The year 2020 Nobel prize in Medicine or Physiology has been awarded jointly to 3 scientists Harvey J. Alter and Charles M. Rice from America and Michael Houghton from United Kingdom for their discovery of Hepatitis C virus.  Harvey J. Alter, Charles M. Rice, and Michael Houghton (From left to right)   According to the World Health Organisation, there are 70 million people infected across the globe and 400,000 mortality each year due to the chronic virus. The disease caused by the virus is responsible for liver failure and liver cancer.  How was the Hepatitis C virus discovered? Hepatitis B virus was already discovered in the 1960s and was considered to be responsible for blood transmitted hepatitis, therefore, blood used to be tested for the virus before blood transfusions to avoid blood transfusion-related Hepatitis B disease.  Although the number of patients infected through blood transfusion was reduced post Hepatitis B screening, but a large group of individuals were still contracting the disease after blood transfusions. It meant that apart from Hepatitis B another unidentified causative agent responsible for the disease existed.  Harvey J. Alter and colleagues showed that blood from these infected individuals transmitted the disease to chimpanzees (only susceptible animal apart from humans). As the disease neither spread from Hepatitis A nor B so it was then called “non-A, non-B” Hepatitis.  Later almost after a decade, Michael Houghton in 1989 finally identified the unknown causative agent as the Hepatitis C virus. The final question of whether Hepatitis C virus can on its own cause hepatitis was answered by the work of Charles M. Rice. Rice’s lab works to understand virus replication and innate immune responses that fight infection.  Mode of transmission of Hepatitis virus Both Hepatitis B and C virus are found in the blood of infected individuals, therefore before blood transfusions, one is now checked for the presence of both the viruses. It can also be transmitted by sharing needles, sexual contact, contaminated medical or tattooing equipment’s. If a mother is infected with the virus she can also transmit it to her child during birth. It is important to note that Hepatitis A is not transmitted by blood or blood products. What is the significance of the Hepatitis C virus discovery? Identification of the causative agent of an infectious disease is the most basic and crucial step in fighting a successful battle against the disease. Due to their discoveries, today we have developed advanced testing kits to identify the Hepatitis C virus and have been able to prevent the blood transmitted hepatitis during blood transfusions.

The emergence of Coronavirus and Diagnosis Coronavirus disease (COVID-19) has emerged as that superstar who needs no introduction. Coronam is the Latin term for crown, and coronavirus has the crown-like appearance. As of now, global Covid-19 deaths have exceeded over 8 lakhs with total cases at 25.1 million and still counting. There was only one lab for testing on January 23, 160 labs on March 23 and 1370 labs by August, in India according to the health ministry.  Previously known as a 2019-novel coronavirus (2019-nCoV) was officially renamed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by International Committee on Taxonomy of Viruses (ICTV) on February 11, 2020. This name was selected because the virus is genetically related (70% similarity) to the coronavirus responsible for the SARS outbreak of 2002. Diseases are officially named by WHO in the International Classification of Diseases (ICD) while viruses are named by the ICTV.   Diagnosis of COVID-19 involves multiple phases from sample collection to submitting data of individuals in the ICMR portal. Sample collection-  Most commonly, nasal and throat swabs are collected and transported to the nearest testing facility in the Viral transport medium. Each individual is assigned a unique Specimen Referral Form (SRF) ID at the time of sample collection, and details of the individual are recorded. Samples are received in the above cooling boxes Labelling and neutralization-  Once the samples reach their destined labs, they are allotted lab IDs which are meant to easily distinguish among different samples. Further, neutralization is carried in Biosafety cabinet (BSL-2 and above required), during which virus-cell lysis is done using lysis buffer. This procedure requires maximum preventive measures, as mishandling could result in virus spread. Post neutralization, samples are sent for RNA isolation. Post neutralization remaining sample volume is stored at -80 ֯ C RNA isolation-  Coronaviruses are a large family and its genetic material is a single-stranded positive-sense RNA (+ssRNA); thus, RNA isolation is must to detect its presence in an infected individual. This RNA is then converted to complementary DNA (cDNA), using an enzyme called reverse transcriptase and PCR is done. Internal controls are used as an indicator of perfect nucleic acid extraction, quality of samples, quality of PCR.  For a quicker screening of individuals pooled sample approach for RNA extraction is being employed. Under this approach, samples from individuals (3 or 5 samples are generally pooled into 1) are mixed into a single tube and RNA extraction proceeds.  If a pool appears negative for E-gene after RT-PCR (Real-time PCR), then all individuals are considered negative. However, if a pool is detected positive then, RNA extraction is individually done for the pooled samples and tested for E-gene as well as RdRp gene (RNA-dependent RNA polymerase). RdRp is essential for the replication of the virus genetic material once it infects a host. Thus, individuals are diagnosed positive or negative based on the results obtained from two different genes. Real-time PCR and data analysis-   In real-time PCR, a positive reaction is determined by the accumulation of a fluorescent signal. Generally, real-time assays undergo 40 cycles of amplification. The cycle at which the fluorescent signal can be detected is termed cycle quantification (Cq) value.  Some instruments show a cycle threshold (Ct) value instead, which is the number of cycles required for the fluorescent signal to cross the threshold value. Not to be confused, both the Cq and Ct values are technically similar. These values show how many cycles it took to detect a real signal from samples.  CY5 (cyanine5) is a synthetic dye which detects the internal control and thus confirms the quality of RNA and PCR. If Cq or Ct value for CY5 dye is nil, then the RNA extraction should be repeated. The other dye FAM (fluorescein amidite) is specific for coronavirus specific gene called E-gene. E-gene encodes a small membrane protein (E protein), necessary for the assembly of virions.  If a person tests positive, then there must be a Cq value for FAM (values greater than 36 are considered as false positives while between 22 to 36 are considered positive), while a person negative for the presence of the virus has no Cq value for FAM. Similarly, FAM is also capable to detect RdRp gene, which is another coronavirus specific gene. Furthermore, positive and negative controls provided with the real-time kit are also run simultaneously to verify the assay. Real-time PCR amplification curve showing Cq value of different samples The relation between Cq value and amount of target gene-  Cq values are inverse to the amount of the target gene (here E and RdRp gene) expression in samples. Lower Cq values indicate high amounts of target sequence while higher Cq values correspond to weak expression of the target gene. Other commonly available methods for testing  Rapid diagnostic tests based on antigen detection–  Rapid antigen tests account for about 30% of the total Covid-19 test conducted in India. The rapid test requires sampling similar for a molecular test (real-time PCR), but detects viral proteins (antigens) on the surface of the virus instead of genetic material and also provide results within 10-15 minutes.  However, antigen tests are not as sensitive as molecular tests but are useful due to their quicker output. Rapid antigen tests have been found to provide more false negatives and intermittent false positives. Due to these irregularities in the results, ICMR (Indian Council of Medical Research) has issued an advisory on antigen detection test, individuals who detect negative must be tested successively by RT-PCR to rule out infection, while a positive test should be considered true positive if the symptoms of the patients also, confirm so and does not require confirmation by RT-PCR. Rapid Antibody detection tests–  Antibody detection test is different from the other diagnostic tests, as it identifies people who were infected and have recovered. Unlike RT-PCR or antigen test, antibody detection requires a blood sample. It depends on the detection of antibodies against SARS-CoV2 in a blood sample, which can be easily obtained through a simple finger prick. Two different antibody isotypes, IgM and IgG are detected by this test. If a

1. Mosquitoes and their incessant buzz From vexatious buzz to itchy bites and their exceptional ability to escape from our murderous actions and buzzing around again as if chanting what a loser we were.  Yes, mosquitoes have been a threat to humans as they are vectors or carriers of deadliest diseases that have affected people across the globe. There are over 3,000 species of mosquitoes in the world and at least 400 of them can be found in India.  Before the female mosquito dies, it can produce up to 500 eggs. The only pleasant thing about mosquitoes is that they do not live long, but in the meantime, they contribute to the death of millions of individuals over the world. Yes, millions you read it right, according to World health organization every year mosquitoes result in deaths of over 1 million people around the world. Fun fact- Only female mosquitoes feed on blood, without which synthesis of yolk and development of eggs is implausible, while male mosquitoes get energy from plant nectar, rotten fruits and honeydew. Male mosquitoes are not able to suck blood from humans due to shorter mandibles. According to research published in the journal Frontiers in physiology, when male mosquitoes were allowed to feed on a blood-soaked cotton roll, they did not survive for too long. Thus concluding that blood is toxic for males. Below is the list of commonly caused diseases which utilize mosquitoes as a vector.     2. What research says?  In the latest research by Baik and colleagues, it has been found that preference of light or darkness by mosquitoes is dependent on the spectrum, time of day, sex and species of mosquitoes.  Dengue and malaria are the most widely heard diseases associated with mosquitoes, with dengue being caused by day-biting (diurnal) Aedes aegypti. In contrast, malaria is caused by anopheles which targets the vulnerable host at night (nocturnal). Aedes mosquitoes are more active and attracted to light during the day and become inactive and hide in dark locations once they have had their blood meal. In contrast, Anopheles coluzzii, Anopheles gambiae are inactive and specifically avoid ultraviolet (UV) and blue light during the day. 3. Circadian rhythm and mosquito response towards the light In a simple sense, the circadian rhythm is a 24-hour internal clock within almost every living organism which tells them when to do what irrespective of the external environmental conditions. These clocks are essential in determining the sleeping, feeding patterns and many more functions in all individuals, including humans.  However, these clocks can also become disrupted if natural external conditions are continuously manipulated, thus ultimately affecting once behavioural aspects. Similarly, in mosquitoes circadian clock functions to help diurnal/nocturnal mosquitoes in differentiating between day or night. While disruption of the clock by continuously exposing the mosquitoes towards artificial light for long hours confuses them and does not allow to distinguish between the day or night.  Thus, disruption of the circadian clock severely interferes with light-evoked attraction/avoidance behaviour, biting, flight and egg-laying activities in mosquitoes. 4. Why conditional differentiation of mosquito species occurs? Throughout evolution, different organisms of the species have evolved to differentiate in their abilities to avoid competition for survival. Similarly, different mosquito species might have evolved to occupy unique time-related differences (diurnal and nocturnal), so they can reduce inter-species competition and increase their opportunity of biting, mating and overall existence. It has been found that multiple behavioural changes occur in mosquitoes with respect to time of the day, which includes flight activity, mating, egg-laying, and biting. 5. Why light-based approaches can be substantial to target mosquitoes? Light-based strategies to control mosquitoes are relatively safer in comparison to toxic pesticides which are harmful to the environment. Moreover, mosquitoes and the disease-causing pathogens have become increasingly resistant towards currently available treatment regimen.  Thus, an environmentally friendly and innovative method against mosquito control is necessary. With the application of the light-based approach, specific species can be targeted if the timing and the light spectra used is efficiently monitored. For example, if high-intensity UV light is employed during the day, it will be ineffective against nocturnal mosquitoes. However, the same approach, when utilized appropriately, has produced significant results. A research stated that an exposure of female anopheles mosquito (nocturnal) which causes malaria towards light up to 10 minutes during the night or dusk period can retard its biting and flying ability. Thus, disruption of the circadian clock by using artificial light may be applied toward species-specific control of harmful diseases like dengue and malaria.