What is Coronavirus and 2019-nCoV?
This article will use a time-line of science studies in date order to explain how the understanding of coronavirus has evolved. It will explain the basic understanding of viruses and show that they are classified into families. It will explain how viruses spread and the impact they can have of society and also explain the long lasting neurological damage that can be done when we catch a common cold.
It will explain the details of the 2002 SARS outbreak and how attempts to develop a SARS vaccine failed because after a vaccine, any second exposure from the wild virus caused a multiplication in severity of illness and a worse outcome for sufferers. It will show that receiving an influenza vaccine leaves a person more susceptible to contracting other respiratory viral infections like the coronavirus.
It will explain the details of the 2013 nCoV outbreak and explain that this version of the virus did not transmit from human-to-human very well.
It will show that the latest 2019-nCoV infection transmits from human-to-human better, however, because the genome sequence has too many changes from the original, it did not originate directly from bats. It will show that either an intermediary animal version must exist that has not yet been seen by scientists, or, as one study suggested after matching it exactly, the gene changes come from the HIV-AIDS virus using "unconventional evolution" that is "unlikely to be fortuitous."
It will show that 2019-nCoV has caused person-to-person transmission to occur and that the originating patients have never been in contact with freshly killed produce.
It will show that Thailand successfully treated patients using negative pressure isolation facilities and not positive pressure ventilators and it will show that Korea has successfully treated patients using HIV-AIDS antiviral drugs and that both these treatment regimes gained a quick recovery for the patients involved.
Lastly it will describe a coronavirus test that is easy to use and gives more accurate results than the PCR test that is being currently used.
It will be helpful to first understand what viruses and a bacteria are. Although both move in body fluids, bacteria are mobile and can move and replicate of their own free will, without aid, while viruses are immobile and can only move in the flow of body fluids, like a stick in a river and they are not able to replicate without aid.
Our bodies are able to recognise all intruders and mount an attack using white blood cells. There is a war going on in our bodies every day, as our bodies strive to keep us clean and free from unwanted invaders. White blood cells are like our guard dogs and one can be seen in this video chasing a bacteria to engulf it and kill it. The same thing occurs with viruses but they are very small and difficult to see through a microscope in the same way as a bacteria.
There is lots of misinformation going around on social media claiming that viruses are not real, while claiming that it is impossible to catch one because they develop spontaneously and originate inside a person. These ideas are not supported by observations or science and the idea is damaging to everybody’s understanding of life. We have all observed children catching the measles virus from other children, in fact, measles parties were a normal way of life until the 1960s. Additionally, science is able to sequence the genome of each virus and they are not made from the DNA of the people they live in and are proven genetically to be separate entities. Therefore, this notion of viruses developing internally can easily be dispelled.
Furthermore. please don’t get viruses confused with exosomes, they are completely separate things. Viruses carry genetic material that has nothing to do with the host and definitely originates externally. Whereas, exosomes are important internal workers, carrying out many important jobs but most importantly they contain a direct copy of the persons DNA, this proves that they originate internally. Exosomes can even be used to determine the health of a person because they contain up-to-date DNA copies that can be examined for the latest genetic expression markers (Kahlert et al. 2014).
With regard to viruses, they are small packages of proteins that have no ability to move or replicate without hijacking a host. Viruses are usually transmitted from person to person in body fluids such as droplets of water in coughs or sneezes. Once the virus is inside the body it travels around aimlessly in the body fluids until it is able to lock onto the receptors positioned on the exterior of a cell membrane. Once locked on, they inject their genome of proteins into the interior of the body cell. Once the viral proteins are inside the cell, it hijacks the cell mechanics and uses its energy to replicate itself many times over. The new copies of the virus are released, to go forth and infect many other body cells that will cause a systemic infection.
The coronavirus is no different. It is a virus that has been studied many times in the past because it is known as a common cold virus. A cure for the common cold has been a topic on scientist’s mind for centuries because it would generate such a large revenue stream.
A study by Siddel et al. (1983) explains that the coronavirus family consists of 11 viruses and explains its clinical and economic importance because of its ability to cause respiratory and gastrointestinal disorders. The virus is described physically as being generally spherical, with club-like projections, a form that we have all become familiar with seeing in the media. The virus is further described as having a large single strand RNA genome sequence containing about 15 - 20,000 nucleotides. Nucleotides are the basic building blocks of life and this sequence is complex because it is said not to repeat very often.
A protein kinase is an enzyme that modifies others and this section of the virus can alter the host mechanics to replicate its own genome. The virus has a surface that projects peplomer proteins that can lock onto host cells to gain access and the virus also has the ability to neutralise antibodies in order to stave off attack. It is, therefore, a fully armed virus that has many tricks in its making that enable it to infect people while defending against attack. It is a formidable opponent for the immune system to deal with, however, it is the common cold virus and therefore our bodies are up to and complete this task many times in our lives.
Interestingly, this study notes that persistent infections of the central nervous system can result if an attenuated virus has been used to cause that infection. Therefore, this observation should be tested carefully before considering an attenuated virus vaccine for human use. Additionally, the study notes that the disease develops faster in animals that have high antibody titres which may explain why coronavirus challenge tests have found that the person is more likely to suffering an infection after receiving the influenza vaccine (Cowling et al 2012; Wolff 2020).
A study by Winther et al. (1984) investigated the bacterial change in the nasal passages and nasopharynx, which is the upper part of the throat behind the nose. After analysing the swabs it was found that the bacterial infections and dynamics did not change during the course of the viral infection. This was contrary to the understanding of bacteria normally taking advantage of changes in the environment during a viral attack by multiplying unchecked.
To apply this to our present 2019-nCoV predicament it would mean that the respiratory problems sufferers are encountering come solely from the virus and not from secondary bacterial lung infections as would be expected.
A study by Payne et al. (1986) examined the stools of patients suffering from gastroenteritis during an 8-year period. It found that of the viral induced conditions, coronavirus particles were present in 69.8% and it was determined that coronavirus was the main cause of infectious diarrhoea. The study explained that infectious diarrhoea is the main cause of infant mortality in the developing countries.
This has serious implications for any world-wide coronavirus vaccine strategy because the shedding through faeces would mean that many children in the developing world would succumb to intestinal disease without continuous vaccinations in the same way as the vaccine-derived polio problem in the Philippines and Africa.
A study by Yeager et al. (1992) explained that there are two groups of human coronavirus HCV-229E and HCV-OC43. It found that the coronavirus HCV-229E uses human aminopeptidase N as a receptor and that antibodies directed against the active site of this enzyme prevent virus infection of human cells but was unable to determine the route of entry for HCV-OC43.
A review by Myint (1994) explained that the Rockefeller institute was the first to discover the common cold was indeed caused by a virus in the 1930s and it was in the 1950s that it was discovered that viruses could be grown on human kidney cells. Swabs were taken from students suffering from cold symptoms and these were grown on human embryo kidney cells and after examination by electron microscope they were found to be identical to avian infectious bronchitis virus and mouse hepatitis virus. It was these viruses that were named coronaviruses because of the crown of surface projections and given the family name of Coronaviridea. All of the viruses in this family infect one species of animal and are termed as being “host-specific” and they all cause respiratory or gastrointestinal disease, while some are also responsible for disease of the nervous system even though the basic structure of all Coronaviridea viruses are the same. Experiments found that each time an animal was subjected to the Coronavirus the incubation period shortened and the disease became worse. After the fourth exposure the animals died.
It was discovered that the virus grew best at a temperature of between 32 and 34 degrees C and raising the temperature of the growth medium to 37 degrees C resulted in an absence of virus growth. It was show that it would not replicate in Humans who lacked the q11-qter region of human chromosome 15 and this genetic susceptibility was further born out when studying twins with different phenotypes.
It was found that patients suffering from lower respiratory tract infections also had antibodies reacting with Epstein-Barr virus and it was therefore implicated in the resulting disease. it has also been shown that human foetal brain cells are susceptible to coronavirus and that it is detectable in the brains of sufferers of multiple sclerosis and that a sequence of genes from the coronavirus is identical to the sequence that is important to Human myelin basic protein, this is suggestive of a relationship between children born with multiple sclerosis and coronavirus infection during pregnancy.
Additionally, the virus can bind to Human blood group O red blood cells. This binding to red bllod cells might deminish their ability to carry oxygen and therefore, may be of importance when considering the difficulty doctors are having in dealing with the problem of poor oxygenation in patients suffering in the present 2019-nCoV pandemic.
A study by Falsey et al. (1997) investigated the impact of coronavirus and rhinovirus on old people in day-care. It explained that there are more than 100 types of rhinoviruses and two major types of coronavirus that have been identified. It noted that while the young do not usually suffer dire consequences from these infections the old represent a special population at increased risk of infection and at risk for complications. Coronavirus infections were more common in the winter and early spring whereas rhinovirus activity was sporadic but tended to be more frequent in the summer and autumn. It was noted that when coronavirus was circulating rhinovirus nearly ceased. 50% of illnesses were associated with lower respiratory tract infections and 36% complained of feeling short of breath but oxygen saturation only dropped a small amount from an average of 95.3 to 94.2 during illness, however, one patient dropped from 95% to 89%. No deaths occurred during this study time-frame and when sufferers of underlying cardiac or pulmonary disease were compared with those without, no significant difference in the severity of rhinovirus or coronavirus infections was noted.
A study by Makela et al. (1998) found that 63% of cold sufferers were struck by a viral origin and the largest number (105) came from rhinovirus infections, with coronavirus second (17).
A study by Eleouet et al. (1998) explained that coronavirus infections usually attacked the organ lining and can cause extensive cell damage. Cell death (apoptosis) is used by the body as a way of destroying cells that are infected, however, viruses have evolved proteins to delay this cell death action until they have used the cell machinary to replicate.
A study by Arbour et al. (1999) found that the coronavirus was neurotropic, in that it attacked the nervous system. Receptors for the coronavirus were found on human neuron cells, that do our thinking and also on the neuronal supporting cells oligodendrocytes, that supply the protective sheathing and astrocytes that supply the physical scaffold support. It was also discovered that coronavirus infections were present in the brains of most people who had been subjected to the cold virus at one time in their lives. This led the study to surmise that chronic infection of the brain can occur even after the cold symptoms had passed. Confirmation of the coronavirus’s ability to hide within the central nervous system (CNS) was made using rodents as subjects. It was found that the route and dose of infection and host factors such as age, species, strain, and immune system status as well as the genetic constitution of the virus influence the ability of an infection to penetrate the CNS. However, once an infection had gained entry it host cells it persisted at a constant level.
Further investigations found that the virus is made from 4 structural proteins - spike (S), membrane, small membrane, and nucleocapsid. As previously explained by Siddel et al. (1983) the spike is responsible for locking onto the host cell. Further to this understanding Arbour et al. (1999) found that Vaccination with the S protein, or even only peptides of it, protected mice from a lethal intracerebral infection of coronavirus. Moreover, important determinants of neurovirulence reside in regions of the S glycoprotein of the coronavirus and this would suggest the vaccine containing only the spike protein would protect against coronavirus and therefore CNS attack.
This study explains that whole viruses are not necessary in a coronavirus vaccine in order to confer protection and only the spike protein needs to be included. For good measure the membrane proteins could be included but there is no need to use whole viruses. It has been shown many times that whole viruses, including their DNA, can recombine to form vaccine-derived versions of the original virus which presents a danger to humanity.
A study by Xu et al. (2004) looked at the SARS outbreak in China that occurred in the first week of February 2003. There were 1,454 confirmed cases and 55 deaths. This outbreak was found to occur in five different municipalities and occured near a produce market, however, patient 1 did not travel outside his own area in the 2 weeks prior to his infection. It is said that he prepared food including chicken, domestic cat, and snake. Patient 2 was not infected by patient 1 and did not kill any animals. Further investigation of other patients found that many of them had no contact with dead animals or contact with other sufferers.
Although the SARS virus was found to be a coronavirus, its origin could not be determined because it was not closely related genetically to any other known coronavirus. The only clue to its origin comes from immunity to SARS in traders of palm civets, however, the species of animal that SARS belongs too could not be determined. It was found that dealers in wild animals were more prone to disease and not farmers, which suggests the origin is more likely a wild animal and not livestock.
The provincial health department introduced a range of public health control measures, including guide-lines on epidemiologic investigation of cases and contacts (February 3) and on hospital admission, clinical management, and infection control arrangements for patients (February 9). Subsequently, the department issued guide-lines on community prevention and control, including mandatory home quarantine of contacts.
A study by Masters (2006) explains that the enormous size of the coronavirus genome has previously hampered genetic investigations because it has the largest genome of all RNA viruses. The study describes the 2002 SARS pandemic as a “sudden appearance” that “came as quite a shock” to the science community and it stemmed new investigations into the coronavirus family. From those investigations a further 2 human versions and 3 bat versions of the coronavirus were discovered and they were positioned into their genome family groups.
However, genome sequencing of the SARS coronavirus (SARS-CoV) found characteristics that positioned this virus roughly equidistant from each of the three previously established groups. This led scientists to presume it was the first example of a new 4th group. These investigations have led to the acknowledgment that many of the SARS-CoV components come from group 2, however, it was also found that many other parts of the virus have their origin in groups 1 and 3. Leading to the conclusion that although SARS-CoV is more closely related to group 2 its origin must be from all 3 coronavirus groups.
Angiotensin-converting enzyme 2 (ACE2) is a zinc binding enzyme that is involved in heart function. ACE2 receptors on human cell membrane was found to bind to the SARS-CoV S protein that is responsible for cell docking, therefore, it was thought interfeering with the ACE2 body receptors could be a possible method to interfere with the infection.
Aminopeptidase N (APN) is also a cell surface, zinc binding protein receptor found in the human respiratory system, the CNS and in digestive tissue that is used by SARS-CoV to initiate attack.
The study found that only a slight change in the S protein spike allows the coronavirus to jump from one species to another but asks why two very different, zinc-binding, cell-surface peptidases, APN and ACE2, should serve as receptors for such a substantial number of coronaviruses and explains that this situation can currently be ascribed to "an amazing coincidence, but it may later be found to have deeper significance."
A study by Yasui et al. (2008) found severe pulmonary inflammation occurred after infection with SARS-CoV in animals that had previously been vaccinated with a virus that had related characteristics. It was found that the nucleocapsid (N) protein was responsible for the severe pneumonia observed during a SARS-CoV infection.
The changes in immune response and pathogenesis after SARS-CoV vaccination is not well understood, as almost all the previous studies reported only protection within a few days of SARS-CoV infection. In the present study, we demonstrate that vaccinated mice that are again exposed to SARS-CoV infection exhibit an imbalance between T cell activation (high expression levels of IFNγ, IL-2, IL-4, and IL-5) and subsequent suppression (low ex- pression levels of IL-10 and TGF-ß), as well as high-level production of proinflammatory cytokines (IL-6 and TNF- α) and chemokines (CCL2, CCL3, and CXCL10). Meaning that the inflammatory and repair systems are altered by the infection.
An elevation in production of pneumocytes, CD3+ T cells, and monocytes and macrophages of the lungs of patients with SARS was reported and it was suggested that CXCL10 may be responsible for the infiltration of activated T cells and monocytes or macrophages, which is a pathologic finding in SARS patients that may cause a thickening of lung tissue in the air sac where oxygen exchange occurs.
Finally, the N protein of SARS-CoV has been shown to induce both cellular and humoral immune responses. Taken together, these results raise the possibility that a percentage of SARS patients already possess the adaptive immune response elements that can interact with SARS-CoV components, including the N protein, and that their adaptive immune response may be involved in the exacerbation of pneumonia.
In conclusion the study demonstrate that the vaccination of mice with the N protein of SARS-CoV causes severe pulmonary inflammation upon subsequent SARS-CoV infection, probably via the imbalance created between T cell activation and suppression, as well as by massive proinflammatory cytokine production. These results provide new insights into the mechanisms involved in the pathogenesis of SARS and it is said this understanding may help in the development of safe vaccines.
A study by Bolles et al. (2011) found that SARS-CoV vaccines using double-inactivated viruses provided incomplete protection and actually left the subject more susceptible to increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication when exposed to the SARS-CoV after vaccination. Eosinophils are a type of white blood cell that are normally present during a parasitic infection, an allergic reaction or cancer.
The study explains that when unvaccinated animals were exposed to SARS-CoV their lungs were less affected than vaccinated animals. This proves that vaccination make the problem worse.
Although these tests were using animals, the study concludes by saying that these findings raise significant concerns regarding SARS-CoV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing SARS-CoV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.
A study by Cowling et al. (2012) found that trivalent inactivated influenza vaccine (TIV) given to children made them more susceptible to non-influenza infections. They found a slightly lower number of influenza illnesses suffered by the unvaccinated group, however, there was a four-fold increase in other respiratory illnesses suffered by the vaccinated when compared to the unvaccinated.
They concluded that no significant difference was noted in influenza rate between the vaccinated and the unvaccinated although serology evidence pointed to a lower infection rate in the vaccinated. However, they observed a significant increased risk of non-influenza infections in the vaccinated and suggest that virus interference is responsible.
A study by Tseng et al. (2012) evaluated 4 different types of SARS-CoV vaccine.
The animals were vaccinated and then challenged with SARS infection. In all cases of the 4 different types of vaccines the animals developed immunopathology even though there was an absence of detectable virus in the lungs. Vaccinated animals were found to exhibit similar infections to unvaccinated but with the addition of immunopathology.
The reaction was attributed to the presence of the nucleocapsid protein (N) in the vaccine. Histopathology scores were high for the N containing vector group and low for the S containing group.
To be certain the Th2 type immunopathology was elicited by the S protein vaccine in our studies and in hopes a greater immune response would result from higher dosages of the vaccine and induce greater protection against infection as well as reduce or prevent the immunopathology, our experiment 2 used up to 9 mg of the S protein for immunization. While increased titers of serum antibody were induced and no virus was detected day two after challenge in most animals, the Th2-type immunopathology occurred after challenge, and the immunopathology seen earlier after vaccination with the whole virus vaccine was seen again in the S protein vaccine.
This combined experience provides concern for trials with SARS-CoV vaccines in humans. Clinical trials with SARS coronavirus vaccines have been conducted and reported to induce antibody responses and to be ‘‘safe.” However, the evidence for safety is for a short period of observation. The concern arising from the present report is for an immunopathologic reaction occurring among vaccinated individuals on exposure to infectious SARS-CoV, which is the basis for developing a vaccine for SARS.
A study by Khan (2013) reports of a new coronavirus outbreak in Saudi Arabia and that it is of the same family as SARS-CoV. The new virus is named NCoV and the genome sequence reveals it is most closely related to the bat coronavirus BtCoV-HKU4 and BtCoV-HKU5 which were first isolated in 2006. The NCoV was found to be able to infect monkeys, humans, bats and pigs but like SARS the NCoV animal reservoir is not known. The cases in both, the Saudi and Jordanian cluster had no reported contact with animals. Similarly, the most recent laboratory confirmed cases from UK, also had no contact with animals or any history of travel to the Middle East.
This outbreak was the first time this form of the virus was thought to be transmitted from human to human however, based on current information, NCoV does not appear to transmit easily or sustainably between people, but it can lead to serious lower respiratory tract infection and renal failure.
It is noted that a number of different bat species resident in Ghana and parts of Europe are infected with coronaviruses very similar to NCoV, in some cases differing by less than 2% at the genetic level, however, many questions remain unanswered because there is no clear understanding of how the virus jumped from the bat family or how did the sufferers acquired the infection.
A study by Khuranda et al. (2013) noted that vaccine-associated enhanced respiratory disease (VAERD) has been reported in multiple respiratory infections in humans and in animals. Seronegative children administered formaldehyde-inactivated respiratory syncytial virus (RSV) vaccine followed by exposure to wild-type RSV demonstrated enhanced respiratory disease. Similarly, atypical measles with severe disease was reported in children vaccinated with formalin-inactivated vaccines. The mechanisms underlying VAERD after respiratory infections are not completely understood and may vary with the disease and/or vaccine modality.
Pigs were shown to be an exceptionally good for the modelling of human influenza and vaccine studies and in this study were used to investigate VAERD when the circulating/ infecting influenza strain does not match the vaccine strain. A failure was noted of an inactivated classical swine H1N1 vaccine to protect pigs after infection with an H1N2 swine influenza virus while enhancing the severity of pneumonia. Further studies demonstrated that pigs vaccinated with whole inactivated virus vaccine containing a human-like H1N2 (WIV-H1N2) and subsequently challenged with pH1N1 had enhanced pneumonia and respiratory disease when compared with nonvaccinated pH1N1-challenged animals, thus providing strong evidence of VAERD after mismatched influenza infection.
The lesions in the lungs of vaccinated pigs was 3 times greater than the lesions in unvaccinated pigs. It was found that the animals developed a heightened immune response to the virus version they were vaccinated against but did not produce immune responses when challenged with the other virus version.
A study by Joyjinda et al. (2019) made the first genome sequence of the bat coronavirus that had infected a bat guano miner in Thailand. It found that the genome sequence was closely related to HCoV-HKU1 from Hong Kong that was isolated in 2006. Therefore, the study concluded that although the patient had extensive occupational exposure to bat faeces, this individual was likely exposed to HCoV-HKU1 due to person-to-person transmission and not via exposure from bats. It went on to inform that, our surveillance strategy and viral characterization pipeline provide valuable insight into the circulation of endemic infectious diseases in Thailand. It is worth noting from this study that although 4 separate families of human coronavirus exist, 38 separate genome sequences within those families were recorded for versions of coronavirus that are able to infect humans. This is testament to how many versions of coronavirus are known to be in existence and how many more probably exist that have not been sequenced yet.
A study by Prashant et al. (2020) mapped the genome sequence of 2019-nCoV.
The study took a comprehensive approach at analysing the 2019-nCoV genome and used approved databases and analytical software.
The study aligned and visualised the 2019-nCoV genome using Multalin software and found the closest relative to be SARSCoV.
Further investigation revealed that the genome of the spike protein contained 4 insertions. Multiple examinations were made of all 55 coronavirus versions available to investigate if these insertions were present in previous genome sequences. It was found that these 4 insertions were unique to 2019-nCoV. They cited Zhou et al (2020) as having previously found 3 spike insertions but noted the study had compared fewer spike protein sequences.
They then analysed all available full-length sequences (n=28) of 2019-nCoV in the genetic database GISAID as on January 27, 2020 for the presence of these inserts. As most of these sequences are not annotated, we compared the nucleotide sequences of the spike glycoprotein of all available 2019-nCoV sequences using the BLASTp alignment search tool. The study reported that, interestingly, all the 4 insertions were absolutely (100%) conserved in all the available 2019-nCoV sequences analysed.
The study then found that the 4 inserts were not in the bat version of the virus. They were Intrigued by the 4 highly conserved inserts unique to 2019-nCoV they wanted to understand their origin. For this purpose, they used the 2019-nCoV local alignment with each insert as query against all virus genomes and considered hits with 100% sequence coverage.
Surprisingly, each of the four inserts aligned with short segments of the Human immunodeficiency Virus-1 (HIV-1) proteins. The amino acid positions of the inserts in 2019-nCoV and the corresponding residues in HIV-1 gp120 and HIV-1 Gag. The first 3 inserts (insert 1,2 and 3) aligned to short segments of amino acid residues in HIV-1 gp120. The insert 4 aligned to HIV-1 Gag. The insert 1 (6 amino acid residues) and insert 2 (6 amino acid residues) in the spike glycoprotein of 2019-nCoV are 100% identical to the residues mapped to HIV-1 gp120. The insert 3 (12 amino acid residues) in 2019-nCoV maps to HIV-1 gp120 with gaps. The insert 4 (8 amino acid residues) maps to HIV-1 Gag with gaps.
Although, the 4 inserts represent discontiguous short stretches of amino acids in spike glycoprotein of 2019-nCoV, the fact that all three of them share amino acid identity or similarity with HIV-1 gp120 and HIV-1 Gag (among all annotated virus proteins) suggests that this is not a random fortuitous finding. In other words, one may sporadically expect a fortuitous match for a stretch of 6-12 contiguous amino acid residues in an unrelated protein. However, it is unlikely that all 4 inserts in the 2019-nCoV spike glycoprotein fortuitously match with 2 key structural proteins of an unrelated virus (HIV-1).
The HIV-1 Gag protein enables interaction of virus with negatively charged host surface and a high positive charge on the Gag protein is a key feature for the host-virus interaction. It was therefore speculated that these insertions provide additional flexibility to the glycoprotein binding site by forming a hydrophilic loop in the protein structure that may facilitate or enhance virus-host interactions.
The study goes on to say that since the S protein of 2019-nCoV shares closest ancestry with SARS GZ02, the sequence coding for spike proteins of these two viruses were compared using MultiAlin software. We found four new insertions in the protein of 2019-nCoV- “GTNGTKR” (IS1), “HKNNKS” (IS2), “GDSSSG” (IS3) and “QTNSPRRA” (IS4). To our surprise, these sequence insertions were not only absent in S protein of SARS but were also not observed in any other member of the Coronaviridae family. This is startling as it is quite unlikely for a virus to have acquired such unique insertions naturally in a short duration of time.
The study explains taken together, our findings suggest unconventional evolution of 2019-nCoV that warrants further investigation. Our work highlights novel evolutionary aspects of the 2019-nCoV and has implications on the pathogenesis and diagnosis of this virus.
However, this study was withdrawn by the authors after they received comments “from the research community on their technical approach and their interpretation of the results” and the authors intend to revise their claims accordingly.
This study appears to have come under fire because it noted the changes in the 2019-nCoV genome were not natural.
A study by Lu et al. (2020) investigated the genome sequence of the 2019-nCoV outbreak by culturing samples from the lungs of nine patients who were infected after visiting the Huanan seafood market in Wuhan.
They managed to gain 8 complete and 2 partial genome sequences of 2019-nCoV from the nine patients that were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%).
It was also found that 2019-nCoV might be able to bind to the angiotensin- converting enzyme 2 receptor in humans.
4 mutations were noted in the spike protein genome sequence that did not match the bat SARS-like coronaviruses, SARS-CoV, nor the MERS-CoV genomes. It was therefore concluded that a recombinant event had occurred.
The mutation had occurred in the spike protein that is responsible for host cell receptor binding and fusion that is critical for transmission. The spike protein can be divided into 2 categories. The S1 domain is responsible for receptor binding and the S2 domain is responsible for cell membrane fusion. The 2019-nCoV S2 protein showed around 93% sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21—much higher than that of the S1 domain, which had only around 68% identity with these bat-derived viruses. This interpretation of this virus adaption is that it has particularly changed its ability to bind with humans without changing any other part of its dynamics.
These facts suggest unknown quantities are also present in that, first, the outbreak was first reported in late December, 2019, when most bat species in Wuhan are hibernating. Second, no bats were sold or found at the Huanan seafood market, whereas various non-aquatic animals (including mammals) were available for purchase. Third, the sequence identity between 2019-nCoV and its close relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21 was less than 90%, which is reflected in the relatively long branch between them. Hence, bat-SL-CoVZC45 and bat-SL-CoVZXC21 are not direct ancestors of 2019-nCoV. Fourth, in both SARS-CoV and MERS-CoV, bats acted as the natural reservoir, with another animal (masked palm civet for SARS-CoV35 and dromedary camels for MERS-CoV)36 acting as an intermediate host, with humans as terminal hosts. Therefore, on the basis of current data, it seems likely that the 2019-nCoV causing the Wuhan outbreak might also be initially hosted by bats, and might have been transmitted to humans via currently unknown wild animal(s) sold at the Huanan seafood market.
A study by Ji et al. (2020) investigated the genome of 2019-nCoV. It found that the virus appeared to be a recombinant version that originated from the bat coronavirus and another unknown animal. It found that the new virus was not closely associated with any other version of the coronavirus.
It found that 2019-nCoV was most similar to the lineage of the bat virus and that of the snake but not close enough for the snake to be the other animal involved in the evolution of 2019-nCoV.
It noted that the spike protein was changed and that there was no other sequence known on the database that matched the change. It concluded that the 2019‐nCoV has most similar genetic information with bat coronavirus and has most similar codon usage bias with snake.
A study by Tang et al. (2020) notes that person-to-person transmission has occurred in people who had no contact with Wuhan wet food market. It also notes that the genome sequence of 2019-nCoV places it in the family of coronavirus that infects bats.
It is noted that of the 2 cases in Thailand. The first could be traced to Wuhan and She reported regular visits to wet markets in Wuhan but not the index wet market from where most cases were reported. However the second case was not
linked epidemiologically to the first case, and she had not visited any market in Wuhan. So far both cases are recovering well in the negative pressure isolation facilities at the Bamrasnaradura Institution in Thailand and may be discharged soon.
A study by Kim et al. (2020) documented the first case in Korea. It notes that on 19th January 2020 a woman arrived from Wuhan and was found to have a temperature of 38.4oC.
On day 4 of the illness the initial chest radiography showed no infiltrations but high-resolution computed tomography (HRCT) showed multiple, ground-glass opacities located in both subpleural spaces. She did not complain of shortness of breath but her arterial oxygen saturation had dropped to approximately 91%. She was put on an oxygen cannular of 3L/min. A pan coronavirus conventional polymerase chain reaction (PCR) assay was positive for the throat swab sample, and sequencing of the PCR amplicon showed that the sequence was identical to that of the 2019-nCoV strain isolated from the Wuhan patient and lopinavir 400 mg and Ritonavir 100 mg was given. Both of these drugs are used to treat HIV-AIDS and are antiretroviral medications.
On day 7 of the illness her oxygen requirement increased to 6 L/min. Her temperature climbed to 38.9oC.
On day 8 of the illness a chest radiography began to show chest infiltrates in the right lower lung field.
On day 11 of the illness her temperature subsided.
On day 14 of the illness her breathing improved, her oxygen requirement subsided and her chest X-Rays showed less lung lesions.
The study noted that the patient had pneumonia from day 4 of the illness but she did not show any clinical symptoms. This was reminiscent of the “walking pneumonia” that presented in the MERS-CoV cases.
A study by Lim et al. (2020) documented the spread of 2019-nCoV in Korea and noted that all of the patients came from or visited China. This 54 year-old patient was the subject of this study because he was the source of secondary transmission to one person and tertiary transmission to 3 more cases. His patient notes from the hospital include test results and these are documented along with his illness. He tested negative for many other possible cause of his respiratory condition and they were ruled out. He was diagnosed using the PCR test as having CIVID-19.
On January 25th just 6 days after the patient documented in the Kim et al. (2020) study above this patient contacted a public health centre and was isolated because of a high temperature.
On day 3 of illness he was admitted to hospital.
On day 4 of illness he was confirmed as suffering from 2019-nCoV.
On day 8 of illness pneumonia started.
On day 10 of illness lopinavir 200 mg and ritonavir 50 mg was given twice per day.
On day 11 and 12 of illness the viral load began to decrease and was undetectable.
On day 13, 14, 15, 16 his viral load increased slightly.
On day 17 of his illness the viral load decreased and he got better.
A study by Lui et al. (2020) examined the transmission of 2019-nCoV.
As of January 9 isolation was occurring after 6.7 days.
As of January 19 isolation was occurring after 0.7 days. This faster isolation was claimed to be responsible for slowing the spread of disease
As of January 23, 2020, a total of 830 confirmed 2019-nCoV cases were identified across China, and 9 cases were reported overseas. A total of 9,507 close contacts were traced and isolated and this also slowed the spread of disease.
As of January 24, 2020, the virus had spread to 29 provinces, regions and
municipal cities across China. A total of 1965 suspected cases and 1287 confirmed cases with 41 fatality had been reported.
The average incubation period was 4.8 days. The average period from onset of symptoms were 2.9 days and isolation occurred after an average of 4.2 days.
Many cases were detected in medical staff and persons without exposure to wildlife or having visited Wuhan within 14 days prior to the onset of illness.
Limited evidence for human-to-human and familial transmission was found and no published reports about the transmission dynamics were found. However, after examination of the available evidence it was concluded that the transmission rate was found to be comparable with the SARS outbreak of 2003. Additionally, 2019-nCoV could be spread by any person in the first stages of the disease who were not showing symptoms. For this reason it was assumed that the 2019-nCoV epidemic could affect a greater number of people than the 2003 SARS epidemic.
A study by El-Tholoth et al. (2020) reports on a closed tube, single stage 2019-nCoV LAMP and 2019-nCoV Closed-Tube Penn-RAMP assays for use at home, in the clinic and at ports of entry that requires minimal sample processing.
The patient or health care provider collects a nasal sample with swab, elutes the swab in water and heats the water to above 65oC. The sample is then inserted into a tube for single stage amplification or two stage amplification (with greater sensitivity). The reaction tube is incubated at 63oC in the LAMP process and at 38oC and 63oC in the two stage Penn-RAMP process.
To simplify our system even further, we have demonstrated that we can detect test results colourimetrically with LCV dye that changes colour from colourless in the absence of dsDNA to deep violet colour in the presence of abundant dsDNA. This colour change is visible to the naked eye and does not require any instrumentation. All reagents can be readily stored in the tube in dry form, providing long, refrigeration-free shelf life.
Although much simpler, our 2019-nCoV LAMP test performs on par with the conventionally used 2019-nCoV PCR, providing similar sensitivity. Our closed- tube, two stage Penn RAMP outperforms both PCR and LAMP providing 10-fold better sensitivity than LAMP alone when purified samples are used and 100-fold better sensitivity when minimally prepared samples are used. This added sensitivity is important since it has been reported that a significant number of 2019-nCoV patients test negative with the 2019-nCoV PCR test.
After reading the information in this article it is hard to understand how the 2019-nCoV came to be in existence without some form of man-made manipulation. The missing information from the evolution is not usual and although the authors of the Prashant et al. (2020) document withdrew their study because of peer pressure it was done to an exacting standard using the best methods and techniques.
Additionally, the changes in the coronavirus spike protein were recorded in other studies but unfortunately, they did not search for the matching sequence in the same way and enthusiasm as Prashant et al. (2020).
Hopefully, it can be seen that cures exist in other countries and that taking a different approach to the treatment regime would give patients in the UK and the US a better chance of survival.
Additionally, a document by El-Tholoth et al. (2020) has been repeated here that outlines a better form of testing that will give more reliable results for those who may need treatment. This faster testing may also be the impetuous to end a lockdown that seems to be an over-reaction to a problem that is nowhere near as bad as it need be, if only a better understanding could have been available and a more suitable treatment plan were in place.
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