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 INFECTIOUS DISEASE

BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

TURKISH


VIROLOGY - CHAPTER   TWENTY FIVE 

CORONA VIRUSES:

COLDS, SARS, MERS AND COVID-19

Dr Richard Hunt
Professor
Department of Pathology, Microbiology and Immunology
University of South Carolina School of Medicine
Columbia
South Carolina

 

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Vaccines
Pandemics
Social Distancing and Masks

 

  
Logo image © Jeffrey Nelson, Rush University, Chicago, Illinois  and The MicrobeLibrary
corona-cdc.jpg (73328 bytes) 

corona2.jpg (52304 bytes)  Figure 1 Coronaviruses are a group of viruses that have a halo or crown-like (corona) appearance when viewed under a microscope
CDC/Dr. Fred Murphy (top) CDC/Dr. Erskine Palmer (bottom)

 

INTRODUCTION

Coronaviruses (figure 1) are the largest RNA viruses and are about 100nm in diameter. They infect humans and animals in which they cause respiratory and enteric disease.

The Nidovirales order of viruses that infect vertebrate (including human) and invertebrate hosts consists of four families:

  • Coronaviridae. There are two sub families - Coronavirinae (Coronaviruses), which infect mammals and birds, and the Torovirinae (Toroviruses) (figure 2) which infect vertebrates, especially cattle, pigs, and horses.

  • Arteriviridae which infect vertebrates, mostly mammals, including equine arteritis virus.

  • Roniviridae which infect crustaceans.

  • Mesoniviridae which infect insects.

          These are large, single-strand positive-sense (mRNA sense), enveloped RNA viruses and there are seven, all coronaviruses, that are known to cause human disease.

This chapter will only discuss the Coronaviruses since they are particularly important in human respiratory disease, causing about one third of "common colds" and the newly recognized severe acute respiratory syndromes: SARS, MERS and COVID-19.

 

 

toro.jpg (51701 bytes)  Figure 2 Torovirus
© Queen's University, Belfast

Coronaviruses

Corona = Latin: crown, Ancient Greek κορώνη - because they look like spiky crowns in an electron microscope.

In the Order Nidovirales, Coronaviruses are the most important in human disease and are divided into four genera:

  • Alpha coronaviruses including HCoV-NL63 and HCoV-229E which usually cause mild upper respiratory tract infections in humans. Porcine epidemic diarrhea virus is also a member of  this class of coronaviruses.

  • Beta coronaviruses including MERS CoV, SARS CoV-1 and SARS CoV-2 (which causes COVID-19 disease) and HCoV-OC43 and HCoV-HKU1 (which usually cause less severe respiratory symptoms) 

  • Gamma coronaviruses which mainly infect birds.

  • Delta coronaviruses which also mainly infect birds although porcine delta coronavirus is important in animal husbandry. It emerged in 2014 and may have spilled over from an avian host.

            Three β-coronaviruses have caused outbreaks of deadly pneumonia in humans since the beginning of this century. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1) was first reported in 2002. The disease that it causes, Severe Acute Respiratory Syndrome (SARS), is highly infectious and has a high fatality rate of 10%. Its epicenter was China and Hong Kong but it was largely contained in 2003 although some additional cases occurred in 2004.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in Saudi Arabia and other countries in the Arabian Peninsula in 2012. Middle East Respiratory Syndrome (MERS) has a very high fatality rate in humans of around 35%.  There have also been travel-associated cases of MERS in Europe, Asia and North America. In May 2014, CDC reported two imported cases of MERS in the United States in people who had traveled from Saudi Arabia.

SARS CoV-2 is the causative agent of Corona Virus Disease-2019 (COVID-19) that has swept the globe in a disease pandemic in 2020. Like SARS, the epicenter of COVID-19 was China (Wuhan, the capital of Hubei Province). It has proved highly infectious and has a fatality rate of around 2% although this may change as more data become available. Mortality occurs more prominently in the elderly with underlying health complications, but people of all ages have died from the disease.

All three of these coronaviruses are zoonotic (animal borne) viruses that crossed species barriers. Bats are the primary animal host for MERS CoV and SARS CoV-1 and -2. SARS CoV-1 may have crossed to humans from bats via palm civet cats while SARS CoV-2 may have infected humans from bats via pangolins. In both cases, the virus probably infected humans who came in contact with wild animals in live-animal markets in China. The intermediate hosts of MERS CoV are dromedary camels.

Several other zoonotic coronaviruses are endemic in the human population. They cause about a third of mild respiratory tract infections (rhinorrhea (runny nose), headache, sneezing, malaise and sore-throat). Coryza (acute inflammation of the upper respiratory tract) with fever and cough is seen in 10 to 20% of cases. In some cases, these otherwise mild infections can lead to severe complications or death in young children, the elderly and immunocompromised individuals. These viruses, which may also cause diarrhea, are HCoV-NL63 and HCoV-229E (alpha) and HCoV-OC43 and HCoV-HKU1 (beta).

Why Bats?

 

 

 

CORONAVIRUS “COLDS”

In humans, about one-third of colds are caused by coronaviruses, the major site of coronavirus replication being the epithelial cells of the respiratory tract. The symptoms are similar to those of rhinovirus colds with an incubation time of about 3 days. Viral spread is limited by the immune response of most patients but this immunity is short-lived. Symptoms may last about a week with considerable variation between patients. Often there are no apparent symptoms but the patient still sheds infectious virus.

Although coronavirus infections are usually localized to the upper respiratory tract, they can spread to other organs. In humans, these viruses have been implicated in infections of the middle ear, in some pneumonias in immuno-suppressed patients and in myocarditis but in animals, systemic infections can be much more severe (e.g. feline infectious peritonitis).

In contrast to the rhinoviruses (Picornaviridae) which are not enveloped, coronaviruses are rather unstable. Transmission is by transfer of nasal secretions such as in aerosols caused by sneezes. Viruses that infect epithelial cells of the enteric tract cause diarrhea. This can occur in human neonates but is common in many young animals where the infection can be fatal.

 

Epidemiology
Most people harbor anti-coronavirus antibodies but reinfection is common indicating that there are many circulating serotypes of the virus in the human population. As with most respiratory infections, coronavirus-caused colds are more common in the winter because of closer contact and lower humidity. Major outbreaks occur every few years with a cycle that depends on the type of virus involved.

Diagnosis
Most coronavirus infections go undiagnosed and the disease is self-limiting. Diagnosis can be carried out using immuno-electron microscopy and serology. There are no anti-virals for routine coronavirus infections but over-the-counter remedies to alleviate symptoms are useful.

 

MOUSE HEPATITIS VIRUS AND MULTIPLE SCLEROSIS

Interestingly, a neurotropic strain of mouse hepatitis virus (a beta coronavirus) can cause a disease in rodents that looks very like multiple sclerosis, leading to the suggestion of their involvement in the human disease; demyelination, a characteristic of multiple sclerosis in the rodent model, is linked to the S protein and it has been suggested that the disease results from molecular mimicry in which an immune response to the S protein results in immune attack on myelin. However, although the virus can be detected in the brain of patients, the link to multiple sclerosis remains unproven.

 

sars-map.gif (67004 bytes) Figure 3
Map of probable SARS cases. June 02, 2003

WHO

sars-case.gif (2757 bytes) Figure 4
.Weekly new cases of SARS
 ©
WHO/BBC

lung-x.jpg (41838 bytes) Figure 8.
Chest radiographs of index patient with severe acute respiratory syndrome (SARS). a, day 5 of symptoms; b, day 10; c, day 13; d, day 15.
Li-Yang Hsu, Cheng-Chuan Lee, Justin A. Green, Brenda Ang, Nicholas I. Paton, Lawrence Lee, Jorge S. Villacian, Poh-Lian Lim, Arul Earnest, and Yee-Sin Leo - Tan Tock Seng Hospital, Tan Tock Seng, Singapore.
Emerging and Infectious Diseases

sars-lung.jpg (80471 bytes) Figure 9 Pathologic cytoarchitectural changes indicative of diffuse alveolar damage, as well as a multinucleated giant cell with no conspicuous viral inclusions.
CDC/Dr. Sherif Zaki

SEVERE ACUTE RESPIRATORY SYNDROME (SARS)

In late 2002, a new syndrome was observed in southern China (Guangdong Province). It was named Severe Acute Respiratory Syndrome (SARS). This disease, which has now been reported in Asia, North America, and Europe (figure 3), is characterized by a fever above 38 degrees (100.4 degrees Fahrenheit) accompanied by headache, general malaise and aches. Respiratory symptoms are initially usually mild but after a few days to one week, the patient may develop a dry non-productive cough and breathing may become difficult (dyspnea). Respiratory distress leads to death in 3-30% of cases. Laboratory tests show a reduction in lymphocyte numbers and a rise in serum aminotransferase activity which indicates damage to the liver.

The initial outbreak of SARS peaked in April 2003 and by June had tailed off (figure 4). By that time, there had been about 8,000 cases worldwide and 775 deaths. In addition, there were billions of dollars in economic losses.

Virus from infected patients was grown on monkey Vero E6 cells in tissue culture and identified as a new coronavirus (SARS-CoV). It has a genome of 29,727 bases and eleven open reading frames. The organization of the genome is very similar to that of other coronaviruses (5’ replicase (rep), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3 and short untranslated regions at both termini). The replicase (RNA polymerase) gene occupies the 5’ two-thirds of the genome and has, like other coronaviruses, two overlapping open reading frames. It also codes for a protease that is part of the RNA polymerase polyprotein. There are nine possible open reading frames that are not found in other coronaviruses and may code for proteins that are unique to the SARS virus. Using antibody tests, SARS-coronavirus has been associated with SARS cases throughout the world.

Diagnosis
The Centers for Disease Control recommend a chest radiograph (figure 8), pulse oximetry (a test used to measure the oxygen saturation of the blood), blood cultures, sputum Gram's stain and culture, and testing for other viral respiratory pathogens, notably influenza A and B and respiratory syncytial virus. A specimen for Legionella and pneumococcal urinary antigen testing should also be considered. People with suspected SARS should be isolated and quarantined.

Infection by SARS-CoV-1 shows changes indicative of diffuse alveolar damage, as well as a multinucleated giant cell with no conspicuous viral inclusions (figure 9).

Treatment
There is no agreed treatment for SARS other than management of symptoms. Drugs are under development and of particular interest are drugs that may block the protease function since this is crucial to the virus. There is no approved vaccine against the SARS virus although some have been developed. Veterinary vaccination programs of modest success exist for a number of economically important coronaviruses.

 

MIDDLE EAST RESPIRATORY SYNDROME

In 2012, a disease caused by a novel Coronavirus appeared in the Middle East, particularly in Saudi Arabia. Initially, all patients lived in or had visited the Middle East even though some had subsequently traveled to Europe where cases appeared in France, the UK and Germany. After an initial infection, the virus spread to close contacts indicating human to human transmission. The patients develop pneumonia and sometimes kidney failure with a fatality rate of up to 50% although this high fatality rate may reflect the failure to diagnose less virulent cases. The virus was first named Novel Corona Virus (nCoV) and then named “Middle East Respiratory Syndrome Coronavirus” (MERS-CoV) and is distinct from the SARS Coronavirus. It can be treated with interferon α2b and ribavirin.

Once again the origin of this novel coronavirus is bats, specifically the pipistrelle bat, the intermediate host being dromedary camels. From phylogenetic analysis, it appears that the virus entered the human population around 2011.

 

 

CORONAVIRUS DISEASE 2019 (COVID-19)

On December 31, 2019, there was a case of pneumonia of unknown etiology admitted to hospital in Wuhan, China. It was quickly established that this was neither SARS nor MERS or any other common respiratory pathogen; others followed. They had the hallmarks of a coronavirus infection and were quickly shown to result from a hitherto unknown coronavirus which was initially termed 2019-nCoV but has now been renamed SARS-Coronavirus 2 (SARS-CoV-2) because it also causes a severe acute respiratory syndrome.

By January 11, there were 41 cases of this illness in Wuhan; most were benign but seven were serious infections and by that time, there had been one death. The disease, spread by human-to-human transmission, was highly infectious and was soon found outside Wuhan in surrounding Hubei Province and then elsewhere in China. The first case outside China was in Thailand and the patient (a 61 year old woman) had come from Wuhan. As a result of severe restrictions on movement in Wuhan and the surrounding Hubei province, the Chinese government was able to get the epidemic under control but COVID-19 has spread worldwide. At the end of the first wave of infections by SARS CoV-2 (April 2020), China had reported 81,865 cases, 3,335 deaths and 77,370 recovered patients. Overall, the case fatality rate in Wuhan was 0.9-2.1%.

By the end of the second week of April 2020, there had been 1.5 million cases of COVID-19 worldwide with 91,000 deaths and 342,000 people had recovered.

 

Symptoms of COVID-19

Two to fourteen days after exposure, according to CDC, the initial symptoms are:

  •  Fever

  • Cough

  •  Shortness of breath (dyspnea)

  • Fatigue

  • Muscle and body aches

  • Headache

  • Congestion

  • Nausea

  • Vomiting

  • Diarrhea

  • Often the loss of smell and taste

Subsequently, more severe symptoms may appear including:

  • Trouble breathing

  • Persistent pain or pressure in the chest

  •  Confusion or inability to arouse

  •  Bluish lips or face

In some children, there are symptoms similar to Kawasaki Syndrome.

Kawasaki Syndrome

However, many infected people either show no symptoms at all or only mild symptoms, such as cough and fever.

Mortality

SARS and MERS have a high mortality rate with approximately 10% of SARS patients and approximately 37% of MERS patients succumbing to the disease. COVID-19 seems to have a much lower mortality rate of around 2% although this increases with the age of the patient and the presence of underlying complications.  

Age                 Death Rate (%)

  •  80+                         14

  • 70-79                        8

  • 60-69                      3.6

  • 50-59                      1.3

  • 40-40                      0.4

  • 30-39                      0.2

  • 20-29                      0.2

  • 10-19                      0.2
         

Males have a higher fatality rate than females

Female                   1.7

Male                       2.8

Certain comorbidities greatly increase the death rate including heart disease, diabetes, hypertension and chronic respiratory disease.

Transmissibility
As of March, 2020, the transmissibility (reproduction factor, R0) of SARS-CoV-2 appeared to be around 2 to 3. This means each infected person has spread the virus to an average of 2 to 3 other people.

 

 

 

corona-diag.gif (28742 bytes) Figure 10 Coronavirus structure. Adapted from Lai and Homes. In Fields' Virology. Lippencott

 

Figure 11
Ultrastructural morphology of a coronavirus viewed by electron microscopy. The spikes (S protein) give the appearance of a crown, hence the name. The E and M proteins are also located on the outer surface of the virus.
CDC

Figure 12
SARS-CoV-2 genome showing the order of the genes and the proteins encoded by them

 

 

nested.gif (5478 bytes) Figure 13 Messenger RNAs of corona viruses. A nested set of RNAs with a common 3' end are formed. The mRNA for the polymerase (pol) is the same length as the genomic RNA. The remainder are truncated at the 5' end although all have a common leader sequence

CORONAVIRUS STRUCTURE

Coronaviruses are positive-sense single strand RNA viruses. Unusually for RNA viruses, they are rather large with a genome of about 30kb. This large size has consequences for their mutation rate that will be discussed below. The fact that these viruses are positive sense means that their genome is in the same sense as mRNA and the genomic RNA may be used as an mRNA as soon as the cell has been infected. Maturing virus particles bud through intracellular membranes and gain a lipid envelope (i.e. coronaviruses are enveloped viruses). This has consequences for infection control since they are likely to be less stable than non-enveloped viruses and to be sensitive to detergents and organic solvents. The structure of a coronavirus is shown in figure 10 and the external morphology in figure 11.

 

COVID-19 Genome

SARS-CoV-2 has a genome of 29,829 bases and is like a typical cellular mRNA (figure 12).  It has a 3’ poly A tail and is capped at the 5’ end. The latter consists of a guanine nucleotide linked to the mRNA via an unusual 5 to 5 triphosphate bond. This guanosine is methylated on the 7 position by a virus-encoded methyltransferase. In addition, the 5’ end is methylated on the 2 hydroxy-groups of the first two ribose residues.  This cap provides resistance to degradation by 5 exonucleases in the cell.

About two thirds of the genome, starting at the 5’ end, codes for non-structural protein (NSP) 1ab. This is also known as the replicase gene although other proteins besides the replicase (RNA polymerase) are encoded in this gene. Non-structural proteins are virus-encoded proteins that are not part of the mature virus particle but are used in the replication and maturation of the virus. The coding sequences for protein 1a and 1b are not in the same reading frame but the ribosome undergoes a -1 frame shift at the end of the gene for protein 1a so that a long polyprotein is made. This is then cleaved by a virus-encoded protease to proteins 1a and 1b. Proteins 1a and 1b are themselves polyproteins and are cleaved by virally-encoded protease activity to 16 smaller non-structural proteins. Non-structural proteins are needed before the structural proteins since they are involved in viral RNA and protein synthesis and thus must be made shortly after the infection of the cell. This is done by translating incoming positive sense genomic RNA which has all of the characteristics of a cellular mRNA.

The genes for the structural proteins are located in the the 3’ third of the genome. These are transcribed into a set of complementary minus-strand RNAs that are templates for the transcription of a nested set of sub-genomic mRNAs from which the structural proteins are translated (figure 13). 

In SARS CoV-1 and -2 and MERS CoV, there are four main open reading frames in this 3’ region that code for:

  • The spike (S) protein by which the virus attaches to the host cell receptor

  • The membrane (M) protein

  • The envelope (E) protein

  • The nucleocapsid (N) protein

In addition, there are a number of smaller open-reading frames that code for proteins that may be structural or serve some accessory function.

 

Coronavirus Proteins

The non-structural proteins, NSP 1 – NSP16

The function of some of these proteins is unknown but some are involved in controlling the infected cell’s nucleic acid metabolism. In most cases this is inferred from research on other coronaviruses such as the closely related SARS CoV-1 and MERS CoV and is not the result of investigations of SARS-CoV-2.

NSP1 - Reduction of host cell protein synthesis

NSP1 very effectively shuts off host cell protein translation by binding to the 40S ribosomal subunits. The complex of NSP1 and the ribosomal subunit also acts as an enzyme that inhibits host cell protein translation by means of an endonucleolytic cleavage near the 5'UTR (untranslated region) of host mRNA leading to host cell mRNA degradation. This begs the question of why the viral mRNAs are not cleaved in a similar manner since, to all intents and purposes, they look just like cellular mRNAs. It turns out that NSP1 binds to a stem-loop structure in the 5'UTR of SARS CoV-1 RNA and this interaction stabilizes the mRNA carrying the specific stem-loop and enhances viral protein translation.

NSP2 - A protein of unknown function

NSP2 expressed in cells using retroviral transduction was specifically recruited to viral replication complexes. It is not required for viral replication in cell culture although deletion of the NSP2 coding sequence attenuates viral growth and RNA synthesis.  Other than that, the function of NSP2 is not known.

NSP3 - A multifunctional protein that contains a protease

This is the largest protein encoded by the coronavirus genome, with a size of about 200 kD. It is an essential component of the replication/transcription complex of the virus and spans the membrane of the endoplasmic reticulum. As might be expected for such a large protein, it has several domains with different functions:

A ubiquitin-like domain 1 (Ubl1). This binds to the single-stranded RNA and interacts with the nucleocapsid (N) protein. It is essential for viral replication which ceases when the UbL1 domain is partially deleted.

A glutamic acid-rich acidic domain (also called "hypervariable region")

A protease (papain)-like domain (protease 1 (PLpro). This releases NSP1, NSP2, and NSP3 from the N-terminal region of polyproteins 1a and 1ab.

A macrodomain or X-domain. This is not needed for RNA replication but may be involved in counteracting the host innate immune response.

Another ubiquitin-like domain 2 (Ubl2). The function of this is not known.

Another protease (papain)-like domain (protease 2 (PL2pro)).

 NSP3 ectodomain (3Ecto, "zinc-finger domain"). This is the only domain located on the lumenal side of the endoplasmic reticulum in SARS-CoV-1 NSP3. It is thought to bind metal ions and contains an asparagine-linked oligosaccharide.

Domain Y1, the function of which is not known

CoV-Y domain which is also of unknown function. Domains Y1 and CoV-Y are on the cytosolic side of the endoplasmic reticulum.

There are two transmembrane domains in NSP3 which appears to cross the endoplasmic reticulum membrane twice. These, plus the 3Ecto domain, are important for the PL2pro protease domain to cleave the site between NSP3 and NSP4 in SARS-CoV-1. The transmembrane domain may bring PL2pro close to the cleavage site between the membrane-associated proteins NSP3 and NSP4.

NSP3 together with NSP4 and NSP6 are required for the formation of the double membrane vesicles that are characteristic of coronavirus-infected cells.

 NSP4 - Reorganization of cell membranes

NSP4 is a glycoprotein that spans the endoplasmic reticulum membrane four times with three loop regions. Loops 1 and 3 are exposed to the endoplasmic reticulum lumen while loop 2 and the N and C termini are cytosolic. There are two asparagine-linked glycosylation sites in loop 1.

As will be described below, many positive strand RNA viruses, including coronaviruses, modify host cell cytoplasmic membranes that are sites of viral RNA synthesis and the formation of viral replication complexes. Coronaviruses induce double-membrane vesicles and when infected cells are analyzed by electron microscopy, NSP4 mutants have aberrant morphology in their double-membrane vesicles compared to cells infected with wild type virus. Thus, NSP4 may play a role the organization of the membrane vesicles which are important in RNA synthesis and viral replication although their role in these processes is unclear.  A glycosylation site on the lumenal side seems to be important in RNA synthesis.

 NSP5 - A protease

NSP5 (3CLpro, Mpro) is a protease that cleaves other NSP proteins at 11 cleavage sites and is essential for virus replication. NSP5 protease is also an interferon antagonist in that it inhibits Sendai virus-induced interferon-beta production in infected cells by targeting a protein called NF-κB essential modulator (NEMO).

NSP6 - Reorganization of cell membranes

NSP6 is also involved in double membrane formation within the infected cell. It induces perinuclear vesicles localized around the microtubule organizing center. The double membranes are formed as part of autophagy, a cellular response to starvation that generates autophagosomes to transport long-lived proteins and organelles to lysosomes for degradation. Besides being a normal cellular process under starvation conditions, autophagy can be activated by virus infection as part of an innate defense mechanism; however, this anti-viral mechanism is hijacked by some positive strand RNA viruses when autophagosomes are used to facilitate assembly of replicase proteins. NSP6 generates autophagosomes from the ER but limits autophagosome diameter and expansion which inhibit the ability of autophagosomes to transport viral components to lysosomes for degradation.

NSP7 and NSP8 - A primase

NSP7, NSP8, NSP9 and NSP10 are constituents of the RNA replication complex of coronaviruses.

Coronaviruses encode two RNA-dependent RNA polymerase activities. One is primer-dependent and is associated with NSP12 (see below). The other is associated with NSP8, a 22kD protein which is unique to coronaviruses and is capable of de novo initiation of RNA synthesis with low fidelity from single strand RNA templates. Thus, NSP8 has been proposed to operate as a primase, that is it makes oligonucleotide primers that can then be used by NSP12, the major RNA-dependent RNA polymerase. NSP7 and NSP8 form a supercomplex, a cylinder-like structure made up of eight copies of NSP8 and held tightly together by eight copies of NSP7.

NSP9 - A protein of unknown function

NSP9 is a single-stranded RNA-binding dimeric protein.

NSP10 - A scaffold protein

NSP10 is a scaffold protein with two zinc fingers. It interacts with NSP14 and NSP16, stimulating their 3'-5' exoribonuclease (NSP14) and 2'-O-methyltransferase (NSP16) activities. NSP10 is required by NSP16 as a stimulatory factor to execute the latter’s methyltransferase activity and may stabilize the S-adenosyl methionine-binding pocket and extend the substrate RNA-binding groove of NSP16.

 NSP11 - A protein of unknown function

The function of this protein is unknown since a deletion in the NSP10-NSP11/12 site abolished NSP5 protease-mediated processing but allowed production of infectious viral particles suggesting that cleavage at the NSP10-NSP11/12 site is not needed for viral replication in cultured cells.

NSP12 - An RNA polymerase

This protein, the major RNA polymerase, assembles along with NSP7 and NSP8 into a multi-subunit RNA-synthesis complex that carries out replication and transcription of the viral genome. NSP12 has a unique N-terminal extension which has been proposed to contain a nucleotidyltransferase activity whereas replication of the viral RNA genome is catalyzed by a polymerase domain in the C-terminal region.

NSP13 - A helicase

NSP13 is an RNA helicase and 5 triphosphatase that interacts with the RNA polymerase, NSP12. A helicase is an enzyme that catalyzes the unwinding of duplex oligonucleotides into single strands in a nucleoside triphosphate-dependent manner. NSP12 enhances the helicase activity of NSP13 but how NSP12 increases helicase activity is unknown.

NSP14 - An exonuclease and methyl transferase

Most RNA polymerases do not possess a “proof-reading” activity and, as a result, the size  of RNA viruses is normally limited to about 10kb (see below). However, coronavirus genomes are the largest of the RNA viruses with a size around 30kb. Given the error rate of RNA polymerases due to tautomerization of the bases, coronaviruses would seem to need some sort of proof-reading.  

NSP14, which forms a complex with NSP10, is an exoribonuclease and its inactivation leads to a 15-fold decrease in replication fidelity. It hydrolyzes double-stranded RNA in a 3' to 5' direction as well as a single mismatched nucleotide at the 3'-end mimicking an erroneous replication product. The exonuclease activity is also involved in the synthesis of the set of sub-genomic RNAs that encode the structural proteins. In addition to its nuclease activity, NSP14 also has a (guanine-N7)-methyltransferase activity involved in the 5’ capping of mRNAs and the genomic RNA.

NSP15 - An endonuclease

NSP15 is a hexameric endonuclease that preferentially cleaves at uridines. It associates with the primase (NSP9) and the RNA polymerase (NSP12). Mutations in the catalytic site reduced sub-genomic RNA accumulation and profoundly attenuated virus proliferation. Coronaviruses are able to avoid detection by host innate immune sensors that recognize double stranded RNAs. NSP15 is required to evade these dsRNA sensors.

NSP16 - A methyl transferase

NSP16, in complex with NSP10, has RNA ribose 2'-O-methylation activity. In order to mimic cellular mRNA structure, many viruses modify the 5'-end of their RNAs. The 5’ cap is important for RNA stability, protein translation and also viral immune escape. In addition to NSP14 S-adenosyl-L-methionine-dependent (guanine-N7) methyltransferase, coronaviruses have another methyl transferase, NSP16 which is an S-adenosyl-L-methionine (SAM)-dependent ribose 2’O-methyltransferase.

 

 

 

 

Figure 14
The domains of the Spike (S) protein

 

Figure 15
The association of S, M and E proteins with the viral membrane

 

The Structural Proteins

These are the proteins that compose the mature virus particle.

Spike (S) protein
Protruding from the viral lipid membrane is the spike protein, a homotrimer of S proteins that are responsible for attachment to host receptors and for fusion of the viral envelope with the host cell membrane. Each monomer has a molecular mass of 175kD and is N-glycosylated. In SARS-CoV-1 and -2, the trimer binds to the receptor human angiotensin converting enzyme 2 (hACE2) on the cell surface after which the virus enters the cell by endocytosis.

Each spike protein (S) monomer consists of two subunits (figure 14) formed by proteolytic cleavage using a Golgi complex enzyme called furin. These are the S1 subunit (binding/attachment subunit) and the S2 subunit (fusogen subunit). S1 first binds to the cell plasma membrane receptors and then dissociates from it. This is followed by a change in S2 conformation inside the endosome that is needed for membrane fusion.

The S protein spans the viral membrane once with the glycosylated N-terminus on the exterior of the virus (figure 15).

Membrane (M) protein
The M protein has three transmembrane domains (figure 15). It promotes membrane curvature and forms a link between the envelope inner structures and the nucleocapsid. It has a small N-terminal domain located outside the virus particle (which is equivalent to the lumenal side of the intracellular membrane vesicles) and a large C-terminal domain, comprising half of the protein, inside the virus particle (this is equivalent to the cytoplasmic side of the vesicle). M proteins of some alphacoronaviruses contain an additional hydrophobic segment that functions as a signal peptide. The M protein is glycosylated on its N-terminal extracellular domain. It appears to be the catalyst for virus assembly since it can interact with all of the structural proteins including itself. In some coronaviruses M and E proteins can form virus-like structures but in SARS-CoV-1, N protein is needed as well to form these structures.

Envelope (E) protein
The E protein (figure 15) plays a role in virus assembly and release, and is involved in viral pathogenesis since the virus is attenuated in vitro and in vivo when the E gene is deleted. E protein is a small multifunctional integral membrane protein that forms an ion channel that is important in virus-host interactions. Since E protein is small, oligomerization is needed to form the channel. It is not known why coronaviruses need an ion channel but several other viruses have a similar protein. In the case of influenza virus which also enters the cell by endocytosis, it appears that upon acidification of the endosome, the influenza M2 protein allows transfer of hydrogen ions into the virus particle to aid uncoating of the genome.

E protein has one membrane spanning-domain with the N terminus on the outside surface of the virus particle and a palmitic acid anchor on the inside linked to a cysteine residue (figure 15). Mice infected with coronaviruses that possess E protein ion channel activity rapidly lost weight and died whereas those infected with E protein mutants that lacked ion channel activity recovered from disease and most survived. E protein is involved in several aspects of the virus' life cycle, such as assembly, budding, envelope formation, and pathogenesis.
 

 

Nucleocapsid (N) Protein
This internal protein has two domains each of which can bind virus RNA by different mechanisms. It binds to NSP3 to attach the genomic RNA to replication-transcription complex, and packages the encapsidated genome into virus particles. The N protein binds to the viral genome to form the ribonucleoprotein core of the virus and interacts with carboxy-terminal domain of the M protein.

Hemagglutinin-esterase (HE)
A gene on the 5’ side of the S protein gene encodes a fifth structural protein, the hemagglutinin-esterase. This is present in some of beta-coronaviruses including mouse hepatitis virus but not SARS-Cov-1 or -2. HE binds to sialic acids on surface glycoproteins and acts as a hemagglutinin. It contains acetyl-esterase activity which may enhance S protein-mediated cell entry and virus spread through the mucosa. It enhances murine hepatitis virus neurovirulence but the HE gene is lost in tissue culture for unknown reasons.


HE forms spikes (shorter than S spikes) on the virus surface. It is a dimer and does not appear to be essential for replication in those types that possess it. The esterase activity of HE protein can cleave the sialic acid from a sugar chain, which may aid the virus in escaping from the cell in which it was replicated. Antibodies against HE protein can also neutralize the virus.
 

 

Other open reading frames (ORFs)
Inspection of the SARS-CoV-1 and -2 genomes shows a number of additional open reading frames (ORFs). An ORF is a region of the genome that has the ability to be translated into a protein. To be translated, it needs a start codon (usually AUG) and a stop codon (usually UAA, UAG or UGA). There is some information about the functions of the proteins encoded in these ORFs but the role of others is, as yet, unknown.

ORF3a and 3b
Proteins 3a and 3b are encoded by ORF3a and ORF3b and make up the second largest sub-genomic RNA encoded in the SARS-CoV genome. Protein 3a has been shown to be present in the plasma membrane in a punctate pattern, as well as intracellularly. The N-terminal region of protein 3a consists of three transmembrane domains. The protein forms a homotetramer and since all ion channel proteins form homo- or hetero-polymers and associate with the membrane, it has been proposed that protein 3a may form an ion channel.

Protein 3b may play a role in immunomodulation. In addition, it may act as an interferon antagonist.

ORF6

ORF6 protein is found associated with the membranes of the endoplasmic reticulum and Golgi complex in SARS-CoV-1 infected cells. This protein is dispensable in viral replication in vitro and in vivo but functions in viral escape from the innate immune system, particularly inhibition of type I interferon production and signaling.

ORF7a
This is a minor structural protein that is not essential for virus replication. It may be involved in viral assembly and in the detachment/escape of virus particles from the cell surface. It may do this by inhibiting the glycosylation of bone marrow stromal antigen 2 (BST2, also known as tetherin) which is thought to restrict virus release from the infected cell surface by physically tethering the budding enveloped virus particle to the plasma membrane.

ORF7b
This is another minor structural protein found in the Golgi complex. It is not essential for viral replication in vitro and in vivo.

Orf9b
This protein interferes with ubiquitination of cellular proteins and, like ORF6, alters host innate immunity.
 

 

 

 

corona-er.jpg (101548 bytes) 

  Figure 17
Coronavirus within cytoplasmic membrane-bound vacuoles and cisternae of the rough endoplasmic reticulum. Thin section electron micrographs of infected cells

 
CDC/C.S. Goldsmith/T.G. Ksiazek/ S.R. Zaki


 

 

Life Cycle

The life cycle of a coronavirus is shown in figure 16

         

Figure 16. Coronavirus Life Cycle

Positive (sense) strand RNA is black

Negative (anti-sense) strand RNA is green

Proteins are shown in blue and orange (except in last image where S is red, E is purple and M is brown).

1. The virus S (Spike) protein binds to the receptor (angiotensin converting enzyme 2) on the surface of an epithelial cell.

2. The plasma membrane invaginates and the virus enters an endosome.

3. Hydrogen ions are pumped into the early endosome to form a late endosome.

4. The lowering of the pH in the endosome changes the conformation of the S protein so that the S2 subunit (fusogen) allows the viral membrane to fuse with the endosomal membrane. The nucleocapsid enters the cytoplasm and is uncoated.

5. The viral genomic RNA is positive sense (same sense as an mRNA) and has the characteristics of an mRNA (It is 5’ methyl guanosine capped and has a 3’ poly A tail). Ribosomes bind to the genomic RNA and translate the 5’ two thirds of the genome to the p1ab polyprotein.

6. The p1ab polyprotein has an endoprotease activity that cuts the primary translation product to p1a and p1b.

7. p1a and p1b are cut by the same protease activity to 15 or 16 non-structural proteins (NSPs).

8. NSPs 7, 8 and 12 associate to form the viral RNA polymerase (replicase).

9. The polymerase binds to an initiation site near the 3’ end of the genomic RNA.

10. The positive sense genomic RNA is copied (3’ to 5’) into full length negative sense genomic RNA. This is NOT capped or polyadenylated as the negative sense RNA has no capping or adenylation signal.

11. The negative sense RNA is copied 3’ to 5’ into positive sense genomic RNA that is 5’ capped and 3’ polyadenylated.

12. The genomic RNA is copied by the RNA polymerase to a nested set of negative sense sub-genomic RNAs (sgRNAs).

13. The negative sense sgRNAs are copied to positive sense sgRNAs that are 5’ capped and 3’ polyadenylated and act as mRNAs.

14. The positive sense sgRNAs are translated into proteins which are mostly structural proteins of the virus.

15. The N protein binds to the positive strand viral genomic RNA to form the nucleocapsid and to the M protein which is embedded in the intracellular membranes.

16. The nucleocapsid buds through the endoplasmic reticulum/Golgi complex membranes into the lumenal space.

17. The virus leaves the cell along the secretory pathway.

 

Receptors for SARS-CoV-1, SARS-CoV-2 and other Coronaviruses
S and HE proteins bind to sialic acid which is found on the surfaces of all cells; however, all coronaviruses have a restricted tissue tropism and so binding is likely to be more complicated. Moreover, some coronaviruses do not bind to sialic acid at all and some, including SARS CoV-1 and -2, do not have an HE gene.

Both SARS-CoV-1 and SARS-CoV-2 use the same cell receptor although there is some suggestion that they bind to a slightly different region of the receptor. The spike protein trimer binds to human angiotensin converting enzyme 2 (hACE2) after which the virus enters the cell by endocytosis. It is been found that transforming Hela cells so that they express hACE2 makes them susceptible to SARS-CoV-2 infection.

MERS-CoV uses a different receptor, binding to the cell surface CD26 antigen (dipeptidyl peptidase 4).
In the case of murine hepatitis virus, the receptor is a member of the immunoglobulin superfamily, carcinoembryonic antigen-related cellular adhesion molecule (CEACAM), and antibodies against this protein block virus attachment.

Many alpha coronaviruses utilize aminopeptidase N (APN) as their receptor.



Entry into the cell
Some coronaviruses, such as MHV, can fuse at the plasma membrane and do not require endocytosis but lysosomotropic agents show that entry of the SARS-CoV-1 and SARS CoV-2 nucleocapsid into the cytoplasm is via endocytosis. Once in the acidic endosome, the S protein is cleaved by an acid-dependent protease cathepsin or another protease. This alters the conformation of the spike protein so that the fusogen (S2) can promote the fusion of the viral and cellular membranes.

Studies usings cryo-EM of the SARS-CoV-1 spike and its interaction with the hACE2 showed that in the late endosome there is dissociation of S1 from hACE2 followed by a change in S2 conformation that is needed for membrane fusion thereby ejecting the nucleocapsid into the cytoplasm.

Phosphoinositides are also involved in endocytosis. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is synthesized by phosphatidylinositol 3-phosphate 5-kinase in the early endosome and controls the transition of early endosomes into late endosomes. When cells expressing the ACE2 receptor were treated with an inhibitor of this enzyme (apilimod), there was significantly reduced entry of the virus, whereas it had no effect on entry of VSV-G pseudovirions, which occurs in early endosomes.



Inside the cell
Coronaviruses, like many other positive-stranded RNA viruses, rearrange cell membranes which are then used in viral genome replication and transcription (figure 17). Specifically, coronavirus NSPs induce the formation of double-membrane vesicles in infected cells.

Since the virus is positive strand, i.e. in the same sense as mRNA, the genes at the 5’ end of the viral RNA are translated immediately into a large polyprotein p1ab that is then cleaved by a virally encoded protease activity present within the polyprotein to p1a and p1b. These two polyproteins are processed into 15 or 16 (depending on the virus type) NSPs by multiple viral protease activities also present within the polyproteins. Some of these NSPs form the replication-transcription complex (RTCs) that synthesize viral RNA.

The RTC proteins are found in association with convoluted membranes and double-membrane vesicles which, along with host factors, copy the genome to a full size negative strand genome-length template from which new positive strand genomes are transcribed. This is genome replication. Alternatively, the genome is transcribed to smaller negative strand sub-genomic RNAs which are then copied into sub-genomic mRNAs that encode the structural proteins (S, N, E etc.) and other accessory proteins. The sub-genomic mRNAs encode both CoV structural proteins and accessory proteins of unknown function.

The spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins are the major proteins of the new viruses. The N protein binds the genomic RNA and allows its encapsulation into nucleocapsids. The S, M and E proteins are integral membrane proteins that are made in association with rough endoplasmic reticulum-associated ribosomes. The S protein, at least, is cleaved by host cell signal protease. The C-terminal domain of the MERS coronavirus M protein contains a trans-Golgi network localization signal and protein 7b has a Golgi retention signal. The membrane-embedded proteins migrate through the endoplasmic reticulum to the Golgi complex where the virus is assembled on the cytoplasmic surface. M protein organizes the components of the viral membrane and interacts with the nucleocapsid (N) protein to drive virus assembly and budding.

The nucleocapsids bud intracellularly into the ER-Golgi intermediate compartment or the Golgi complex and travel to the exterior of the cell along the secretory pathway. This is in contrast to most other membrane-bound viruses which bud through the plasma membrane.

 


Proof Reading

Most RNA viruses are small with a genome size of 10kb or less. As noted elsewhere in this book, their RNA polymerase lacks a mechanism to recognize and correct errors (mutations) that arise during genome replication. As a result, quasispecies are formed giving rise to adaptation and pathogenesis. Such mutations are inevitable because tautomeric forms of the nucleotide bases will lead to a low rate of misincorporation by the polymerase, however accurate it is. There is clearly a need for a functionally perfect full length genome but in a small virus (<10kb), genome instability (mutation load) leading to non-viable virus particles is not a great problem. However, the larger the genome, the more likely are deleterious mutations to arise.

It is not known how viruses encoding large viral RNA genomes such as the coronaviruses (up to 32 kb) deal with potential genome instability but some kind of proof reading, as found in DNA polymerases, is likely to be needed. Coronaviruses have a number of NSPs that are involved in RNA synthesis and the formation of RTCs which are unique to this type of virus. These enzymes include a 3′-5′ exoribonuclease, NSP14. Inactivation of the coronavirus exoribonuclease results in a decrease in genome fidelity. Thus, NSP14 may enhance RNA synthesis accuracy by correcting errors in nucleotide incorporation made by the virus’ RNA-dependent RNA polymerase.

 

Coronavirus Drugs

 

 
  Figure 18
A plot of cumulative counts of D614 and G614 sequences by day in Snohomish county. Orange represents the original form, blue the form with the G614 mutation. 2020. King County, Washington state, USA
Tracking SARS-CoV-2 Spike mutations (lanl.gov))

Figure 19
Maps showing the relative frequency of sampling D614 and G614 in different time windows. The size of the circle indicates the relative sampling in a given country within each of the four maps. The proportion of the original D614 Wuhan virus is shown in orange and the proportion of the D614G mutant is shown in blue. Top left, distribution prior to march 11, 2020; top right, distribution March 11-20; bottom left, distribution April 11-20; bottom left, distribution June 11-20.
Tracking SARS-CoV-2 Spike mutations (lanl.gov)

 

  Figure 20
Percentage of positive Covid-19 cases as detected from one laboratory in the south of England between the beginning of October and mid-December, 2020. The blue bars show the percent positive tests of all tests done. These rose from 7% to around 20% in mid-December. The orange bars show the proportion of tests that detected the B.1.1.7 variant. The variant was hardly present at the beginning of October whereas, by mid-December almost all tests revealed the variant.

  Figure 21
The domains of SARS CoV-2 S protein showing the Furin-cleavage sites

Figure 22
The important mutations in the B.1.1.7 variant found originally on southern England. Three are deletions and 14 are point mutations. They are clustered in the 1a, S (spike protein), 8 and N open reading frames.

  Figure 23
The distribution of important mutations in the S gene of the B.1.1.7 variant are shown in red in the bottom half of the figure. The mutations in the South African variant 501Y.V2 as detected up to October 15, 2020 are shown at the top in orange. By the end of November, the variant had acquired additional mutations shown in blue. All these variants have the D614G mutation. Shadowed amino acids are in the receptor binding domain.

Figure 23a
The distribution of important mutations in the S gene of the P.1 variant (top) and the B.1.1.7 variant (bottom)
 

  Figure 24
Presentation of S protein of SARS CoV2 to the immune system by an mRNA vaccine
The capped and polyadenylated mRNA is encapsulated inside a lipid nanoparticle (A) which is taken up into an endosome (B and C). The mRNA is released into the cytoplasm and associates with ribosomes. The translated protein contains an N-terminal signal sequence so that the polysome associates with the signal receptor on the cytoplasmic surface of the endoplasmic reticulum and translocates into the intra-endoplasmic reticulum space. It acquires post-translational modifications, including glycosylation, folds in the normal way and trimerizes (D). The S protein follows the normal exocytic route via the Golgi Body and passes into exosomes (E). From here, the protein may go to the cell surface (F) and be endocytosed into a proteolytic endosome (G) or it may go directly to a proteolytic endosome without secretion (H). The protein is degraded by endosomal proteases and the resulting peptides bound by major histocompatibility (MHC) antigens (I). CD4+ and CD8+ T cell activation then occurs via the presentation of the peptides on MHC class II and class I respectively (J).

  Figure 25
Pseudouridine and uridine structure

  Figure 26
Transcription of an mRNA vaccine molecule from a DNA plasmid construct

  Figure 27
1-methylpseudouridine. An extra methyl group is added enzymatically to the base of the pseudouracil

 

 

See also:

VIROLOGY CHAPTER 8

VACCINES

SARS COV-2 VARIANTS

SARS CoV-2 belongs to the Coronavirus family which have the largest genome of any RNA viruses. Despite having some proof-reading capacities, these viruses, like all RNA viruses, are subject to rapid mutation as they replicate. Most mutations are either deleterious to the virus or are silent (i.e. have no effect). The latter either do not alter the amino acid sequence (they just alter the nucleotide sequence) or are conservative mutations in which the properties of the amino acid side chains are similar (e.g. a change from alanine to leucine or aspartic acid to glutamic acid). However, some mutations give the variant virus a selective advantage; these will proliferate more rapidly and will rapidly become the dominant variant in the population.


D614G mutation

The original Wuhan virus has an aspartic acid at amino acid 614 in the receptor binding region of the S1 subunit of the spike protein (614D). This mutated to a more infectious form with glycine at that position. The mutant virus (known as D614G, or just G614) increased in frequency relative to 614D in a manner consistent with a selective advantage. This amino acid mutation has become increasingly common as SARS-CoV-2 viruses spread around the world. In fact, the original G614 SARS-CoV-2 viruses differed from the original Wuhan form by 4 mutations and almost all of the time G614 is found linked to the other 3 mutations.

The Wuhan D614 form of the virus rapidly spread around the globe in early 2020 but where D614 and G614 co-circulated, the G614 form usually showed a rapid increase in relative frequency and came to dominate the population of viruses. D614G is now clearly the dominant form of the virus globally and the transition took about 4-6 weeks (figure 18 and 19).

The question arose as to why G614 seemed to out-compete D614. This could be due to what is known as a founder effect such as the mutant form arising in a super spreader so that there were more of these viruses available to infect other people. Alternatively, the mutant form might just be more infectious (i.e. transmissible) than the D614 form. The latter appears to be the case since the frequency of G614 increased everywhere throughout March 2020, including in many areas where G614 appeared in well-established local D614 epidemics. An in-depth investigation of transmissibility in the United Kingdom found that G614 increased in frequency relative to D614 in a manner consistent with a selective advantage in the virus.


UK Variant B.1.1.7 - December 2020

Variants arise all the time as the virus mutates but in December 2020 a variant, called B.1.1.7 was identified that appeared more often in samples in the south of England, although this variant had in fact been circulating for some months. When compared to the Wuhan virus, this variant contains 23 mutations. Some are silent but some could affect the interaction of the virus S protein with the cell ACE2 receptor. This variant displaced other variants as it spread across southern England, suggesting that it is more easily transmissible (more than D614G which was itself more transmissible than the original Wuhan virus) although other explanations of the displacement are possible. It is estimated that B.1.1.7 has an increased transmission rate of 50 to 70 percent compared with other variants. Although the variant spreads more rapidly, there is no evidence that it causes more severe disease or that it will not be susceptible to vaccines that originally targeted D617G.

The S protein consists of 1273 amino acids. In the Golgi Body it is cleaved by a protease called Furin into the S1 and S2 subunits. There are two Furin cleavage sites and a small part of the protein is lost. The S1 subunit contains the N-terminal signal peptide and the receptor binding domain. The S2 subunit contains the fusion sequence that allows the viral envelope to fuse with the cell membrane, the transmembrane domain and the cytoplasmic domain (figure 21).

B.1.1.7 has 23 non-synonymous mutations (mutations that cause an amino acid change or stop protein synthesis). Figure 22 shows the mutations of importance in the variant. Eight of these mutations are in the S protein gene including two small deletions. Two are in the receptor binding domain of the S protein; these are N501Y which causes a change from Asn to Tyr (both neutral polar amino acids though the side chain of Tyr is larger) and A570D in which the change is Ala to Asp. This is a non-conservative change in which a neutral polar amino acid is replaced by an acidic polar amino acid. This mutation might be the basis of the greater transmissibility of the variant as it may alter the S protein - receptor interaction. Three mutations are in ORF (open reading frame) 8, one of which is a stop mutation leading to an inactive truncated protein. However, the ORF8 deletion has only a small effect on virus replication compared to viruses without the deletion.

By late December, 2020 this variant was identified in several counties in Europe and in the United States. Neither of the first two variant-positive Americans had traveled internationally in recent weeks.


South African Variant 501Y.V2

At the same time as B.1.1.7 was emerging as the dominant, more transmissible variant in southern England, another more transmissible variant was arising in South Africa. This is known as 501Y.V2 and has eight lineage-defining mutations in the spike protein, including three at important amino acids in the receptor-binding domain that may have functional importance. These mutations are shown is figure 23 where they are compared with those in B.1.1.7. The three important mutations in 501Y.V2 are K417N (Lys to Asn, basic to neutral polar), E484K (Glu to Lys, an acidic to basic change) and N501Y (Asn to Tyr, both neutral polar amino acids). The N501Y mutation is also seen in the S protein gene of B.1.1.7 and is part of the binding loop in the contact region with human ACE2 where it forms a hydrogen bond with ACE2 tyrosine 41. It also interacts with lysine 353 in the virus-binding region of ACE2 and may enhance the binding affinity of SARS-CoV-2 for human ACE2. There is some evidence that the E484K mutation may also modestly increase receptor binding affinity but the K417N mutation has little effect on the binding affinity to ACE2.

The variant, as might be expected, accumulated more mutations over time. On 15 October, the South African variant had, in addition to D614G, five other non-synonymous mutations in the spike protein: D80A, D215G, E484K, N501Y and A701V. Three additional spike mutations emerged by the end of November: L18F, R246I and K417N (figure 23).
 

Alterations in the B.1.1.7 variant - January 2021

The original B.1.1.7 does not contain the escape mutation (E484K) that makes the South African variant more resistant to vaccines. E484K makes it more difficult for antibodies to attach to the virus and prevent it from entering cells. In late January, 2021 some B.1.1.7 variants in Britain seem to have acquired the E484K mutation.


P.1 (B1.1.248) variant

In April 2020, Manaus, a city in the Brazilian Amazon, experienced a severe first wave of SARS-CoV-2 infections but the population resisted lockdowns and social distancing was not enforced. As a result, so many people were infected (76% of the population) that it was thought that the city could have reached herd immunity since they were assumed to have protection against the virus. As a result of herd immunity resulting from three quarters of the population being infected in the initial wave of the virus, it was expected that there would not be a great spread of the virus in a second wave in which the Rt number would be lower than 1. However, in January 2021, Manaus suffered a second wave of COVID-19 infections that overwhelmed its hospitals leaving oxygen supplies exhausted and dozens of people to die in their homes and intensive care hospitals. Sequencing showed that a new variant of SARS-CoV-2, known as P.1, accounted for about half of new infections. P.1 was also found in a few cases in Japan among people who have recently traveled from Manaus. Like some other variants such as those first identified in the UK and South Africa, P.1 appears to be more transmissible that the original D614G virus that spread across the world, raising concerns about a greater risk of spread. The virus has 17 unique amino acid changes, 3 deletions, and 4 synonymous mutations, plus one 4 nucleotide insertion compared to the most closely related viruses. As with other variants, mutations in the S protein receptor binding site are those that give rise to most concern. These are K417N, E484K and N501Y (figure 23a). In the case of the lysine at position 417, there is a change to Asn in the South African variant and to Thr in the P.1 variant. N501Y is found in both the UK and South African variant and changes at 417 and 484 are also in the South African variant. By late January 2021, the P.1 variant had spread as far as Japan, Germany and the United States.

So why the second surge in infections? Could it be that P.1 is not recognized by antibodies in people who were infected during the first wave of infection? It may be that people thought to be immune because of a previous infection had become reinfected suggesting that the immunity they developed during the first wave was not able to suppress the new variant. This is very concerning for vaccine efficacy. Nevertheless, we do not know (January 2021) whether people are being reinfected or whether the more highly transmissible virus is spreading through the remaining quarter of the population since, as of late January, 2021, there had only been one confirmed case of reinfection; it could be that the increased transmissibility raised the Rt and hence the threshold for the onset of herd immunity.

Besides making the virus more transmissible, it does appear that P.1 mutations decrease the immune system’s ability to recognize and neutralize the virus. This seems also to be the case with the South African variant that is so similar to P.1 at three important sites in the S protein. Studies on whether the South African variant could be neutralized by antibodies from patients infected with older versions of SARS-CoV-2 showed that in about half of cases the new variant was resistant to neutralization by the plasma serum; however. it should be noted that while P.1, like the other recent variants, is more highly transmissible, there is no current evidence that it causes more severe disease.

Mutations in P.1 (figure 23a)

N501
The mutation at N501 allows the virus S protein to bind more easily to the ACE2 receptor on the cell surface. This makes the virus more infections (up to 70% more infectious in some studies).

E484
The mutation in P.1 at amino acid 484 (Glu to Lys) is more worrisome. It has been referred to as the escape mutation and is also in the South African but not the UK variant. It seems to allow the virus to escape at least partially the antibodies generated in a previous non-P.1 infection and also possibly the antibodies in the therapeutic monoclonal antibody cocktails made by companies such as Regeneron. Of much more concern is that this mutation may allow the virus to escape antibodies generated by the current vaccines which would require the alteration of the DNA sequences used to generate those vaccines. It is probable that the vaccine will work against the new variants avoiding serious COVID-19 disease which may be replaced by milder symptoms.

K417
Both P.1 and the South African variants have a mutation at amino acid 417, although the altered amino acid differs in the two variants (Thr in P.1 and Asn in the South African variant). That mutations at this point should have risen independently suggest that it confers some advantage on the virus but its significance is unknown.

P.2 variant

On 12 January 2021, researchers in Brazil reported on the detection of a variant of the P.1 lineage that, like the P.1 variant, has the E484K mutation. It probably evolved independently of the P.1 variant.


Are these variants neutralized by the current vaccines?

So far, the answer seems to be yes, although the South African variant may be less susceptible to both the antibodies produced in a natural infection and by the first vaccines but at least they should prevent serious illness.
 

Co-infections

Two COVID-19 cases have been discovered in Brazil in people in their mid-30s who were infected with both the P.2 variant and a different strain circulating in Brazil. It is possible that these co-infections could lead to the creation of additional hybrid variants.

 

COVID-19 VACCINES

Until the Covid pandemic, all successful vaccines had been based on attenuated viruses, killed viral particles or purified proteins (subunit vaccines). These present viral proteins with or without the viral context to the immune system. They require a lot of development, take time to produce in large quantities, require substantial purification and usually do not present the antigen to the immune system in the same way as a natural infection resulting in a virus-infected cell. Until December 2020, no vaccine for human use had been approved that was based on injecting nucleic acids even though these vaccines are easier to produce in large quantities and can be rapidly tailored to changes in the circulating virus using molecular biology techniques.

Nucleic acid-based vaccines can be either DNA or RNA. DNA vaccines consist of the appropriate gene inserted into a viral vector that can be taken up by the cell, transcribed to mRNA and translated into protein. RNA vaccines omit the first stage and directly insert translatable mRNA into the cytoplasm of the cell. Both types of vaccine cause the cell to produce and process viral protein or proteins in the same way as occurs in a natural infection. The surface protein encoded by the nucleic acid passes through the cells’ export pathway acquiring the post-translational modifications that also occur in the natural infection. The antigen may also be passed through the proteosomal or proteolytic endosomal pathways resulting in peptides that can be presented at the cell surface is association with class I and class II histocompatibility antigens and so mediate a strong cell-mediated immune response as well as an antibody-mediated response.


MRNA VACCINES

The first two vaccines approved in late 2020 are based on a protocol in which mRNA coding for the antigen of interest surrounded by a lipid carrier (lipid nanoparticle) is injected into the vacinee. The lipid protects the mRNA from ribonucleases and facilitates its entry into cells. The mRNA is translated to protein, processed and presented to the immune system in the usual way. The protein of interest is usually that which binds to the cell receptor and antibodies to this protein which block virus-cell receptor interaction will prevent infection and are called neutralizing antibodies. In the case of vaccines against SARS-CoV-2, this is the S antigen that binds to the human ACE2 receptor.

A major problem with mRNA vaccines is their stability in transit from the site of manufacture, outside the cell at the site of injection and within the cell. DNA Is inherently stable within the cell since it must pass the genetic code from cell to cell indefinitely. In contrast, mRNAs have a very short life compared to DNA. The amount of a mRNA depends on the balance between the rate of synthesis and the rate of degradation. Many proteins are required only for a very short time, and if their mRNAs were very stable the protein level could not be controlled. Hence, although all mRNAs have short lives, many are degraded very rapidly after translation, facilitating rapid responses to the conditions in the cell. The mRNAs are degraded by ribonucleases (RNAses). Different mRNAs have different degrees of stability resulting from their secondary structure and the nature of the ends of molecule. These are known as cis elements. In addition, their stability is also regulated by RNA-binding factors or trans elements. Cis elements include the 3’ poly A tail and the 5’ methyl guanosine cap. The 3’ poly A tail is bound by poly A-binding proteins that stabilize the RNA. These proteins require a certain length of poly A tail to bind and so the longer the poly A tail, the more of these proteins can bind to the RNA. mRNA is degraded from the 3’ end by 3’-5’ exonucleases and the 5’ end by removal of the 5’ cap and 5’-3’ exonuclease activity. Endonuclease activity also degrades mRNA and this can be regulated by other RNA binding proteins. AU-rich sequences in the 3’ untranslated region (UTR) are also involved in stability.

MRNA may also be stabilized by chemical modification of the bases of the nucleic acid itself. Such modifications include methyl adenosine, N-1-methylpseudouridine and pseudouridine (made from uridine by pseudouridine synthase (figure 25)), a base modification that is common in tRNA and enhances its stability. In mRNA these substituted bases enhance translation. Pseudouridine and N-1-methylpseudouridine repress intracellular signaling triggers for protein kinase R activation which is involved in mRNA stability. Of course, such modifications must not alter the fidelity of the translation of the message.

MRNA vaccines are made by the transcription of a plasmid encoding a protein recognized by a neutralizing antibody, in the case of a Covid-19 vaccine, this is the S protein. The plasmid, which contains the appropriate promoter sequences, is linearized and transcribed in vitro using a T7, T3 or Sp6 phage RNA polymerase. The resulting product contains an open reading frame that encodes the S protein flanked by 5’ and 3’ UTRs, a 5’ methyl guanosine cap and a poly A tail. This is what is used as the vaccine.

Figure 26 shows one way that this might be done in a system from AmpTec. The S protein gene is cloned into an insertion site in an m13 plasmid along with a T7 promotor (A). A forward primer complementary to the end of the M13 sequence (Pri) and a second reverse primer complementary to the end of the S gene are used (B). The latter primer includes a poly T sequence, usually around 120 nucleotides which does not hybridize to any m13 sequence. Using PCR, the DNA structure shown in C is produced. This is then used in in vitro transcription from the T7 promoter to form the polyadenylated mRNA shown in D. In vitro transcription can be carried out in the presence of modified nucleotides such as pseudouracil and/or N6-methyl adenosine, 5-methyl cytidine and others. These modified mRNAs are much more stable than normal mRNAs and are highly translatable giving the vaccine much increased efficacy.

The resultant protein is processed in the normal way through the exocytic pathway with all the usual post-translational modifications including glycosylation and transported to the cell surface. As described above, the protein may also be cleaved by proteases to form small peptides that can be presented at the cell surface to the immune system. The cell has anti-viral mechanisms to detect and degrade foreign RNAs and steps are taken to minimize this.

Even with nucleotide modifications, naked mRNA is likely to be rapidly degraded when injected into the vaccinee. In addition, the mRNA must cross the cell membrane to gain access to the cell protein translation machinery. Both of these problems can be solved by encapsulating the mRNA in a lipid envelope (a lipid nanoparticle or liposome) that helps the mRNA vaccine enter the cytoplasm from the endosome before it is degraded in a lysosome.

The initial Covid mRNA vaccines from BioNtec and Moderna use a technology similar to the above. A modification that may well be used in future mRNA vaccines is to make an mRNA vaccine which contains not only mRNA for the protein of choice (e.g. the SARS-CoV-2 S protein) but also mRNA for a viral RNA-dependent RNA polymerase (replicase). When this type of mRNA vaccine is injected into a vaccinee and enters a cell, it will be translated into S protein and into the replicase (which may be encoded on the same mRNA or a second mRNA). The viral replicase can recognize viral replication signals included in the vaccine mRNA(s) and can then amplify the input vaccine mRNA, making more copies of the mRNA and therefore more of the protein. Since there is now more of the vaccine mRNA in the cell than was originally delivered to the cytoplasm, this is known as the self-amplifying (SA) mRNA approach.
 

Tozinameran (BNT162B2. Brand name: Comirnaty) Pfizer-BioNTech Covid-19 vaccine

Tozinameran was the first mRNA vaccine to be approved. In clinical trials its efficacy is around 95%, 28 days after the first dose and is well tolerated. In one of the initial trials, there were 170 confirmed cases of Covid-19 of which 162 were in the placebo group and only 8 in the vaccine group. It is given in two doses, three weeks apart. It was not evaluated for asymptomatic infection. It appears to be effective against the variants described above. This vaccine must be stored and transported at -70 C.

It contains (WHO Non-proprietary names Program):

A modified 5’-cap1 structure (m7G+m3'-5'-ppp-5'-Am)

5´-untranslated region derived from human alpha-globin RNA with an optimized Kozak sequence. The latter ensures that the protein is correctly translated by the ribosome and functions as the translation initiation site in most eukaryotic mRNAs.

S glycoprotein signal peptide necessary for directing the nascent protein/ribosome complex to the signal receptor on the cytoplasmic surface of the rough endoplasmic reticulum membrane. This guides protein translocation to the correct orientation in the endoplasmic reticulum.

Codon-optimized sequence encoding full-length SARS-CoV2 S protein that contains two mutations: K986P and V987P. These alter the folding of the S protein so that it adopts an antigenically optimal pre-fusion conformation. All of the uridines are replaced by 1- methyl-3’-pseudouridine residues (Ψ) (figure 27) that are nevertheless efficiently translated.

At the end of the coding sequence are two ΨGA stop codons

The 3´ untranslated region comprises two sequence elements that confer RNA stability and high protein expression.

A 110-nucleotide poly A-tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues.

In addition the vaccine contains lipids that make up the solid lipid nanoparticles that encapsulate the mRNA (ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 1,2-Distearoyl-sn-glycero-3-phosphocholine; and cholesterol. In addition, the vaccine contains water, sucrose, dibasic sodium phosphate dehydrate, monobasic potassium phosphate, potassium chloride and sodium chloride.

Moderna Vaccine. mRNA1273

The Moderna vaccine is also an mRNA consisting of a synthetic message encoding the pre-fusion stabilized spike glycoprotein of SARS-CoV-2 virus. Pre-fusion stabilization is achieved by the substitution of two prolines as in the BioNTech vaccine.

Again, the mRNA is made by transcription from a T7 promotor in a reaction in which UTP was substituted with 1-methylpseudoUTP. In addition to the mRNA, the vaccine contains lipids to form a lipid nanoparticle: (SM-102, 1,2-dimyristoyl-rac-glycero3-methoxypolyethylene glycol-2000 [PEG2000-DMG], cholesterol, and 1,2-distearoyl-snglycero-3-phosphocholine) and, tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, sucrose and water.

The efficacy of m1273 is around 94.1%, similar to the BioNTec vaccine. In an initial trial, there were 196 confirmed cases of Covid-19 of which 185 were in the placebo group and 11 in the vaccine group.

It has the advantage over the latter in that the different lipid nanoparticle formulation allows it to be stored and transported at 2-8C, rather than the -70C of the BioNTec vaccine. It is administered in two doses, three weeks apart.

 

ADENOVIRUS-BASED DNA VACCINES

AstraZenica/Oxford University Vaccine: AZD1222, CHADOX1 NCOV-19

The ChAdOx1 nCoV-19 vaccine (AZD1222) consists of the replication-deficient simian adenovirus vector ChAdOx1, containing the full-length S glycoprotein gene of SARS-CoV-2, with a tissue plasminogen activator leader sequence. ChAdOx1 nCoV-19 expresses a codon-optimized coding sequence for the S protein. A simian adenovirus rather than a human one is used because the use of human adenovirus is limited by pre-existing immunity to the virus within the human population that significantly reduces the immunogenicity of vaccines based on the human virus. This is not a problem with the simian virus because, although simian adenoviruses are closely related to human adenoviruses, the hypervariable regions of the main immunogen are significantly different from the human virus thus avoiding preexisting immunity.

The simian adenovirus vectors lack the E1 region encoding viral transactivator proteins which are essential for virus replication and the E3 region encoding immunomodulatory proteins. The latter deletion allows incorporation of larger genetic sequences into the viral vector.

The vaccine adenovirus is taken up by cells and is transcribed in the nucleus to give mRNA which is translated to S protein.

Efficacy is up to 90%, depending on the dosage. Higher efficacy was found in a subgroup in which the first of two doses was halved. The average efficacy was 70.4%.

AD5-NCOV, Convidicea (Cansino Biologics, China)

This is another adenovirus-based vaccine. It is based on recombinant replication-defective human adenovirus type-5 vector to induce an immune response. Again, the virus has been rendered replication-deficient by deletion of the E1 and E3 genes. It encodes an optimized full-length S protein gene based on Wuhan-Hu-1 virus sequence with the tissue plasminogen activator signal peptide gene.

GAM-COVID-VAC, Sputnick V (Gamaleya Research Institute of Epidemiology and Microbiology, Russia)

Gam-COVID-Vac is a two-vector vaccine based on two modified human adenoviruses containing the gene that encodes the S protein of SARS-CoV-2. The first inoculation uses adenovirus 26 (Ad26) as the vector for the S protein gene while the second uses adenovirus 5 (Ad5). This vaccine was shown in January, 2021, to have 91.6% efficacy against symptomatic Covid-19.

AD26.COV2.S, JNJ-78436735 (Janssen/Johnson and Johnson, United States and Belgium)
This vaccine is based again on a recombinant modified adenovirus vector. Like the Sputnick vaccine, it uses human Ad26 expressing the S protein, in this case in a single inoculation. It raises a strong neutralizing antibody and cell-mediated response. It uses AdVac technology which increases stability so that the vaccine may be stored at refrigerator temperatures for at least three months.

 

SUBUNIT VACCINES

NVX-CoV2373, Novavax

The Novavax vaccine (NVX-CoV2373) is based on older technology using purified SARS-CoV-2 S protein with a Matrix M  adjuvant. In clinical trials, it produced high levels of anti-S protein antibodies and has been ordered by several governments as part of their anti-Covid-19 strategy. The gene for the S protein is inserted into a baculovirus. The Baculoviridae are a family of double-stranded circular DNA (80-180 base pairs) viruses that infect insects and arthropods. The modified baculovirus is then use to infect insect cells (usually Sf9 cells, isolated from Spodoptera frugiperda, the fall army worm) which make the S protein. This assembles into native trimers on the surface of the infected cell. These proteins are extracted and associated with lipid nanoparticles so that the S protein is presented to the immune system in a manner similar to that on the surface of an infected cell. Included with the vaccine is an adjuvant extracted from Quillaja saponaria, the soap bark tree (which, as its name implies can be used as a soap). In the case of vaccines, it stimulates the attraction of immune cells to the site of the injection where they respond more effectively. The adjuvant properties come from saponins (triterpene glycosides). The nanoparticles containing the S protein are taken up by antigen-presenting cells, cleaved into peptides and presented on the cell surface in association with MHC antigens to T and B cells.

Phase 3 trials have shown that this vaccine has 89% efficacy against Covid-19 and appears to provide strong immunity against the UK and South African variants.

 

INACTIVATED VIRUS PARTICLES

Valneva vaccine (VLA 2001)
This uses an even more established vaccine technology similar to that used in the Salk polio vaccine, that is the use of inactivated whole virus particles. Virus is grown on African Green Money Kidney (Vero) cells, purified and inactivated with an agent such as formalin. The vaccine also contains alum and CpG 1018 adjuvants. CpG 1018 is a toll-like receptor 9 (TLR9) agonist.

 

OTHER VACCINES

TMV-083, Pasteur Institute
This is an attenuated live virus vaccine using the measles vaccine virus as a vector expressing the S protein antigen of SARS-CoV-2 virus. Because of low efficacy, the development of this vaccine has been abandoned.

There are a number of other SARS-CoV-2 vaccines in phase I and II trials including older technologies such as formalin-inactivated whole virus particles (Sinovac and Sinopharm).

 

WHY DO WE NEED TWO INOCULATIONS?

Most of the vaccines that have been developed against SARS-CoV-2 require two inoculations. This is because of the way that the immune system responds to a foreign pathogen such as an infecting virus.

Initially, it is important to suppress infection by stopping the invading pathogen entering cells and replicating. Infection by a virus binding to its receptor on the cell surface (ACE2 in the case of SARS-CoV-2) triggers an initial response in which plasma B lymphocytes produce neutralizing antibodies that bind to the surface of the invading organism thereby, in the case of SARS-CoV-2, blocking virus S protein binding to ACE2. The initial antibody response, however, quickly declines but some of the B cells differentiate into memory B cells that survive for a long time and relocate to the periphery of the body. Here, they will be more likely to encounter more antigen during a second exposure. When this happens, they proliferate and differentiate into more plasma B cells, which then respond to the antigen by producing more antibodies. Memory B cells can survive for many years so that they are able to respond to multiple exposures to the same antigen.

During the first phase of the immune response, the immune cells also secrete cytokines that recruit other immune cells to the site of infection, among which are CD4-positive helper and cytotoxic T cells (killer T cells) that recognize and kill virus-infected cells. As with B cells, some T cells differentiate into memory cells that can reactivate and proliferate in response to new exposure to the original antigen. These memory T cells can also remain in the body for many years (and perhaps for a lifetime).

Since only a small number of memory T cells are made as a result of the initial exposure, a second exposure to the antigen (infection or inoculation) is required to boost their levels. Thus, with the mRNA SARS-CoV-2 vaccines, protection starts about 12 days after the first inoculation and rises to about 50% effectiveness. After a second injection three to four weeks later, the second phase of the immune response starts, memory B and T cells increase and effectiveness rises to around 95%.

 

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