clinical studies have shown that vitamin C can effectively prevent this process. In addition, vitamin C can help to eliminate alveolar fluid by preventing the activation and accumulation of neutrophils, and reducing alveolar epithelial water channel damage.
At the same time, vitamin C can prevent the formation of neutrophil extracellular traps, which is a biological event of vascular injury caused by neutrophil activation. Vitamins can effectively shorten the duration of the common cold. In extreme conditions (athletes, skiers, art workers, military exercises), it can effectively prevent the common cold.
And whether vitamin C also has a certain protective effect on influenza patients, only few studies have shown that vitamin C deficiency is related to the increased risk and severity of influenza infections. In a controlled but non-randomized trial, 85% of the 252 students treated experienced a reduction in symptoms in the high-dose vitamin C group (1g / h at the beginning of symptoms for 6h, followed by 3 * 1g / day).
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C19 tests may show cross reactivity or interference with other viruses
CR: human coronavirus 229E, human coronavirus OC43, human coronavirus NL63, MERS coronavirus
IF: Influenza A, B; RSV, Protein A-positive Staphylococcus aureus
Is there any laboratory which is capable to test these?
Kind regards,
Dietrich Doll
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Another slovak company, Medirex, commercially tests "A rrspiratory panel Covid/flu/rsv PCR.
But I hope you find something in Germany.
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Do we need adapt influenza viruses in MDCK cells before conducting serum neutralization test with influenza viruses in MDCK cels?? Thanks
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Thank you..
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From 40 to 64 years of age, about 92,000 people have died or at least their deaths are attributed to Covid in the United States alone. https://www.statista.com/statistics/1191568/reported-deaths-from-covid-by-age-us/ If the new variants are as reported this disease is evolving to a more lethal version that kills younger patients https://www.cnbc.com/2021/03/11/covid-variant-in-the-uk-appears-to-be-64percent-more-deadly-than-other-strains-study-finds.html This begs serious questions did our defensive measures hand washing, masks, and quarantines encourage this trend to appear. Certainly, the modern practice of clustering or warehousing elderly members of society allowed the virus to specialize in rapid reproduction very similar to what happened to Spanish influenza in the trenches of WW1. Reports of unmitigated stress, binge eating, monthly weight gain lend credence that confinement might have been counterproductive. Indeed, some might argue that had we done no quarantining we would have gained herd immunity by now. Instead, are we giving Covid the time to evolve each time it breaks into a protected sector of the population? Please feel free to debate this question in the replies.
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I think there is evidence that the measures put in place against the virus are working. The vaccines are also helping a lot, although some of them have adverse side effects.
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Dear Fellows
With the developing stories of the spread of COVID-19 all around the world, which has been declared as a pandemic by WHO recently, I am wondering, in how much time this virus would vanish from the surface of the Earth? Is there any scientific study available for this?
Please share your opinions.
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Todavía no se puede predecir q desaparecerá el Covid 19. Al parecer tendremos q convivir con él e inmunizarnos con las vacunas aprobadas por la OMS.
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Hello,
I have recently started to do some Plaque assays to determine Influenza titer. However, my plaques dont really look nice and are therefore hard to count, so I wanted to ask if someone has some suggestions to improve that.
Some Info: I use MDCK-II cells in 96-well plates for infection and after a 1 hour infection period I incubate these for 28 hours with an 50/50 mixture of Avicel/2xMEM + Trypsin.
I have attached a picture of one of my assays so you can see that sometimes the layer of cells seems to be disrupted, making me unable to count properly.
Thanks for your help.
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I would like to suggest to do it 12- or even 6 well plate to get attached monolayer. After infection, incubate the plate for 1 hr, take out the solution carefully with tips without touching the bottom surface. Add respective medium with 5% FCS and incubate 48-72 hrs to get nice plaque. for details you may check the following article; https://www.nature.com/articles/s41598-019-44220-4
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Hi all,
I wonder if anyone know where I can find the plasmid map for pHH21 that is commonly used in Influenza research?
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Hello Dat Nguyen
Please refer to the link below.
I hope this helps.
Best.
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Should we give steroids in every covid-19 patient with elevated inflammatory markers?
Can we demarcate between appropriate immune response and dysregulated immune response?
Does the answer lie in Interleukin-10 (IL-10)?
Dr. Prashant R. Wankhade
Hypothesis: Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine which can guide regarding optimal use of steroids and immunosuppressants, allowing immune system to fight at its best.
Recovery trial has shown positive effects of steroids in COVID-19 patients who were receiving either invasive mechanical ventilation or oxygen alone but not among those receiving no respiratory support1.
Now-a-days steroids are among most widely used drugs in COVID pneumonia and we know that ‘steroid’ is a double-edged sword. If these drugs are given when patient is actually going through cytokine storm phase then it has beneficial effect but if we are administering them in appropriate immune response then it may be hazardous as it can cause suppression of immunity which in turn can escalate viral replication and subsequent disease progression.
Cytokine storm?
There is no widely accepted definition of cytokine storm.
Cytokine storm is an umbrella term encompassing several disorders of immune dysregulation characterized by constitutional symptoms, systemic inflammation, and multiorgan dysfunction that can lead to multiorgan failure if inadequately treated2.
IL-1, IL-6, ferritin, CRP are the part and partial of immune response irrespective of appropriate or dys-regulated.
Elevation of interleukin 6 and CRP is a part of cytokine storm but it’s not the absolute marker. We need to corelate these markers with overall clinical scenario to label it as cytokine storm.
So, which is the crucial moment to start steroid?
Does the answer lie in interleukin 10 (IL-10)?
Justification for the hypothesis:
Interleukin 10 (IL-10) is a potent anti-inflammatory cytokine that plays a central role in limiting host immune response to pathogens, thereby preventing damage to the host and maintaining normal tissue homeostasis. It is also essential for regulation of immune responses3.
The generation of an effective immune response along with limiting tissue damage requires a delicate balance between pro- and anti-inflammatory responses3.
Impaired IL-10 expression and/or signaling can enhance clearance of pathogens during an acute infection, but also exaggerate inflammatory response, resulting in exacerbated tissue damage4.
Some pathogens can harness the immunosuppressive capacity of IL-10 to limit host immune response, resulting in persistent infection5.
Though absence of IL-10 is often initially beneficial, it’s prolonged deficiency can be detrimental in the long term. Prolonged and increased production of inflammatory cytokines can lead to septic shock in the context of viral, bacterial, or fungal infections6,7.
Inflammatory molecules can often be potent activators of cell death and increasing levels of IL-10 can moderate the extent of apoptosis that is induced in response to infection.
For instance,
1. In a Chlamydia pneumoniae model, where bacterial clearance is enhanced in the absence of IL-10, mice also develop severe inflammation and experience elevated levels of apoptosis3.
2. During Mycobacterium avium infection, early IL-10 production is correlated with the failure of control of infection; ablation of IL-10 signaling led to enhanced pathogen control, demonstrating a causal relationship between IL-10 and the lack of pathogen control8.
3. In acute influenza infection, blocking the action of IL-10 results in enhanced pulmonary inflammation and harmful injury9.
It is not clear whether elevated IL-10 levels during infections are a cause or a consequence of high pathogen burdens, but studies indicate that resolution of infection requires a coordinated response in which initial pro-inflammatory mechanisms clear the pathogen and are subsequently limited by IL-10 before pathology occurs3.
A unique feature of the COVID-19 cytokine storm is the dramatic elevation of interleukin 10 (IL-10) in severe/critically ill patients which has led to speculation that IL-10 might play a pathological role in COVID-19 disease progression10.
Possible conclusions:
1. If IL-10 levels are elevated along with IL-1, IL-6, CRP, ferritin etc.: This indicates that IL-10 is already trying to control the inflammation by acting as anti-inflammatory agent and in-turn might be helping virus to proliferate. If in such situation we are administering steroids to the patient which can further suppress immune response leading to further viral proliferation.
2. If IL-10 levels are suppressed along with elevated levels of IL-1, IL-6, CRP, ferritin etc: If patient is showing clinical symptoms and signs of cytokine storm then this is probably the best indicator of exaggerated inflammatory response and such patients are probably the best candidates to be benefited from steroids and immunosuppressants.
3. If IL-10 levels are normal along with elevated levels of IL-1, IL-6, CRP, ferritin etc: Use of steroids should be guided by clinical symptoms of signs of cytokine storm.
Needs to test the hypothesis.
References:
1. Lin WS, Emberson JR, Mafham M, Bell JL, Linsel L, Staplin N et al. Dexamethasone in Hospitalized Patients with Covid-19. N Engl J Med 2021;384:693-704. DOI: 10.1056/NEJMoa2021436
2. Fajgenbaum DC, June CH. Cytokine storm. N Engl J Med 2020;383:2255-73. DOI: 10.1056/NEJMra2026131.
3. Iyer SS, Cheng G. Role of Interleukin 10 Transcriptional Regulation in Inflammation and Autoimmune Disease. Crit Rev Immunol. 2012; 32(1): 23–63.
4. Li C, Corraliza I, Langhorne J. A defect in interleukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice. Infect Immun. 1999 Sep; 67(9):4435–42.
5. Brooks DG. Interleukin-10 determines viral clearance or persistence in vivo. Nature Med. 2006;12:1301–9.
6. Gazzinelli RT, Wysocka M, Hieny S, Scharton-Kersten T, Cheever A, Kühn R, Muller W, Trinchieri G, Sher A. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J Immunol. 1996 Jul 15; 157(2):798–805.
7. Li C, Corraliza I, Langhorne J. A Defect in inter-leukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice. Infection and Immunity. 1999; 67(9):4435–42.
8. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. 32 clones secrete a factor that inhibits cytokine production by 31 clones. J Exp Med. 1989; 170:2081–95.
9. Sun J, Cardani A, Sharma AK, Laubach VE, Jack RS, Müller W, Braciale TJ. Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus. PLoS Pathog. 2011 Aug.7(8):e1002173.
10. Lu L, Zhang H, Dauphars DJ, He YW. A Potential Role of Interleukin 10 in COVID-19 Pathogenesis. Trends in Immunology, January 2021, Vol. 42, No. 1.
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Dexmethasone 6mg for six days or until hospital discharge is recommended for patients require supplemental oxygen. However, it is not recommended for patients that not require oxygen treatment.
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Croup is a viral infection affecting the upper respiratory tract. Its causative organisms includes Influenza and Parainfluenza viruses. This question aims to know the role of antiviral agents in the management of disease.
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there is no antiviral role in treatment of croup
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The Spanish flu, also known as the 1918 influenza pandemic, was an unusually deadly influenza pandemic caused by the H1N1 influenza A virus. Lasting from February 1918 to April 1920, it infected 500 million people – about a third of the world's population at the time – in four successive waves. Deaths: 17–100 million (estimates)
One hundred years later - 2019 COVID-19 pandemic.137 million cases, 2 million 960 thousands deaths.
The Spanish flu started on hundred years ago and continued for two years. Had four waves.
The COVID-19 started 2019 and continued for now. Had two waves, but pandemic continues.
Can we expect third and forth wave of COVID-19 in shorth time future?
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YES I AGREE
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Hi, I am looking for a test lab that can do tests on anti-viral activity of formulations. Strains to be tested are HSV-1, and for other formulation typically HRV, Influenza, and covid-19.
test labs may be from the west, say US, EU, but also from India, China, indonesia.
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There are two Anti_viral tests known for screening :1_Microneutrilization test(MN).2_Virus neutralization assay(VN).
These tests used to determine the presence of functional antibodies to prevent viral infection,where dilution of antibody sample(either purified antibodies or animal serum samples) are prepared in test tubes.Thanks
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We used to do the H1N1 WSN plaque assay without TPCK-trypsin, and it works fine. Now we are trying the H1N1 PR8 and H3N1 plaque assay, but the PR8 failed to form plaques, and the plaques of H3N1 was tiny and not clear.
I'm following the Virapur protocol, using DMEM and MDCK cells and my virus does make strong CPE effect in MDCK so I was wondering if adding TPCK-trypsin can optimised the result?
thank for the response 
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Luciana Tavares Are you using the same culture of MDCK or have you tried starting a fresh culture? I ran into a hiccup with mine because my cells weren't taking to the infection because they were old. (They looked fine, grew fine, but wouldn't infect!)
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Dear all,
I have a naive question. When we want to study Type 1 response, we infect mice with influenza for instance, if we want to study Type 2 response we use for instance T. Bruecei. Does anybody know which type of response we induce when we immunize with our classical protocols such as SRBC or NPCGG/NPOVA/NPKLH?
Thank you very much
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From an old hand at immunology, a Type 1 response is what we used to call a T-dependent antibody response, and that is triggered by viruses, SRBC and hapten-carrier conjugate. Basically, the B lymphocyte binds the relevant antigen [in your case, NP], endocytoses it and processes the carrier protein for display on its' surface class II MHC molecules. If a T helper cell recognizes the carrier protein antigen that is displayed, it binds to the B lymphocyte and stimulates it to produce antibody. For naive B lymphocytes, you get IgM initially, but IgG later in the response. Classic immunology.
Type 2 responses are T-independent; the carrier is LPS, or Ficoll or T bruecei. These are or have repetitive structures that bind to the B lymphocyte and crosslink the surface immunoglobulin [IIRC, you need a complex of at least 10 hapens to trigger the response and Toll-like receptors are involved]. For T-independent responses, you get primarily IgM, and some IgG3 in mice.
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As widely accepted presently, early childhood education and development are closely intertwined and Impossible to truly set apart. Adults, living in pandemic Times, are pivotal actors in the way this challenging Times integrate children's development.
Educators, parents, political leaders and other stakeholders how should we go about this to assure the best developmental outcomes for the new generation ranging from 2 to 6 year old, within this important frame of socialization and development stages? Should we look to what happened during the great influenza for lessons?
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I think we should take away some lessons from the great influenza. however, we do have more resources to provide children with appropriate support that they need now.
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Previous research has suggested an involvement of meteorological conditions in the spread of droplet-mediated viral diseases, such as influenza. However, as for the recent novel coronavirus, few studies have discussed systematically about the role of daily weather in the epidemic transmission of the virus.
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With the appearance of COVID-19 vaccines on the market, many available choices are there.
Which one is good?
Which one is safest?
Which one is most expensive?
Which one is easiest to store?
How many doses are required?
Other than intramuscular injection, any other forms?
How to check immune response after?
Do we need post-injection blood test?
Do we need annual booster dose?
Do we need new vaccines every year by prediction as if flu vaccines?
Any contraindications?
Any allergy from vaccination?
Do we need to mask after injection?
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Agreed with dear Arvind Singh
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Hello,
Most hand sanitizers contain 60-80% alcohol. Alcohol destroys organisms including bacteria, fungi, protists, and enveloped viruses by breaking down the plasma membrane. In recent years, microbial communities on the skin, in the airways, and in the gut have been shown to play an important role in health. Does daily use of hand sanitizer negatively impact the diversity and strength of the hand microbiome? If so, what are the implications of this to overall health? Do skin microbes play beneficial roles such as helping to prevent transmission of other pathogens?
Thank you.
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All literature data I came across showed that Ribavirin had equal if not greater effect in titer reduction, survivability, and body weight loss compared to Oseltamivir. The biggest difference I found was that Ribavirin caused lower levels of interleukins but I'm not sure if that correlates to that rarity of its practical usage.
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doi: 10.1007/s13238-016-0287-0
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Can the formation of drugs against coronavirus also be useful against the Influenzas virus or HIV? This is because these viruses shared many common proteases for viral replication.
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Dear Talha Bin Emran, the answer is no, the targets and the mechanisms of action aren't the same. My Regards
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Kindly share any research outcomes related to the above topic or virus /influenza based on the contemporary situation.
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Thanks, Koki.
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Antibiotic resistance and the emergence of many new viral strains due to climate change, animal farming and deforestation are increasing the usefulness of pharmaceutical agents, incl. antibiotics. We are currently dealing with yet another viral pandemic. Are there any natural plant extracts that are being studied that are effective disinfectants for the whole suite of fungal, viral and bacterial infections that act as pathogens to humans ? Particularly interested in those effective against coronaviridae ?
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Or not?
In The Great Influenza by John Barry at p. 340, about government controlling fear, writes: "They could not control it because every true report had been diluted with lies." At page 460, he writes: "For if there is a single dominant lesson from 1918, it's that governments need to tell the truth in a crisis. Risk communication implies managing the truth. You don't manage the truth. You tell the truth."
An article touching on these issues is, The Only People Panicking Are the People in Charge, by Malka Older, September 16, 2020.
Statistics, lies and the virus: Tim Harford's five lessons from a pandemic, Sep 10, 2020 Financial Times magazine, includes: "Carefully gathering the data we need, analysing it openly and truthfully, ... this is the only chance we have to defeat the virus ...."
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Yes..
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I am looking for your opine
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As the clinical syndromes associated with SARS-CoV-2 and influenza are broadly similar it will be difficult to distinguish these infections in winter when the seasonal increase in influenza typically occurs.
However, social distancing measures should reduce the incidence of both influenza and COVID-19.
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I am looking for protocols and/or information as to how to perform sucrose gradient ultracentrifugation to purify samples of influenza A that have been propagated in egg allantoic fluid. Does anyone have any protocols that includes the speeds used? (Protocols that do not cause lysis of the virus after purification are greatly appreciated as well)
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Interesting question. Following the discussion.
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Does anyone know what the immunogenic similarities of the two mentioned viruses are (I mean the specialized similarities needed to make a vaccine, such as a specific genomic sequence)?Please give me more information.
Thanks.
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During the popularity of covid-19, I think it can be achieved by increasing the test site, test frequency and the distance between people. At the same time, candidates and invigilators need to wear masks to enter the examination room.
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UVGI technology has long been known to be an effective tool to fight against airborne infectious viruses. However, lower wavelength UV light source (<240 nm) is also known to induce Ozone production from oxygen in the air. In addition, UV light is known to have carcinogenic and cataractogenic effects. UV lamps of wavelengths in far-UVC range (207 to 222 nm) and low dosage (2 mJ/sq.cm) have been studied to have an effective germicidal effect by neutralizing viruses and bacteria without adverse health effects to humans. Far UVC has emerged as a promising technology in the recent years ( https://www.nature.com/articles/s41598-018-21058-w ). While the use of wavelengths higher than 240 nm can be incorporated in enclosed systems like air purifiers and HVAC which can minimize direct skin and eye exposure of humans to UV light, far UVC has additional advantages of usability in open configuration in public places.
What are some other known real-life challenges and other important issues associated with UVGI technology to be cared for while designing systems integrating HEPA/ULPA filtration to be used in portable air purifiers, HVAC systems or upper room UVGI lamps in hospitals, commercial and residential buildings specially in the context of fighting the rapid spread of infectious viruses like the coronavirus associated with COVID-19?
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The following may have useful information
Deleted research item The research item mentioned here has been deleted
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Does anyone know of a panel of serum/plasma with antibodies to known pathogens such as Influenza A, HBV, HCB etc, that could be purchased for testing? or through ethics application to biobank?
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The NIBSC (https://www.nibsc.org/) has various antibody panels for different infectious diseases, but doubt there is a single collective panel for widely different pathogens.
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Inhalation of steam from the decoction of mint and oregano can be effective in treating lung infections and coughs caused by COVID-19 or influenza, especially during the recovery period. This has been explored in a very small statistical community.
These plants can act as inhaled antibiotics and can be applied directly to the infected areas, especially the inactive parts of the lungs, and can be effective in treating the infection.
These herbs also have anti-inflammatory properties and can be effective in treating cough and easier breathing of patients.
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In addition, these plants have a warm nature, while these viruses have a cold nature.
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The NS1 allows that patients asymptomatic can infect.
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There does not appear to be any such interaction between SARS-CoV-2 and host ACE-2 receptor to block the interferon system. In fact reverse is true.
To see why, have a look at the attached articles:
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Hi everyone,
We are synthesizing a virucidal material that will kill SARS-CoV-2 on contact.
In these preliminary stages, we want to use enveloped surrogate viruses that are the easiest to propagate and safe to handle. For example, Phi6 bacteriophage and its easily cultured host, Pseudomonas syringae.
Other CoVs (299e, OC43, etc.) and influenzas have been considered, but as I said for now we want to keep complications to a minimum.
Any advice on possible enveloped viruses to use?
Thanks,
Max Bueckert
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COVID-19 is a worldwide threat to humanity and spread fast. There is no effective vaccine yet, the combined antiviral therapies are performed and have a higher success rate. But we need additional treatments and approaches to this disease.1
I and my colleague realized that the levels of high-density lipoprotein (HDL) and non-HDL were very important for sepsis fatality in a small observational study. We tried to predict mortality with lipoprotein levels and ratios. We have believed that there is a balance between HDL and non-HDL, and it is an immunological actor for infectious disease.2 Our study was about bacterial infections, but a similar relationship may exist in viral infections. Hepatitis-C infections may alter lipoprotein metabolism. Statins have used for adjuvant treatment for chronic hepatitis-C infections.3 HDL may be an innate immunity actor, modulate macrophage functions and inhibit thrombotic events. LDL decrease was shown with inflammation.4
A meta-analysis showed that total cholesterol and low-density lipoprotein (LDL) levels were good predictors for Dengue virus severity. The investigators thought the lipoprotein levels should be screened routinely and the approach should be reconsidered with these levels.5
Another study showed that statin users with Influenza had fewer complications and 30-day mortality than non-users. But this protective association was demonstrated for some seasons and subtypes.6
The lipoprotein levels and ratios may be explored in COVID-19 screening and follow-up may be a predictor for prognosis. These levels and ratios may be targets for treatment.
References
1. Yan Y, Shin WI, Pang YX et al. The First 75 Days of Novel Coronavirus (SARS-CoV-2) Outbreak: Recent Advances, Prevention, and Treatment. Int J Environ Res Public Health. 2020 Mar 30;17(7). pii: E2323. doi: 10.3390/ijerph17072323.
2. Karahan I, Cifci A. Are Lipoprotein Levels and Ratios Able to Predict Mortality due to Sepsis? J Coll Physicians Surg Pak. 2020;30:272-275. doi: 10.29271/jcpsp.2020.03.272.
3. Aizawa Y, Seki N, Nagano T, Abe H. Chronic hepatitis C virus infection and lipoprotein metabolism. World J Gastroenterol. 2015;21:10299-313. doi: 10.3748/wjg.v21.i36.10299.
4. Levine DM, Parker TS, Donnelly TM, Walsh A, Rubin AL. In vivo protection against endotoxin by plasma high density lipoprotein. Proc Natl Acad Sci USA 1993; 90:12040-4.
5. Lima WG, Souza NA, Fernandes SOA, Cardoso VN, Godói IP. Serum lipid profile as a predictor of dengue severity: A systematic review and meta-analysis.
Rev Med Virol. 2019;29:e2056. doi: 10.1002/rmv.2056. Epub 2019 Jun 6.
6. Atamna A, Babitch T, Bracha M Statins and outcomes of hospitalized patients with laboratory-confirmed 2017-2018 influenza. Eur J Clin Microbiol Infect Dis. 2019;38:2341-2348. doi: 10.1007/s10096-019-03684-y.
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As a LT statin-using patient, I am selfishly interested in this research. Best of luck!
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JAMA article published today states that SARSCOV2 is at least 40 times as deadly as the Influenza virus. Why are most people not concerned? Do they not know this information? Do they think that they are immune? Do they not trust the Health authorities? Do they know that the data, assumptions made, results and conclusions are somewhat suspect of comparing "apples to oranges?" Do they not see evidence to support the studies? I went out today, it seems like I was the only person taking precautions. Why?
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July 24, 2020: As shown by CT Scan that 97% of people who are positive for COVID-19 by RT-PCR developed pneumonia; in the following study:
Hope, MD, Raptis, CA, et al: A Role for CT in COVID 19? What data really tells us so far. Lancet 2020: 395; 1189-1190.
So, almost invariably, anyone positive for the virus by PCR will have "pneumonia" by CT Scan of the chest, and thus some degree of pulmonary injury, loss of function, lung scarring, and perhaps metaplasia formation. This was even present in non-clinical and sub-clinical cases. More evidence that one should not promote "herd-immunity" before the vaccine is given. Stay safe, thank you; Gary Ordog, MD
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Due to their small size, viruses were shown to dry and die quickly, especially on porous (hydrophobic) surfaces and in dry environments.
Could drying the air be a method of treatment / prevention for colds and influenza?
Deleted research item The research item mentioned here has been deleted
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Definitely sunlight has a disinfection effect, as shown in studies using artificial ultraviolet light. The problem with solar or artificial radiation is that it propagates only in straight line, thus it does not reach the shadowed areas.
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In this review I will elabotely study the complete spectrum of antiviral compounds used in different Viral Diseses - applied to the latest Covid19 Pandemic .The review is mainly based on to point out the Older to Newer antiviral medicine -from Medicinal Synthetic background .
Your openion will be cordially welcome - Thanks
Jaydip Datta
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But earlier anti-viral medicine is Amantadine.hydrochloride - from where we have started the anti-viral synthesis . It is antiviral ,antiparkinson as well as antiallergic
compound .
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I am interested in measuring the underlying inflammation associated with viral infections (corona virus or influenza) to test the hypothesis that a inflammation-associated severity index might have clinical utility in treating patients. The prototype can be seen at: https://cslide-us.ctimeetingtech.com/actrims/attendee/eposter/poster/66
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Good topic
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Is the absence of decision by UK and NL to enforce social distanciation against Covid19 (unlike China, Italy, France, Spain, Germany, USA, etc) caused by memory loss of the positive effects of proactive enforcement of social distancing by US cities in the 1918 influenza pandemic? Death rates were reduced by 50%, source:
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Many thanks to all contributors. What emerges is that although the viruses of 1918 and 2019 are different, human behaviours in these two pandemics can be compared. Is it not notable that the portfolio of NPIs (non pharmaceutical interventions) is the same: handwash and reinforced hygiene, self-isolation, quarantine, lock-down, social distancing, gloves, face-masks?
100 years for not inventing better, and not learning from 1918 or from other countries (China/Wuhan) how to detect and implement early?
100 years to forget!
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Which markers are suitable for assessing the effect of influenza on the heart (FABP or NTproBNP, ELISA)?
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I want to see new markers.
Troponin was taken. I want to see in the complex
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The 1918 H1N1 Pandemic Influenza did not have a vaccine. In fact, physicians and scientists didn’t even know what a virus was or how it worked. They knew it was not bacteria and that it did not seem to be alive, but that was about it. But for the transmission, most scientists thought it was a chemical agent of some sort.
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There was no vaccines for the Spanish flu of 1918. It ended in the summer of 1919, mostly due to deaths and higher immunity levels.
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Given the scale of this pandemic, it is possible that 2020 is just the first (and worst) year of an annual coronavirus outbreak, similar to how we have an annual flu season.
Coronavirus mutates at a faster rate than influenza (Nextstrain was researching flu when the pandemic started and they are currently tracking mutations in SARS-CoV-2 too). By the time billions of people have been infected, SARS-CoV-2 (the novel coronavirus) may have mutated into a few distinct strains, different enough to each require its own vaccine.
It is also possible that a strain appears that is far less lethal. If this disease was killing 1 in 10,000 instead of 1–5 in 100, we probably wouldn’t even have noticed it until tens of millions had been infected and we’d probably just let it run its course without any actions, just as we do every year with the other coronaviruses that cause the common cold.
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If the SARS-CoV-2 Coronavirus causing Covid-19 disease would never disappear again, then man would have to learn to live with this virus. It is highly probable that this virus, just like any other virus previously created for many years to come, will remain. However, on the other hand, it is also very likely that man will obtain a natural collective resistance to Coronavirus and Coronavurus through constant mutations will come back in the following years just like the influenza virus.
Greetings,
Dariusz Prokopowicz
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As we know, Australian researchers have found that body's immune response to COVID-19 is similar to influenza. Now my question is: "Does getting influenza give some immunity against COVID-19?"
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No, different virus altogether. Chances of cross immunity would be exceedingly small as the protein structures the immune system would have stored to react to would not match the coronavirus
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I am interested in the robustness of this technique. I have seen so far publications only from the lab of founders of the technology. Therefore, my curiosity if it is used in the field of vaccine development too
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I have not personally used the VaxArray technology and have not reviewed it thoroughly. For measuring potency of influenza vaccines using HA antigen, you need to make sure that VaxArray measures antigenicity of HA that is measured by SRID (HA trimer). You may also like to speak with the regulatory agencies about their experience with VaxArray and whether VaxArray technology would be acceptable as an alternative to the SRID potency method. I am sure that if the scientific basis of VaxArray technology to measure HA antigen is same or similar as the SRID and there is data to show that the VaxArray measures the HA trimer, the regulatory agencies would accept the VaxArray technology. Of course, you would need to generate appropriate method validation and comparability data between VaxArray method and SRID. Alternatively, if the the VaxArray method and SRID method do not measure same form of HA antigen and there is no correlation between these methods, you need to demonstrate the the VaxArray potency method is a surrogate for clinical effectiveness of the influenza vaccine. Best wishes.
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Referring to research papers and general advice are welcomed as part of the discussion
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Zeyad T. Al-Rrassam Yuan-Yeu Yau many thanks for participating in this disuccsion.
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Which markers are suitable for assessing the effect of influenza on the heart (FABP or NTproBNP, ELISA)?
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Which markers are suitable for assessing the effect of influenza on the heart (FABP or NTproBNP, ELISA)
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Can either rhinovirus or Dengue virus, for instance, coexist with coronavirus in the same infect cell?
It is know that multiple viruses can coexist in the organism, like HIV and C hepatitis, B and C hepatitis, syncytial respiratory and influenza viruses, Dengue and Chikungunya viruses, etc.
SARS-Cov, SARS-Cov-2, rhinovirus, Dengue virus, among others viruses, are Group IV RNA viruses that affect the respiratory tract, and could target to some extent the same epithelial cells. Peace and blessings.
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I am no expert in that area, but my guess is that Dengue, Zika and other flaviviruses don't really go for lung cells so much. I have never seen much research or speculation on dual infections with influenza plus Dengue, or common cold coronavirus plus some other virus. HIV plus HCV is not good, and has been researched a lot. HIV plus tuberculosis is not good. I suspect that dual infections in general are more additive than synergistic, but it would not matter much if the pathogens are in the same cell, so much as that they are in the same host body and the immune system can only fight so much at once.
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We want to study about influenza viruses and producing vaccines. In vaccines, 15 mcg HA / 0.5 ml units are determined for each strain. Which methods are used to calculate this concentration? I am glad if you give me information.
Thank you for any advice.
Regards,
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The potency of inactivated influenza vaccines is determined by the single radial immunodiffusion (SRID) method against monovalent antigen standards and HA specific antibodies obtained from National Control Laboratories (NCL), including US FDA, UK's NIBSC, Australia's TGA and Japanese NIID. These 4 NCLs collaborate to harmonize the strength of standard antigens. Monovalent bulk pools are tested for SRID potency by multiple tests to get the HA antigen content of pools in terms of µg/ml, as determined by SRID. Based on that value the DS or quadrivalent vaccine is formulated to contain 15 µg of HA per dose of 0.5 ml. The DP is finally tested for potency by SRID to confirm the potency and release the DP.
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1918 influenza pandemic caused death more than that of combined death in World war I & II. Since then world has faced four Influenza pandemics. last one in 2009. We are in threat of more Influenza pandemics. What measures could be taken to prevent the development of Influenza pandemics?
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Its quiet difficult to predict influenza pandemic due to continuous antigenic shift which is occurring in this virus.
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Sure, quite well (e.g. see Oncol Rep. 2013 Jul;30(1):462-70. doi: 10.3892/or.2013.2413.)
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Hey there,
I've been trying to purify these influenza samples, however, I've come across 2 problems. I've repeated the procedures twice and have come across the same issues both times (first time thinking some error had occurred).
Sample 1: After PCR, product is visualised on gel to see if there is a correct product size. There is but also some non specifics, so I cut the band out. After gel purification, the band is now larger bp than original. Size of PCR product is 916bp. However, after purification, it is now well above 1000 bp.
Sample 2: After gel purification, the product has disappeared.
I know it is not an error of the kits (i.e forgetting to add ethanol to wash buffer or forgetting to incubate) as I have used these same kits to purify many samples before it. So these are perhaps sample-specific problems.
I have run these samples using both the Qiagen Gel Purification kit and the Intron Purification kit. Both times have encountered the same problem.
Any recommendations on what I should try?
Thank you so much!
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If you use the Qiagen Gel Purification kit it really helps when you warm the Elution buffer up to 37 degree. But this just help to increase the yield of eluted DNA.
For the first question did you use the gel with the same percentage of agarose and the same marker from the same company?
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Hello everyone;
I'm preparing my master thesis it's about influenza prediction using deep learning. The data that I had is the rate of dangerous cases and the suspicious ones per week and per region around the country.
for now I implemented the Random Forest, KNN, DNN, LSTM, CNN, CNN-LSTM and Deep Belife Network. I concluded that I'm faced with time series forecasting so I used the window method to make my problem surpervised with window_size=3, 2 and 1. Calculating the r2_score I got 10 regions under the 70% (which I had read that it's the acceptable threshold).
So I'm writing this question hoping to find a solution or get some idea or another deep learning technique and maybe special architecture of the technique used above to improve my prediction in this region.
and thank you in advance
(You will found attached some picture of the regions that I want to improve and my LSTM model.)
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Spatial autoregressive models are statistical models A good paper is : ``About predictions in spatial autoregressive models: Optimal and almost optimal strategies’’.
The following link for the R codes used to obtain the simulation results included in this paper :
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These are not contaminants. I have checked several times. I have used Gibco high glucose DMEM media with 10% Heat inactivated FBS and 1X anti-anti. I have used gibco TrypLE for trypsinization for around 10 mins. I do not understand what it is?
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Since you have ruled out the contamination or mycoplasma infection, you can try to replace the high-glucose/DMEM by MEM/NCS 5%.
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Interested in bio security aspect of disposing of horse faeces in an Equine Influenza 2 outbreak. Cannot find any research regarding if the virus is or is not shed in droppings. Also, how long would the virus remain infectious in the bedding?
Many thanks
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Many thanks, Kelli, this is very relevant and helpful. Kind regards Janine
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Canine influenza viruses (CIV) are contagious respiratory diseases found in dogs. These Type A influenza viruses and the H3N2 (canine and human) virus is known to infect dogs, cats and people. There is a concern that the disease can mutate with dogs being the ”mixing bowl” and potentially produce a pandemic. See: https://www.ingentaconnect.com/search/article?option2=author&value2=Daesub+song&pageSize=10&index=1#
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Most recent publication (April 2019) of Daesub Song and collaborators. See: https://acmi.microbiologyResearch.org
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I am attempting to perform a qPCR standard curve using viral DNA (Adenovirus and MCMV) and cDNA (Influenza A) to be used to determine relative abundance of these viruses in primary samples. Viral DNA and RNA was extracted from viral stock using the QIAmp MinElute Virus Spink kit and vRNA was converted to cDNA using the ImPromII Reverse Transcription System. 6 point 10x dilutions were performed using this viral DNA and cDNA (undiluted-1:100,000) and a negative water control was included. For the qPCR, I am using TaqMan Universal qPCR Master Mix along with virus specific primers (forward and reverse) and probes whose sequences were provided from literature review or provided by neighboring labs but have not previously been used by our lab with this particular master mix. I believe that all of the input DNA/cDNA concentrations are fine (ranging from 17-180 ng/rxn note: master mix prefers concentrations <250 ng) and being that I have repeated these experiments a number of times with great care, I do not believe that it was a pipetting error with the dilutions. The amplifications curves, especially for Adenovirus, do not follow the natural curve progression seen in most standard curves and for all of the viruses the points do not fall on the slope of the standard curve line. I have attached photos for reference. I have tried repeating these experiments many times and adjusting the input concentrations of the viral DNA and cDNA. All experiments were for each virus was done separately, there was no multiplexing. I am wondering if it is possible that the primer efficiency may be the problem. The annealing temperatures for these primers are slightly higher (~3-6 degrees) than recommended by the master mix but a representative from Fisher seemed to believe that the reaction should still work. Also the target regions for these primers seem very short (all <150nt). Is it possible that I need to design new primers or is there possibly another obvious issue. If anyone could provide any feedback on what they believe the possible problem is or has further questions, please let me know.
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Did you try to quantify your cDNA? Ferralita Madere
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I m using dh5 alpha cells to insert some specific segments from influenza A . I ligated the cells using TA cloning kit and transform them . I cultivated the cells on an LB media with ampicilin , Xgal and IPTG for the blue white screening. The screening worked perfectly , I cultivated them overnight and after this I used the Qiagen mini prep kit. I used ecor1 to digest the mini prep and after this i run a gel electrophoresis (agrose at 1%) for 45 minutes at 70V . I can't understand why I got the top bands like this , is it super coiled DNA ? Is something normal or I didn't do any of the steps properly . Does somebody know where I failed ?
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Hi Sandu,
First please consider the bellow suggestions:
1. Dilute your digestion samples and also your ladder
2. Let your gel electrophoresis run further so bands separate completely
3. Increase the voltage to 90-100
4. Digest your self-ligated plasmid (without an insert)
5. Load undigested plasmid (harboring insert) as control
What is your plasmid and insert size? Do you have your expected insert released in the current gel?
How long did you treat your recombinant plasmid with restriction enzyme (s)? did you digest it with a single enzyme or two? How many sites are there on the plasmid for enzyme (s) used? Are all the lanes representing the same insert and plasmid?
After successful digestion you have two bands including your linearized plasmid and insert, and if your cloning is not successful then you only see a single band on gel. In case you see other band patterns varying between the lanes would be due to unsuccessful digestion which might be due to short digestion time or missing of any other factors required for digestion.
Further, white colonies could be tricky and not really containing the desired fragments; if this is the case you do not see your insert band after digestion … performing colony PCR before digestion can help you to verify the ligation as well.
I think you have to prolong your digestion reaction to let it have a complete cut and also to check out if other required factors such as temperature, DNA/enzyme concentration etc. are set to optimum.
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Hello,
I was doing a PCR for influenza viruses ( more specifically defective interfering segments) and i had good results beside 2 segments. I store them at -20 degrees and i was thinking to run them 5 cycles more ( initially all the sample were run for 15 cycles) . Do i need to make fresh samples or i can just reuse the 2 stored samples. I'm thinking just to place them in the PCR machine and add 5 more cycles to this 2 samples .
Will this work ?
I know the buffer, dNTPs , Taq polymerase and others are limited and have a specific life expectancy of PCR cycles, but 20 ( 15 already done + 5 which i will run )cycles from my presumption will not exhaust all the dNTPs and other reagents.
Thanks a lot ! Sorry for this stupid question !
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That likely refers to the concentrated stock, usually in 50% glycerol (so it doesn't freeze even at -20): enzymes last longer at cold temperatures, provided they don't actually freeze solid (ice crystals can do bad things to proteins).
Once you've diluted your Taq to make your PCR reaction mix up, it will freeze solid, and this will usually result in a stark drop in viability.
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Hi,
I want to see if my mice have gotten infected, is it possible to coat an ELISA plate with IAV broken up by sonication?
Thank you!
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In case anyone else is wondering if it works, I tried it today and it works!
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Hi, I want a correct protocol for plaque assay with influenza viruses .
I used MDCK CELLS ( 450 000 cell/well ) with different strains and different plates but i didn't see plaque formation but only holes on all conditions ( also control).
What do you think about this problem?
Can i have a correct protocol of it?
thank you
best regards
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Camilla, from your protocol that you sent, you did not add TPCK-trypsin, which is required for your virus in order to visualize plaques. You should use 1 - 2 ug/ml TPCK-trypsin to your cultures. This should allow you to see nice plaques withing 2-3 days.
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Vitamin D status is associated with upper respiratory tract infection and / or influenza.
Of course such association (or causal relation) is stronger when there is :
- initially state of deficiency (lower than 50 nmol/L or 20 mg/nl)
- sufficient high dose of suppletion (e.g. 1000 - 2000 iU / d)
- time of suppletion is long enough (at least 3 month) before the season of cold / influenza etc starts
I point at a metaanalysis of Martineau et al (BMJ 2017) and know of some special RCT's but I would like to discuss this with you and maybe find some extra evidence regarding a positive association / causal relation.
Martineau Vitamin D supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data
BMJ 2017;356:i6583 | doi: 10.1136/bmj.i6583
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Poultry diseases cause increased mortality and high economic losses. Is it possible to breed to produce disease-resistant poultry strains?
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It would be very long and expensive, but not impossible. To achieve this, chicken breeds with a high genetic variability would be required, since many of the current breeds have been improved for productive characteristics, which has caused that the current chickens are more susceptible to some diseases, which has caused a low heritability of the characteristics related to resistance to diseases. Therefore, it is necessary to increase the genetic variability to obtain individuals with high genetic heritability to specific diseases.
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How to seek for the UTR of IAV?
Is there any database to seek for the UTR of influenza?
I need to know the UTR sequence of influenza to rescue IAV.
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You can RACE the UTR sequences or use universal primers (Hoffmann E et al.) for reverse genetics.
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I was using MDCK cells to grow Influenza H1N1 in 12-wells plates. Cells was seeded 1 x 10^6 cells/well and left overnight in media without serum. Cells were then infected with H1N1 at moi of 0.1 and virus titre was tested by HA assay after 24 hrs, and 48 hrs.
Trypsin was added during infection.
Under the microscope, the cells was achieved ~80% cytopathic effect after 48 hours. However, no titre was determined by HA assay (all negative HA) either after 24 hours or 48 hours.
Is there any mistake happening here?
In my point of view, first, I guess its because of the temperature during harvesting, virus was harvested at room temperature from 37C cell culture and spin at 4C and then store at -80C freezer for further HA testing.
Second point, was the virus titre too low? or was the cells density too low?
Does anyone here working in the same field or experiment with me before?
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What strain of H1N1 and what species of RBCs? Not all flu strains have the right sialic acid preference for a given type of RBC. Also, the HA assay is probably the least sensitive routine titration method for flu. If you used an infectivity-based assay you might find you have some replication.
paul
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I'm currently performing the MN assay using A/Hong Kong/4801/2014 (H3N2) strain of influenza. I came across a strange change in cell layer post 3 days of inoculation with the virus. The cells were detached from the surface of the well and formed clumps. Surprisingly, the clumps are found around the corners, so it has generated half-moon shape in the well where half of the cells are attached smoothly and rest half are detached and clumped.
I seeded cells 2 days ago and then used the plates for the assay.
Thank you in advance.
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Hi Aakash, How was your negative control? Did the cells also form a moon-shape like your sample well and positive well? If the negative wells are doing the same thing, there might be something wrong with your culture. Have you checked the supernatant in day 3, was it too low to cover the surface of the cells? Did the half moon-shape in all wells appear in the same side (all left or all right side)? If yes, maybe the place you incubate the plate was not horizontal, you can check using a gradienter. Hope it will be helpful.
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The viral RNA obtained from a patient with influenza symptoms was analyzed and the results for a real-time PCR were negative for the influenza A virus and positive for the AH1NI virus.
The positive and negative control were fine.
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In logical terms, it is indeed impossible because A/H1N1 is an influenza A virus. If all the assay controls are OK (and this includes being sure that the primers used for "influenza A virus" and "AH1N1" are still good) then it's most likely telling you that the virus the patient was infected with has changed so that one of the primers used for "influenza A virus" no longer binds to the viral RNA. In the multiplex diagnostic screen I used to use, the generic flu primers were chosen to match conserved regions of segment 7, which changes more slowly than some other parts of the viral genome, but it can happen. If you see this again with other samples then it might well be telling you that the assay needs to be updated.
Paul
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Hi everyone,
I am currently trying to work out inactivation procedures for influenza virus. I am interested in using heat treatment for cell culture supernatants, but I'm concerned that heating a sample used for ELISA may negatively impact how well the antibodies will be able to recognize potentially denatured proteins. It's been shown previously that heating samples containing influenza at 70C for 30 minutes will completely inactivate the virus, but do you all think that heating at the temperature for such a lengthy period of time might potentially make the samples useless for ELISA, etc?
I recognize that this is far from an ideal situation, but have any of you ever heated supernatants before using them for ELISA, etc?
Thanks for any input!!
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heat inactivation could denature your proteins and affect binding by ELISA. However, it depends on what you are testing for in your ELISA. As for antibodies, we inactivate our serum samples for 30min @56C which works well and does not affect antibody binding in our ELISAs. Several groups have looked at the effect of heat inactivation on influenza viruses. Zou et al. Virol. J. 10:289 (2013), have shown that heat treating avian influenza viruses (H7N9) @56C for 30min or UV irradiation for 30min resulted in complete loss of infectivity. You can treat your supernatant with heat or UV and then look for infectious virus to make sure that you have inactivated the virus prior to running in your ELISA. As a control, you can spike a positive sample in culture supernatant, treat with various heat or UV conditions, and compare to untreated to see if this effects your ELISA.
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0 📷 9 hours ago by krc3004 • 0
Recently I have been very interested in calculating the dN/dS ratio to estimate rates of nonsynonymous and synonymous substitutions. Since I've just started learning the theory, I have a few basic questions:
  • One of the applications I'm interested in is estimating selection pressure on various viral genomes. Does it make sense to use dN/dS across coding sequences from different viruses; e.g. an analysis considering HCV, HIV, and influenza all together?
  • relatedly, what is the best way to estimate dN/dS within species, and is there an existing implementation? I ask because of this paper, which explains why we shouldn't rely on dN/dS within species...http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1000304
  • is it possible to use dN/dS on whole genome coding sequences within species? I obtained CDSs for 4 strains of HCV from NCBI (NC_009823, NC_009824, NC_009825, NC_009826) and ran them through FEL in data monkey (http://www.datamonkey.org/fel), but the results said that there were no regions under positive or negative selection. This seems wrong to me..so it is likely that I'm doing the analysis incorrectly.
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Hi Chirag,
I'll respond to each question point-by-point.
- Estimating dN/dS and detecting positive selection across such highly diverged viruses, due to their high mutation rate and recombination, will be difficult. I guess this depends on what your question is - is this about molecular evolution across RNA viruses as a whole? Additionally, detecting positive selection can be done site-wise, gene-wise, or for a proportion of sites on specific branches of a phylogeny. My gut reaction is that alignment across all of these is going to be messy and lead to a lot of false-positive signatures of positive selection. But, if the question is compelling enough, there might be reasonable ways to deal with these sources of uncertainty.
- For within-species, there is just not enough information to reliably estimate dN/dS. Usually, population-level work applies nonsynonymous and synonymous nucleotide polymorphism, which is just counting and does not rely on some underlying substitution model and phylogeny. This depends on evolutionary distance among individuals within that species though, so viruses like HIV usually have reasonable levels of evolutionary distance to estimate dN/dS between strains and samples within those strains (see work from Sergei Kosakovsky-Pond's lab and colleagues - the HYPHY developers).
-Your results sounds right. There were only 4 taxa, correct? There will be almost no power to detect selection with a site-wise method such as fixed effects likelihood (FEL). You want at least 10, more is better. I forget where that number comes from, it might actually be specific to some simulations from MEME (another type of test), but the point is that you should not be surprised by your result.
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I tested different influenza strains in vitro on TLR7 reporter lines and saw differences but want to know if the differences are due to the sequence.
I was thinking something similar as with MHC epitopes but with the RNA sequences. 
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Great, Thank you.
We are measuring the the viral entry and also making sure the amount of RNA is equal.
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Hi everybody,
I am a new user for BEAST software and I would like to estimate tMRCA for influenza viruses. However, after following the instructions of the official website (http://beast.community/workshop_rates_and_dates), I couldn't get the root age or tree model root age at the log file results.
Could anybody help me to figure this out?
Thanks,
Ahmed.
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Don’t know
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I am thinking about using the PCR targeting all influenza viruses, and found the method which was patented (US9803251B2). However, I actually have no idea where to start, can I just use? contact the inventor? or no way I cannot use?
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Dear Prakit Saingam
please, see the attached file. Hope to be benefit. Best regard
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First Issue is : Patient viral load for Influenza flu virus (according to clinical case studies). What is the initial viral load patients come to doctor with (especially for diagnosis in the current times for H1N1 or H3N2 cases).
Second issue is : Most used RT-PCR primers for detecting the viral particle number in Influenza H1N1 and H3N2 cases.
Third is : Reference paper to convert HA units of Influenza virus to viral particle number. How are both correlated?
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Thank you so much Paul. Very much appreciated.
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I have been encountering false positive results while processing the samples for influenza typing and subsequent subtyping. I was wondering if real time rt pcr (without calibration) could be the possible reason behind it because i have tried almost everything as a part of troubleshooting.. need suggestions.
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AA Anjorin....Thank u so much brothr....my pipettes wr contaminated....everythng is gng well nw
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Does anybody have an antibody that can neutralise influenza C in vitro ? We are happy to collaborate on this via joint authorship.
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Oh..Sorry. Got it
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I am doing a TCID50 for influenza. The cells I am using need serum to survive. I plan to wash the serum off of the monolayer prior to infection with the influenza, use serum free media for infection, and add serum after back after. I think this should be ok, but wanted to get opinions.
Thanks.
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we don't add serum back. We use 0.1-1% BSA. Serum has factors which can inhibit virus replication In vitro. the BSA will provide factors which will allow your cells to survive throughout the infection
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Hello dear collegues,
can anyone please recommend a protocol for infection of cells (MDCK and A549 to be precise) by Influenza A (H1N1) virus in "solid medium" (cover with agarose)?
I would like to perform tests with H1N1 infecting cells but I need virus medium to be solid.
I believe there is possibility to cover cell monolayer infected with virus with medium that contains agarose...
Can anyone provide protocole? Or article that consists exhaustive protocole?
Thank you all in advance
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This paper should be very helpful for you. Use of Avicel will be better for plaquing flu.
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Respiratory infectious agents exploit multiple modes of transmission. For example, influenza and tuberculosis appear to be spread mainly by airborne droplets. However, while influenza can infect the majority of contacts, tuberculosis infects only one-third of exposed individuals, making a primary, asymptomatic infection. From these latently infected individuals, about 10% will eventually develop tuberculosis during their life-time, most likely due to a decrease of the fitness of the immune system as well as old age. Even though influenza is transmitted by a virus that causes an acute infection and tuberculosis by a bacterium that leads to a chronic infection, their transmission rates are inversely proportional to the length of infection. Can you elaborate on this question and contribute with your views on this theme?
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Good question.  To my way of thinking, the main difference is likely the sheer numbers of virions that are shed when a person with influenza coughs/sneezes.  It is also fairly effectively spread by hands when they are contaminated with mucus that contains the virus.  
Everybody with symptomatic influenza virus infection is shedding the virus in large quantities; many people with active TB are shedding only very small amounts of the bacterium.  Sometimes we can't even find it in sputum smears even in people we know have active pulmonary disease.  
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I know many Influenza studies use HEK-293 and A549 cell lines instead of healthy lung cells since influenza infects the respiratory tract. Was just wondering the reason to this.
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Here healthy lung cells mean the freshly isolated cells from lung? If that is the case the first hurdle is isolating and maintaining them. And it needs lives. However, with the cell-lines like HEK-293 and A549, it is easy to maintain and they are pretty robust with gene modifications, transfections or other drug treatments. With fresh lung cells, the cells may be sensitive to this kind of experiments.
And, It really depends on what people would like to study with Influenza infection.
If it is studying its replication and effects of inhibitors or gene regulation during the infection etc, the cell lines are better. But studying the virus or cell behavior during the infection needs fresh cells, which is much closer to the reality.
I hope this clarifies your query. Goodluck
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We know for example that influenza combination of serotypes in animals could acquire determinants for adaptation to human respiratory tissues.
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Dear Jorge Fernandez de Castro, i think this is an interesting article ablout your question
regards
Intervirology. 2017 Aug 4. doi: 10.1159/000478729. [Epub ahead of print]
Evolution and Emergence of Pathogenic Viruses: Past, Present, and Future.
Parvez MK1, Parveen S.
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In the last 117 years the population of the US has only once declined against a steady background of population growth.  The world was hit by a massive influenza pandemic during that period but could that alone have accounted for this anomaly or was there another factor operating
July 1, 1919        104,514,000          1,306,000              1.26 
July 1, 1918        103,208,000            -60,000               -0.06 
July 1, 1917        103,268,000          1,307,000              1.27
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Barry,
I think that the July 1, 1918 U.S. population number is unreliable. Prior to 1940 the U.S. Census Bureau did not count U.S. citizens that were stationed overseas in the military. Since over 4 million U.S. soldiers were mobilized between August of 1917 and the summer of 1918 the U.S. Census Bureau had to estimate how many were overseas versus living but not counted in the States. I'd say that the margin of error probably covers that supposed decrease.
There also could have been a decrease in births in due to an immediate anticipation of U.S. involvement in the war. While 1917 looks like a negative growth, there was a substantial decrease in population growth during the Great Depression. It was only after the economy crossed the threshold of where it was at the time of the start of the depression in 1929 that the population continued its more regular trend. Interestingly, from an historic perspective, there was an increase in the U.S. birth rate in 1940 that continued through World War II. This increase in U.S. population growth probably consisted of wartime babies in families that were recovering from the Great Depression and had elected not to have children due to the economic conditions at the time.
JAG
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The diseases of focus are malaria, dengue and influenza
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Hi John!
For influenza, since it is a contagious respiratory disease, it should follow the standard notion indicated by the model Rp=NBL, wherein the density of the population (N), transmission rates(B), and average time one stays infectious(L) affects whether or not the the disease spreads as dictated by the number of infected hosts (Rp). I am not familiar with vector borne diseases but surely, it should follow with something similar. Of course as what Muhammad had said, hygiene (as well as vaccines) is very important but there are of course other factors such as these to consider with the spread of diseases. 
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I am trying to find an antibody that allows immunofluorescence staining of flu antigens on formalin-fixed mouse tissue. This is a common procedure, but I had a hard time finding specific information on what antibodies work (ideally with clone information, or detailed source of polyclonal antibodies). If somebody can share personal experience with antibodies that work, this would be greatly appreciated. Thank you.
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Do flu viruses infect mice? May be nobody has produced abs for such very specific viruses
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 I saw 3 patients and myself whom have vaccinated on December's first week and have fever, myalgia, flu  clinics one month later with laboratory confirmed influenza. How can it be explained to see flu  in vaccinated people?
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Not much of the activity of influenza virus in our part of the world
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As far as I know, Influenza viruses gene (minus-strand RNA) can be replicated in the nucleus of the host cell.
How many copies of these RNA strands can be made in a host cell? and for how long?
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I do not have an exact answer, however,  the virus RNA can be produced wrapped in a nucleoprotein then packaged as a viron.  I believe the cell size governs the capacity /number of virons.  The virus produces  9 different RNA's particles however can package between 7-12 RNA particles. I believe a perfect Viron (infectious virus) requires 9 different RNA's that are packaged into a viron - the number of viruses per cell could range between 100 - 1000.
Hope That Helps - good luck
Dan
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I would like to fluorescently label the RNA in influenza and NDV viruses in vitro using red/far red dyes compatible with GFP imaging. I have seen the SYTO-62 from ThermoFisher. Does anyone have experience with it? Or are there other reagents that would work in fixed viruses?
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Thanks a lot, George ! I'll look into that. But I was initially looking at commercial product for now.
Thanks !
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there was some problem with my influenza A probe as it was giving a peak (>35 ct) in each well, even in confirmed negative samples... i thought that probe must have degraded.  So i ordered a new set of primer/probe, but the problem persists. Negative and Positive controls are working fine. Even Primer/probes for other respiratory viruses are working well...Could anyone please help me with this.....?
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The probe is not degraded as per your statement. I think the problem lies in the RNA extraction/elution buffer. If there would have been a problem with machine then the  results would have interfered with the positive  as well as negative control. Try to extract the nucleic acid with new kit/plastic ware. Place negative and positive control at different positions to ascertain the machine calibration is alright.
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Hello everyone! I am stucking in plaque assay on Influenza virus B study.
  • In my case, A549 were infected by influenza virus B (Yamagata lineage), culture supernatant were collected in day 2 of post- infection. And then,  these culture Sup. used for plaque assay Using MDCK cells. I also checked with positive control( Original virus B) at that time. Finally, only plaque formed from positive control, nothing plaques from culture Sup.I have checked several times but all the time failed. 
  • I also checked 3 cell line ( A549, MDCK, H292 cells) at the same time. They were infected by Influenza virus B and collected the culture Sup for plaque assay ( Using MDCK cells). However, only culture Sup. of MDCK Cells formed plaques. Nothing plaque from A549, H292 culture Sup.
  • All the results means that Influenza Virus B did not propagate in culture medium of A549 ,H292 cells. Is that true ?
  • Could anyone give me some experiments about this issue?
  • what type of Human host cells is best for influenza virus B study?
Thank you very much!!
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Tran, I believe influenza B is also best grown at 33 deg C instead of the typical 37 deg C for influenza A. Thomas's recommendations are also spot on. I have messaged you a protocol that I have used for propagation of influenza B. Joyce 
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I have thousands influenza sequences and tried so many times. But failed. Is there any detailed demo from the begaining of sequence aligenment to phylogenetic tree building? I hope someone could show me the detailed protocol of building ML phylogenetic tree by RAxML-HPC2 on XSEDE (8.2.8) on cipres.
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Hello Li,
Here is the help page for RAxML on cipres:
But here is a quick tutorial.
First of all I suggest you run jModelTest to test the for the best model and to see if GTR (RAxML default) is really the best model. I also suggest building a phylogeny with a Bayesian program as well (MrBayes is on Cipres)
1) Click the data folder in your project and upload a phylip file (i.e. .phy or .phylip) by selecting the upload data button and following the steps.
2) Click the tasks folder in the same project and click the select input data button to select your data.
3) Click the select tool button and select RAxML-HPC2 on XSEDE (8.2.8).
4) Click the 36 parameter set button and adjust the parameters to fit the analysis you wish to preform. One aspect I would suggest changing is to is under the configure the analyses section. Under the select the analyses type pull down menu change from default to Rapid Bootstrap Analyses (-f a). Without this you will not get a bootstrap tree which is what most people want. You also will need to select Rapid Bootstrapping under the configure bootstrapping section. You can also change the number of bootstraps below, but the default is 100 which is pretty normal (although some journals want you to run 1000, but this is rare). If you want to run a different analyses then consult the user manually for RAxML which is in the link above. Also make sure you set an appropriate amount of time, a quarter of an hour is usually more than enough for single gene or other small datasets. If you are using NGS data I would suggest setting this higher. You can also just change it to one hour and they will only charge you for the time used and you do not change much in the queue.
5) Give your task a name and run.
6) Once complete click view output and download one of the RAxML_bipartitions files. There is one that is RAxML_bipartitions.[whatever name you gave, default name is result] and one that is RAxML_bipartitionsBranchLabels.[what ever name you gave]. Different programs for visualizing your tree will require one format or the other, FigTree for example will open the first but with throw up an error with the latter.
If you do not have the files listed above then an error has occurred and you should view the STDOUT to see what error killed the program.
Hope this helps.
Best Regards