Reverse Transcriptase Polymerase Chain Reaction for Covid-19 and Zika testing

PCR 

Polymerase Chain Reaction is an in vitro diagnostic technique. This is being used to amplify the DNA or RNA sequence of any source material. 

In the PCR process a target DNA sequence and DNA Polymerase enzyme is used in lab conditions, usually passing with following cycles in stages:

1. Converting dsDNA to single stranded DNA,  

2. formation of new dsDNA  with one old strand and one new strand,

3. using new dsDNA as an template to for synthesis in next cycles.

RT-PCR 

Reverse Transcriptase Polymerase Chain Reaction is also an in vitro diagnostic technique with a variation of PCR. In this process a mRNA sample with reverse transcriptase enzyme, a cDNA copy is obtained. Here onward a standard PCR process is followed in sequence to obtain the dsDNA sequence. 

RT-PCR and Covid-19 

Often RT-PCR is combined with the real time PCR (qRT-PCR) to quantify the transcript levels in cells/tissues.

Originally radioactive isotopes were used in this nuclear derived method; now replaced by the isotopic labelling with special markers like fluorescent dyes.

Conventional RT-PCR allows to see the result at the end of the process while in qRT-PCR one can see result immediately while the process is still on going. This technique has been used successfully in diagnosing Ebola and Zika virus and is now adapted for testing the Covid-19 virus.

Further reference for:

Virus, genome and Covid-19

Mutation and Strain Biology of Covid-19 Virus

Spanish flue of 1918 

World is still fighting with the third wave of Covid-19 pandemic. Humans do have short memories as we forgot Spanish flu pandemic; a 100 year ago with fatalities in tens of millions at that time. Mutation in influenza, the RNA virus was behind the infamous pandemic. Understanding how virus mutates is critical for making decisions about the vaccines. RNA viruses mutate relatively quickly because they lack a proofreading mechanism to look for and repair errors during replication.

Actually there was no learning from that history, as our initial reaction with this new Covid-19 virus was of utter amazement. Still a lot lessons could be learned if we could visualize the after effects of the last pandemic of 1918.

Viruses fall on the borderline of living and non living.

They are strange as:

1. Do not have cell, a basic structural & functional unit of life to claim it as living organism,
2. Do not have their own metabolism of any kind, as do not possess any cell organelle,
3. Do not reproduce by any form of cell division,
4. Do self assembly only in a host cell, creating their multiple copies on the expense of host cell,
5. The only living characteristic they possess is gene, either in RNA or DNA strand form. They can not synthesize protein as they lack Ribosome, a cell organelle for the translation of viral mRNA,
6. The nucleic acid in RNA or DNA form alone is capable of infecting host cell even after being crystallized and stored for years in abiotic conditions. If and when that comes in contact with any host cell, starts behaving as a copy machine, producing of their own kind and consuming/killing the host cell.

Virus name in various Indo-European languages like Latin, Ancient Greek, Avestan means poison or poisonous substance, originating from Sanskrit root 'Visa'. 

Much before the discovery of structure of virus, it was identified as the poisonous substance rather than some biological entity.

Some specific features of viruses:

1. Viruses are submicroscopic in structure, shape ranges from helical to icosahedral to many more complex forms, invisible in the light microscope, as they are one-hundredth of the size of bacteria.
2. In nature their number is 10 times higher than the Bacteria.
3. Bacteriophage is a virus infecting to bacteria. 
4. Tobacco Mosaic Virus was the first virus discovered by Dmitri Ivanovsky in his 1892 experiments just at the end of the nineteenth century.

Wendell Stanley later studied the virus, managing to extract it in the form of pure crystals and show that it is composed of protein and ribonucleic acid (RNA) in 1935 and was awarded Nobel Prize in 1946.

Viruses consist of a long chain of either DNA or RNA, a protein coat Capsid surrounding and protecting the central genetic material and in some cases a lipid envelope surrounding the nuclear capsid.

Genomics of Viruses:

Of all the viruses found, about 70 percent are of RNA types genome. Due to possible higher rate of error in enzymes responsible for replication in RNAs type, they show much higher rate of mutation than to DNA viruses. That causes much higher rate of variant and that means the great adaptability to the new host.

The viral RNA may be of two types:
Single stranded (ss) or Double stranded (ds), they further vary as occupying on single RNA segment or two or more segments.

Genomes on single stranded RNA may further be of 2 types:

1. a sense strand (plus strand), which can function as mRNA; they alone can replicate if injected into host cell.

2. anti sense strand (minus strand), a complementary to sense strand, can not function as mRNA, no translational function, can not produce viral component.

The retrovirus genome comprises two identical plus-sense ssRNA molecules.
Most DNA viruses contain a single genome of linear dsDNA.

SARS-CoV-2, a corona virus causing Covid-19, is a RNA virus, and it must infect a host cell to replicate itself.
Error during the replication of viral RNA resulting in similar but not exact copies of the original, causing mutation. These mutant viruses called variants, which differ by single or many mutations.
A variant is called strain which shows distinct physical properties, behaving differently from the parent virus.

 
Following are the common variants of Covid-19 which are also strain; each containing several different mutation:

1. UK variant (B.1.1.7)

2. South African variant (B.1.351)

3. Brazilian variant (P.1)

Delta plus variant of Covid-19 is a warning that SARS-CoV-2 is continuing to evolve.

 

When new organisms enter the human race, they learn and adapt quickly to us. Unless we learn and adapt too, we're going to have more problems in the coming months and years. The virus is not done with us yet.

WHO says: 'When a virus is widely circulating in a population and causing many infections, the likelihood of the virus mutating increases. The more opportunities a virus has to spread - the more replicates and the more opportunities it has to undergo changes.'

Note: On the site of  WHO , this has been explained with a nice illustrative video to understand the mutational change in Covid-19 virus genome to form the newer variant. 

Following screenshots are shared thankfully from the above said video put in public domain to understand the possible change in four bases of the RNA's; i.e. Adenine, Guanine, Uracil, Cytosine. 

Delta wave of Covid-19

Delta, a variant of concern

India saw an unparallel devastation of human life, in the living memories of present generation, in the second wave of Covid-19 of Delta variant infection, which ran from mid April to mid May 2021. We are a nation of multitude of population. So in such a demanding situation, health infrastructure and system remained fail. We may have 'many' research institute, many oxygen plant, many hospital and serum institute in 'number', but when demand rose for providing it to 'per capita'; to our dismay, it proved a failure. And it was supposed to be. We are in no way capable of providing 'health needs' to such a 'large number' of people at any given instance, if a sudden pandemic rose even in future. Unfortunately disposal of dead bodies have also seen unprecedented scenario, which put humanity on shame.

People suffering, in desperate need of medicine, oxygen, hospital bed, doctor; and there was none. Disposal of dead bodies saw such painful and shameful visuals. Business of hoarding medical needs and market of fake medicine were on high. There is no 'actual number' of lives lost, because there was no one to record to in such a messy situation. Bonds of accountability of any kind was broken. A time of shame; pity, pain, tears ran high on faces of masses, those who lost their dear ones or children who lost their both parents; not to mention the neglect and negligence by the system to which we are proud of as most religious and democratic nation. Holy values saw a deepest dip of our time.

Delta plus variant acquired a spike protein mutation called K417N, the sub lineage of Delta, is on rise now. No one knows, what are in the store as mutation causes more transmissibility. Precaution measure and vaccines are the only answer at present.

First report from China was of 30th December 2019, later WHO issued disease outbreak news on 5th January 2020. Ever since, this virus has shown tendencies of a quick and efficiently evolving variants. Since one and half year, the cases and deaths are on rise, every minute. As of June 22, the country wise variant reported are; Alpha in 170, Beta in 119, Gamma in 71 and Delta in 85 countries. 

Lab leak theory of Covid-19

WIV image credit: Kyodo News via Getty

There is a huge debate these days about the origin of Covid-19/Corona/SARS-CoV-2; that this virus either has emerged from some zoonotic host in nature or it is bioengineered in some Virology lab. Before coming to any conclusion, it would be better to evaluate both possibilities in the light of available evidences. 

Possible spillover from nature: 

1. Most scientist believe that SARS-CoV-2 has a spillover from nature, because

bats are the known carrier for coronavirus. Horseshoe bat (Rhinolophus affinis) is commonly found in southern China. Genome of virus RATG13 found in this bat is 96 percent identical to the genome of SARS-CoV-2 causing Covid-19 in human.

2. This means that a closer link in between human and bat is missing for the difference of 4 percent genome.

3. Again the spillover from the lab is a remote possibility as lab engineered strain mostly cause small outbreaks. They are well documented and contained. 

4. Same covid-19 like pattern were found for emergence of infectious diseases causing pandemic in previous years; e.g. SARS coronavirus, influenza, Ebola, HIV etc. in other parts of the world.

5. 'WHO' too agreed for investigation in to pandemic's origin in depth (https://www.nature.com/articles/d41586-021-00375-7).

Lab leak theories are basically grounded on:

1. SARS-CoV-2's closest relative is still elusive to scientists.
2. Covid-19 first reported in Wuhan, is not a coincidence where WIV lab is researching on the same virus.
3. SARS-CoV-2 is spreading unusually quickly in humans, that points intention.
4. Furin cleavage site on virus protein spike, absent in it's closest relative. (But opponent to lab spillover theory believe it a convergent evolution for evolutionary advantage as virus adapting to the similar environment; as furin cleavage is also found on common cold viruses)
5. Original host of virus could not be found as yet.
6. CGG encoding for Amino Acid Arginine underlying a segment of furin cleavage site is preferred code for Arginine by humans, not viruses. (https://nicholaswade.medium.com/origin-of-covid-following-the-clues-6f03564c038). (But opposite believer point the 3 percent of Arginine been coded by CGG code in SARS-CoV-2, citing example of Indian variant (B.1.617.2 or Delta) has mutations in nucleotides encoding it's furin cleavage site, making it more virulent variant.)

Spillover theory and word of politics: 

There are many voices echoed in favor of a fair enquiry in this matter. 

1. US President Joe Biden tasked US Intelligence community to find the origin of SARS-Co-2. 

2. Australia, EU, Japan were also in the same league in voicing their concern for a fair investigation. 

3. World leaders in G7 summit were also vocal in a statement issued to the media on June 13, 2021.

(https://www.thehindu.com/news/international/g7-leaders-discuss-wuhan-lab-leak-theory-behind-covid-19-pandemic/article34804805.ece)

It's true that:
The Wuhan Institute of Virology (WIV) in China's central Hubei province is located in the same city from where the first Covid-19 were reported, but there is no substantial evidence to prove the controversial spillover theory from lab as of yet.

A spillover from nature through a bat directly or indirectly seems most likely.

Latest:
Amidst all the suspicion of lab leak theory, the Chinese Academy of Sciences reportedly nominated the 'Wuhan Institute of Virology' for top science award in conducting 'the most comprehensive and systematic identification research on the aetiology of covid-19'.
(https://www.wionews.com/world/wuhan-lab-suspected-of-leaking-covid-19-nominated-for-top-science-award-in-china-393263)

Corona virus outbreak, symptom, medicine, vaccine and lesson learnt

Corona Virus outbreak, causing severe acute respiratory syndrome (Covid-19) was declared a Public Health Emergency of International Concern on 30th January 2020 and a global Pandemic on 11th March 2020 by World Health Organization. Then it was thought as of zoonotic origin of some mammal like bat or other.

Covid-19 was first reported on 31st December 2019 from Wuhan, China. Spreading swiftly on global scale and adapting itself to gradually more lethal strain, surprised the medical community as well because mostly next level of strains are supposed to be less pathogenic, as considered generally.

Disease symptom is most intriguing so far as this affects different people in different ways. Common symptoms of fever, dry cough, tiredness, sore throat, diarrhea, loss of taste or smell to severe symptoms like breathing difficulty, chest pain, speech loss etc., ranging from 5-6 days to 14 days long period.

Getting blood oxygen level down is one point when body seems to be struggling for adequate supply of oxygen to their body parts. It is though lungs and RBCs who are the major player in this mechanism. Either lung being turned to morbid state or RBCs being in clotting phenomenon due to enzymes exudes of the virus, is yet to be fully explored and understood. But post-Covid death is all due to the organ failure at one or many level. Seriously infected patients even after been cured are suffering with many troubles of organ systems as one or more.

Medicines prescribed were mostly meant to take load away from fever and virus with immunity booster pills to fight; are the direction in which medicines were used by prescribing suitable doses of antipyretic, anti viral drugs and vitamin tablets. Plasma therapy in this case is used but with efficacy and validity controversy.

Vaccines in this turmoil came as the only hope, even infection after the second vaccine dose is also on the record. It was and still is a hard task economically and physically to administer to all human population. This pandemic period has shown us as we humans are sitting in the same boat. A person standing last in the line can not be left as he/she will be the asymptomatic carrier. Rich or poor, black or white are the world citizens as deserving equal rights of life, sustainability, health care and education. This is the time to learn this hard lesson for humanity. 

In India by the time of second Covid wave in March-April 2021, every kind of medical system was in total failure. People were asking for medical oxygen for anything and there was none. Death were so enormous that disposal of human dead bodies saw most unprecedented visuals of the century. India was truly devastated. Systems failed at many levels. 

Lessons learnt are to be carried with our life style now and that is cleaning hands often with soap and alcohol based rubs, maintaining safe distance, wearing mask, not going out when it's unnecessary, 

Delta variant is in the corner to meet us, we don't exactly know that what is in store of the Covid strain or some other viruses in future, safety should be the prime concern of every individual as for self and safety of other human fellows too.

Sun sneeze is a heritable trait

Sun sneezing, as name suggests is a photo sneeze reflex or better said a naso-ocular reflex caused by the exposure of an individual to bright light, so Photosneezia is another name came from. Claimed to be affecting 18 - 35 percent of world's population which is quite sizeable number as 1 in 4 persons are sun sneezers, whose exact physiology is yet to be explained.

It is well recorded in history by Aristotle and others but was less understood until recently.

Some believe that it may have evolutionary advantages for the cave dwellers in terms of spreading of pathogens present in their naso-pharynx, to their fellow cohabitants. It doesn't fits that way because we all should have had this in our DNA.

Rock Garden, Chandigarh, India

 

By 1980s it was established as a heritable autosomal dominant trait, is now called ACHOO syndrome (Autosomal-Dominant Compelling Helio-Opthalmic Outbursts), caused by single-nucleotide polymorphism on Chromosome 2, where a single base Adenine (purine) is replaced by Cytosine (pyrimidine) on DNA due to random mutation.

Being of dominant nature (name; the C allele on the rs10427255 SNP), it expresses itself even if being transmitted by the single parent through Chromosome 2 (location; 146,125,523). 

Physiology is simple as Opthalmic branch (Optic nerve) of Trigeminal nerve (longest Cranial nerve) inhances the irritability of the Maxillary branch to get sneeze.

How alien we humans are

In a recent study 19 ancient viral DNA fragments were found, adding 2 to the previously known 17, deep embedded in human genome.(Research article published online on March 21, 2016 in Proceedings of the National Academy of Sciences of the United States of America.)

In a way 8 percent of human DNA is alien, scientifically named as Human Endogenous Retroviruses (HERVs).

More studies are in offing as to prove that how useful or infectious they are to the human beings. 

May be the viral outbreak in the human history have some link to HERVs as if these are capable of causing an epidemic.

These viruses incorporated in our DNAs ever since hundreds of millions of years must have shaped the course of human evolution history, God knows if benefiting to their own ends, is unknown as more studies and speculations are yet to be made. 

We all know that evolution is an endless creative process as what we find useless today, tomorrow it may play a vital role in adaptation course of genetic material. (http://www.nature.com/articles/nature11244.epdf?)

Ancient viruses hibernating in the human genome is a frightening call.

We are viral from the beginning.... 

Now the question is how alien we are ?

courtesy shared screenshot: 

http://www.pnas.org/content/early/2016/03/16/1602336113.abstract

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Some related article reference links:

Viruses are cellular organisms
Mitochondria is a symbiotic bacteria
Chloroplast is a symbiotic bacteria
Viruses hidden in human genes
Human a curious case of symbiont body
Human body and theseus paradox