Genetic sequencing is vital as vaccine will put pressure on virus to mutate, says Dr V Ravi

Since January 10 this year, flights from the United Kingdom have been full with 300-350 passengers arriving in the state daily. So far, 14 such passengers have contracted this new variant. Now that India has started rolling out Covid vaccines, genetic sequencing of Covid samples has acquired new significance, as the virus will be under immune pressure to mutate, says neurovirologist Dr V Ravi, nodal officer for genetic confirmation of SARS-CoV-2 virus in Karnataka, in a chat with Suraksha P. Excerpts:

Can you tell us about Karnataka’s capabilities in genetic sequencing?

Karnataka has three labs in Bengaluru that can do genetic sequencing: National Institute of Mental Health and Neurosciences (Nimhans), The Institute for Stem Cell Science and Regenerative Medicine-National Centre for Biological Sciences (inStem-NCBS), and Indian Institute of Science (IISc).

The other labs are in a research mode and nobody has experience in Covid-19 sequencing. These three labs have sequenced Covid genomes and have contributed sequences to the GISAID database (a global science initiative and primary source that provides open-access to genomic data of influenza viruses).

How do we compare with the other eight labs in INSACOG (Indian SARS-CoV-2 Genomics Consortium) that the Centre has specifically created for the purpose of monitoring any kind of mutations in the virus?

IGIB (Institute of Genomics and Integrative Biology), Delhi, has a high throughput as their primary objective is sequencing genomes, unlike Nimhans which is a diagnostics lab and we had been sequencing over the past four years for research questions. So our throughput cannot match IGIB’s. Nimhans has an Illumina NextSeq machine and if required it can be put to use.

At present, we’re using Nanopore technology (which can process a maximum of 24 samples at a time) because we’re getting five samples or less. Bigger platforms like HiSeq and MySeq require a minimum of 50 samples a day will be put to use.

Once the plan of sequencing 5% samples of all Covid patients who test positive in a day comes into play as routine surveillance takes off, which it should, in the next 10-15 days, once the resources are made available, then high throughput sequencing machines can be used. If Karnataka has 1,000 Covid positive cases per day, 5% of that would mean 50 samples a day.

Can Karnataka handle sequencing Covid samples of a regular stream of UK passengers?

In an unlikely scenario of 10% positivity, we’ll have 33 samples to sequence every day. In those, only those with less than 30 Cycle Threshold (Ct) value can be taken for sequencing. If the Ct value is above 30, they will not be suitable for sequencing as the genome cannot be amplified. IISc is still not a part of the Consortium, which we can use as a standby, but inStem-NCBS and Nimhans can do it.

What is the significance of the GISAID database in the pandemic?

It is an extraordinary initiative, in which each country is contributing thousands of genome sequences. They don’t ask for patient identity but epidemiological information of the patient. God forbid if vaccine failure occurs, and the Covid sample of a patient in whom the vaccine has failed is sequenced, then this genome can be compared with hundreds of thousands of genomes in the international database and we can find out which country this variant was already present in.

We can pinpoint the month and which country it moved thereafter. That is how we came to know in Karnataka that lineages came from Europe, Malaysia, Indonesia, and Iran, among others. The index case in the Nanjangud cluster had a European lineage.

When did we stop sequencing, before starting again, because of the UK variant?

Nimhans had done sequencing until August, 2020. The number of cases started shooting up and we already knew what lineages we have, and curiosity died down. Now, we understand after the UK variant emerged, that we should have continuous surveillance. We cannot do sequencing in spurts across the year, at least till the whole pandemic dies.

This becomes all the more important when vaccination is being introduced. Vaccines put immune pressure on the virus. The theory of how the UK variant emerged is that a chronic immunosuppressed patient who had a Covid infection had received convalescent plasma and had not been recovering. When his sample was sequenced, the UK variant was found.

There are two reasons for viruses to mutate: One is their inherent property -- the RNA polymerases are poor proofreaders. This is an advantage for all RNA viruses. When they copy themselves among the 30,000 nucleotides, one makes a mistake and mutations occur.

Second, when antibodies are being produced and the virus is being neutralised, to escape neutralisation, viruses mutate. We call these immune escape variants. The UK variant is an immune escape variant.

If we vaccinate 30 crore people in India in the next few months and even if the frequency for an immune escape variant is one in ten million, we should be monitoring it. The only way to monitor is through sequencing. Sequencing should now be a part of routine surveillance called molecular surveillance. That is why this whole INSACOG national programme was started.

What is the cost that the government is looking at if it wants to sequence 1,500 Covid samples per month?

The central government is thinking of central procurement and distribution of reagents. It will cost anywhere between Rs 6,000 to Rs 25,000 depending on the platform and volume. We won’t waste money on controls if the volume is high. One control is enough for a large run. An average of Rs 8,000 to Rs 12,000 is the reagent cost, plus we need at least four competent people to run the machine and analyse the data. Conservatively, their salaries alone would cost Rs. 2.5 lakh per month. Cumulatively, it would cost Rs. 1.5 crore per lab per month.