Life in covid19 testing lab and virology research during the times of pandemic - the crazy year.

in StemSocial2 months ago

Well, It has been quite some time, since I had time on my hands to write content. The pandemic changed a lot of things for me, including the amount of time I had in my hands. Ranging from volunteer work to assist in setting up a Covid19 testing facility, to shifting my research gears to antiviral drug development It has. It has been a roller coaster in short. I could have started writing today again discussing some science, but that would be abrupt. Also, I am sure there are many new faces on the platform that I have not interacted with. Hence, I thought why not share my experience with you. How it was working in a Covid19 testing facility during the very early days. How is it inside the walls of BSL3 where we work with the virus?

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Source: my camera

When it all started

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Driving on the empty roads Source: my camera

We had been hearing about Covid-19 cases since January 2020, when it all began in China. However, the cases have not yet come to India (or at least we were not aware of any). Then the reports of cases started coming up in India 1. Soon thermal scanning started on all airports. Our campus started preparing for what now seemed to be inevitable. Weeks before the national lockdown all measures - from masks to social distancing, to dividing each space into sectors (to minimize the interaction between individuals) were put in place. The national lockdown was announced by the prime minister on 25th March 2020 2

Diving in

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Just a sample diving in the liquid nitrogen.Source: my camera, edited on clideo

For the next 15 days, we did not go to the lab. Well except for one volunteer to ensure everything in the lab (the reagents, the animals) was ok. But, they declared that Covid19 related research will be allowed on the campus. Also, the campus was about to start a Covid19 testing lab and we were told that we will be asked to volunteer.

Experiencing drug discovery and virology

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Reading time.Source: A collage of screenshots of abstracds random papers. Created in layout.

It was in those 15 days that I decided that I will start an antiviral screening project in the lab. I did some reading. Maybe about 200-300 papers on virology, assays used and all the new research that was pouring in about Covid19. I and my colleague sent the project proposal to our mentor and got the green light.

The idea

High throughput screening

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Cells expressing GFP as seen under the microscope.Source: my camera

The idea was pretty simple. Design a replication-incompetent virus that expresses Sars-CoV-2's Spike protein on its surface ( a spike pseudovirus)3 but can't cause infection. The pseudovirus will carry a fluorescent gene. When the pseudovirus will infect a cell line that can be infected by the real virus as well, then the infected cells will shine green under the microscope. Well, if any molecule in there inhibits the viral entry or messes up the virus then the number of cells that shine green will be reduced. We will then measure the number of cells shining green in no treatment vs those being treated with 100s of compounds and extracts that we had in the library.
Now, this part was rather easy. We did not have to deal with the infectious SARS-CoV-2 virus at this stage. I did not have to wear that biosafety level 3 (BSL3) appropriate PPE and work in a negative pressure room. This turned out to be real smooth and fast. By end of 2020, we had 5 leads in our hands that could block the viral entry. But now came the challenging part. Will these leads work against the actual virus?

The real deal

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Me in Biosafety level 3 PPE. However this is not an image from BSL3. It was provided in the hospital in the Covid19 ward. Source: my camera

The leads that can block the pseudovirus entry were now ready to be tested for their activity against the real virus. So all geared up I started this phase. Soon I will be testing what we have on the mice model susceptible to Sars-CoV-2 infection. For example, K18-hAce2 transgenic mice (which is a mouse that expresses a human version of Ace2 on its lungs cells)4.
In a nutshell, our idea is to generate a general antiviral drug not just against Sars-CoV-2 but against multiple respiratory viruses that pose threat to us in near future. Hopefully, we will find an antiviral that blocks the entry of the virus. The work is in progress. Not much can be revealed about it but fingers crossed, and hope it works out.

How was it like working in a testing lab

By mid of April 2020, we were asked to volunteer for setting up a Covid19 testing lab on the campus. We formed 5 teams to set up a testing facility.
- The scientific staff-The aliquoting team-The RNA extraction team-The RT-PCR team-The data management team

The scientific staff

Initially, I started in the scientific staff. Our job here was to carefully collect patient samples from the hospital and get them to the aliquoting team in the biosafety lab. It had to be done safely without contaminating ourself, or the environment and surfaces around us. I was trained for Biosafety level 3 protocols. Here we use to work in pairs of staff member type 1 and staff member type 2. Type 1 was responsible for handling the sample in fully covered PPE, while the member from staff 2 would assist to open all the doors and operate the lift so staff 1 member do not touch anything while handling the patient samples. The type 1 member would duff the PPE he used to carry the samples, and then donne the level 3 appropriate PPE. Then he would enter the BSL3 lab and hand over the sample to the aliquoting team.

The aliquoting team

The aliquoting team member would then open the vials with patient swabs inside the biosafety cabinet. He would read the patient ID and name on the sample tube and the scientific staff member would tally it with the list of patient IDs and names we received from the hospital and the central database. If the vial was leaked or if there was a mismatch between the name and ID between the vial and the database then the sample would be discarded. If everything seems to be fine then the aliquoting person would give a green light and a scientific staff member would update the status of all samples for the data team. Meanwhile, the aliquoting person would neutralize the virus, if any, and put the part of the liquid in the mother tube in a new vial which will be sent to the RNA extraction team.

The RNA extraction team.

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Precipitation of nucleic acid on addition of isopropanol. This is a step common in both DNA and RNA extraction. However, its hard to see precipitation clearly when you do RNA extraction as quantity of RNA is relatively low. Hence for purpose of this video I used genomic DNA.source: my camera

The RNA extraction team as the name suggests was responsible for extracting all the RNA in the vial they received from the aliquoting team, using the RNA extraction kit provided to them. I know it sounds simple to put this in words, but their work was the most tedious and essential of all. The quality of extracted RNA mattered for everything that happened downstream. This extracted RNA was then sent to the RT-PCR team.

The RT-PCR team

![JvFFVmatwWHRfvmtd53nmEJ94xpKydwmbSC5H5svBACH81hoEyJnmPheU68pP9P7umDFNu6pJjMRWWFBBQX7G6oH1PeVu34WE3e84iKxQei1KbtLhghcS6kMvZihgFJetnqaVhWs82.png]()
A typical example of a qPCR reaction done using SYBR green dye. The y-axis shows the fluorescence units and the x-axis shows the cycle number. See how the fluorescence increases as the cycle number proceeds. We set a threshold value of fluorescence in the linear phase and derive the cycle number at which the fluorescence reaches this threshold. The point where the the exponential phase meets the threshold is called ct value. If you want to know more about the RT-PCR read my previous [blog](https://hive.blog/steemstem/@scienceblocks/the-lab-notebook-nut-1574077825).

The RT-PCR team was responsible to carefully set the RT-PCR reaction according to the map of 96 well plates provided to them by the data management team. The map instructed which patient sample will go in which well. In the initial days, they would first run the RT-PCR for the human RNAseP gene and viral E gene. The human RNAseP gene was like an internal control. If everything from sample collection to RNA extraction went well, and the quality of RNA is good then this gene should be amplified and detected. If this gene was not detected (which means that ct value > 35) the data management team would classify this as an invalid result. If the ct value for RNAseP was less than 35 but that of the E gene was > 35 then the sample would be considered negative for covid19. However if E gene's ct < 35 it would be declared as a potential positive. The positive RNA would be further confirmed by another RT-PCR for viral orf1a and RdRp genes. If either or both of them showed amplification then the person would be declared covid19 positive 5.

This whole process took at least 2 days to give the result from the date of receiving the sample. That is provided we did not have a backlog. Well, at least that was the case before the multiplex RT-PCR kits came in6, 7. Since multiplex RT-PCR can detect multiple genes in a single run the process was now faster and results can be delivered in a day. Well almost! One limiting step still existed in data management, which was delaying the results.

Just in a matter of a week, we realized that we are not short of people in any other team but the data management team. Given my skills in data analysis, I opted to shift gears and moved to data analysis. Over there our tasks ranged from receiving the data from a central database, uploading the status of each sample, entering information about each patient in the central database, coordinating the flow of process between all teams, designating -80 ° C storage location for each mother vial and its respective RNA, and finally analyzing the RT-PCR results and uploading it in the institute, state and central database. This was in addition to generating hospital reports. The major limiting factor in the initial days was the fact that we had to retrieve and update the databases manually. But we soon recognized the issue and created bots that would automatically update the databases and retrieve the information. This is when the process became really quick and it took just about 24 hours to give the reports from the time of receiving the samples.

Overall, it was a grim time. Nevertheless, there is a lot of science that happened in this period all over the world. I never imagined in my lifetime that vaccine and drug development can move so quickly. But it did. I received my second dose of vaccine in march 2021. While fear from the lurking possibility of all emerging strains of concern is there but with vaccines, we can still breathe in peace.

Anyhow this is my story from last year. I would be happy to take questions. If you want to understand anything related to covid19 research, how drugs or vaccines work, or anything about how testing works please leave your questions in the comments below. Moreover, if you want me to cover any specific topic in this field, I would be glad to do so.
Signing [email protected]

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A video by @gtg

References

  1. First confirmed case of COVID-19 infection in India: A case report
  2. PM Narendra Modi announces a national lockdown for 21 days starting midnight of 24-25 March
  3. Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol
  4. hACE2 transgenic mouse model for CORONAVIRUS (COVID-19) research.
  5. A guide to laboratory diagnosis of Corona Virus Disease-19 for the gastroenterologists
  6. Multiplex Real-Time PCR
  7. Novel Coronavirus (SARS-CoV-2) Real-Time Multiplex RT-PCR Kit.

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 2 months ago 

Dang, I don't even get a hood in the BSL3 room.

Oh we have to book the hood a week in advance too. Plus you have to either do it during office hours or else have a buddy with you. Do you have that buddy thing too?

 2 months ago 

Oh lol I was referring to the head covering not the safety hood.

My work is 24/7. I work for the micro department. The only covid tests we do are the rapid ones using the samples from the emergency room.

The rest are done by the molecular department.

Oh haha, my bad. I assumed you were talking about crowding for biosafety cabinets. Well we do have those reusable head hoods for that, which can be used with a breathing unit. But unless I have work for long hours (3+) in one go, I prefer N95 and face shield over breathing unit.

 2 months ago 

Yeah, I’m well-acquainted with working 3+ hours under the hood. I feel for ya.

was it dangerous?

 2 months ago 

What was?

working there and on top of that without hood didn't you get infected

 2 months ago 

Everything is done under the biosafety hood.

ok got it

May I ask why in first place it is tested for the e gene? Isn´t that common among all Coronaviruses? Why not immediately go for a sequence specific for SARD-Cov2?

Well there is a difference between having similarity with other corona viruses and being identical. Take example of say human actin gene vs mouse actin gene. They are similar but not identical. There are enough differences in the sequence that I can design PCR primers such that I can specifically amplify the mouse gene and not human gene with that pair of primers (check out my qPCR post to have an insight into primers). Anyhow, same goes for viruses, there are similar sequences but you can always design primers specific to your virus of interest. Also, you have secondary test with primers for other genes such as RdRp and N. So even if you did end up getting a false positive due to presence of some virus which shares the sequence with your virus of interest (this issue is ruled out at primer designing step), the probability of this happening in both the cases is lower.

We don't directly go for sequencing because RT-PCR are specific they are robust, sensitive, and high throughput. You can literally carry out a thousand RT-PCR tests in parallel and get result in real time. Sequencing is more useful if you are interested in knowing the strains of the virus in the population than for detection. For instance if you have 1000 posts on hive to scan that are about Covid19, you can search if they contain that word/tag or not by using search or pressing CTRL+F. Why would you want to read 1000s of post manually to say - "ok, two of these are about Covid19". There it is you RT-PCR is ctrl+F applied to 1000s of samples at the same time.

Thanks a lot.
And I meant primer sequence, sorry to be misunderstandingly. Of course sequencing is way too expensive.

This is a lot of information in a single post. Perhaps an infographic would go a long way to simplify things further. I will reserve my questions for now as I can think clearly. But I have got one:

How were you able to hold up mentally during all these?

I think a big chunk of credit for keeping everyone sane also goes to faculty in charges who would order yummy food for all the volunteers twice a week (biryanis, pizza and what not). Well, sometimes tasty food which maybe bad for physical health, somehow tends up keeping people sane under stress. 🙂
But, not going to lie, the year was tuff. And to add to all this my daughter and wife were stuck in different city where they caught covid. I did talk to therapist now and then. Also had really supportive friends to talk to everytime the stress piled up.

Oh well, thanks for pointing out. Will create infographic and update the post later in the evening.

Oh boy, that video needs to be replaced by a Hive flavored one. I'll try to provide one soon.

Haha, yeah. It would be great to have a stemsocial one. 🙂

I don't get it what are you trying to say?
there's only a gig of empty road

I don't get it

Yes

that video needs to be replaced by a Hive flavored one

what are you saying?

There's an old promo video used there. It was made in the pre-Hive era, it contains old logo, and it should be replaced with the updated one.

ok, then plz send me one so I could use it in my future post

I'm so glad I ran into this. I just posted an article on the Covid topic as well, and that I just tested positive for antibodies 10 months after I had Covid. Would love to learn more about your experiences and thoughts on the virus. I will start looking more into your postings, but don't hesitate to reach out!

That's great to know. Hopefully you have neutralizing antibodies and some memory T cells out there keeping you safe and sound. I bookmarked your post, will read it today. 🙂

See that's the thing. My doctor gave me such a hard time about wanting the test, and so after some convincing, it was done, but it was the ab test and I have no idea about my t cell count. Only learning about t cells now. I may go to a private lab instead for another test
Here's my post: https://ecency.com/hive-120078/@swayzilla/antibodies-developed-through-natural-response-to-covid-19

Really like how you have written the post. There is one thing I would like to point out though - we should not assume that a vaccine and natural infection may work the same way. There are lots of aspects to it.

Some infection do coffee life time immunity but this is not universal for every infection.

Then it is bit of an over-strech to assume that virus and a subunit vaccine works the same way. Virus comes with it's whole arsenal. So in fact you produce B cells (antibody producing cells) and T cells to whole range of viral proteins and hence the immunity is much broad spectrum than a subunit vaccine. But the arsenal also contains genes that interact with the immune system such as interferon inhibitors or NLRP3 modulators. In a so how the whole game is going to be played out can only be known over time with enough data. A subunit vaccine is a simplistic system. We know most of what it will do inside. Its a controlled system, designed to prepare the immune system.

I mean there is a lot in that post that I would love to discuss. Maybe I will do a post on this topic, it would be interesting. I esp love your question that someone already infected should get a shot or not and would like to cover that bit.

Anyhow, one more thing in your post that nucleocaspid is a protein that binds to nucleic acid (viral RNA in this case). The antibodies you have are against that protein not the viral DNA.

Have a good day 🙂

Thanks for clearing this all up! I knew I reached out to the right person. No one has been able to explain to me how they would work differently or how the antibodies are to the protein in such a clear way. I agree that not every infection would offer life time immunity. I guess we don't really know until we have done a longitudinal study on it. I look forward to seeing if you do an article on natural immunity and a vaccine afterward.

Thank you once again.

You are the real hero.... Can't imagine doing all of these putting both yourself and your family in danger. God bless you!

Forgive me in advance, I have also felt the effects of covid 19. It starts to fade, it starts to lose any taste of what I eat tastes bland, high fever, whole joints ache. Thanks to the creator I followed the independent isolation for healing. I'd go to therapy in the early morning sun, drink some boiled halian fruit, tangai and finally have some honey. I take it when it's warm. And now it can function as days go by.

you worked hard may god bless you for helping in test lab

Very cool post! Thanks for coming here and sharing what you've been up to. I think what you're doing in the lab searching for antivirals is some of the most important work any human can be doing right now and I salute you for it. The vaccines have done a lot but there's more that can be done to treat it to minimize health impact and spread.

At the start of the pandemic I really hoped that it would spur a large allocation of resources into virology. So many people want to return to the status quo and forget that I'm not sure how much this has happened yet. There's obviously a lot of incentive though, for society at large. Thank you for doing what you can to help all of us.

Thanks for your kind words. I think one of the major blow to us when the pandemic hit us was how small our bag of antiviral drugs was. I mean compare it to the bag of antibiotics that we have to target bacteria. There were some out there for sure like remdesivir, flavipiravir, etc, but the sheer number available was so limited. Vaccines are ultimate preventive measure, but having a counterpart bag of antiviral drugs can save lives not just today from current pandemic but in case something is to hit us in the future.

As for resources going into virology, it's not that bad now. But could get better. Maybe we as society were not prepared to our best this time, but I do hope we don't let it happen again.

@frot @active_truth

Nice - I live in a country where people have started dying like flies from phizzer jabs and the government is spending millions to cover it up.

Yes I do have inside info, and yes i do know some personally... No time to play about in space suits, this country will be completely economically screwed by the end of the year - i'm prepping.

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Hey man!
It's great to connect with you again!
I have so much respect for you and many others during the pandemic.
Teaching hasn't stopped and I've been on the fronline throughout.
Stay safe and DM on Discord sometime,would be great to catch up.
Thanks
Darren Claxton!

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Check out the last post from @hivebuzz:

Hive Power Up Month - Feedback from Day 7

Did you isolate a virus and reproduce it?

That was done by a collaborating lab. Only one lab in the campus has permission to produce and distribute the virus. This is helps in keeping a good inventory of the virus available. The other labs then use this virus produced by the collaborating lab, for their respective experiments.

Hi to you,

I would appreciate it very much in reading your answers on the following questions.

Scientifically, it is not the things that confirm a theory, but the anomalies, those that challenge it.

With this in mind, how do you answer the question that there is little that challenges virus theory in the publications, but a lot that confirms it?

How familiar are you with the history of virus theory science or have you read scientific papers that deal with it controversially?

When you started your career, did you automatically assume that the theory of viruses, as commonly accepted today, was proven beyond doubt?

Within your university or other education, did you discuss anomalies, work that contradicted mainstream science, integrate it into the curriculum? What about outside of it?

Have you personally never had any doubts about virus theory? If not, could you give a reason for this? If yes, what would it be?

I am not really sure if I understand your question clearly. But if you are pointing at the alternative theory where viruses aren't real, then I must say that I have been working with them for over 8 years now. Previously, I use to make replication incompetent viruses for modifying the genetic code of cells and animals for my project. The only difference this time is dealing with an competent infectious virus. Furthermore, you can actually isolate the virus from the patients. That's how we create the repository for different strains. You can look at this virus using cryo electron microscope. Then when you infect the animals with the virus you isolated from the patient and then infect a susceptible animal with it, it gets sick and shows the symptoms. For SARS-CoV-2 this animal can be K18hAce2 mice, or a hAce2 knock-in mice (basically mice genetically engineered to express human version of Ace2 protein on its cells). Given I have done all these experiments myself, including looking at the virus in a CryoEM, I have very little to no reason to doubt the existence of viruses. If I got your question wrong it would help if you elaborate.

Respected Sir ,
I wanted to know , that , is it true that there's another wave coming ( of covid19 ) ?

I am not an epidemiologist, so not the best person to answer that. But from what has happened across the globe and based on few discussions I have been to we do anticipate that. Its best to keep a look out and control things before they get out of hand like the second wave. Also, given that we have been vaccinating people really fast I really hope that the third wave be less in severity the second one.