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10.11: General Categories of Viral Infections - Biology

10.11: General Categories of Viral Infections - Biology


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Learning Objectives

  1. Describe and give an example of an acute viral infection, a late complication following an acute infection, a latent viral infection, a chronic viral infection, and a slow viral infection.

Most viruses that infect humans, such as those that cause routine respiratory infections (e.g., cold viruses, influenza viruses) and gastrointestinal infections (e.g., Rotaviruses, Noroviruses), cause acute infections. Acute infections are of relatively short duration with rapid recovery.

In persistent infections, the viruses are continually present in the body. Some persistent infections are late complications following an acute infection and include subacute sclerosing panencephalitis (SSPE) that can follow an acute measles infection and progressive encephalitis that can follow rubella. Other persistent infections are known as latent viral infection. In a latent viral infection the virus remains in equilibrium with the host for long periods of time before symptoms again appear, but the actual viruses cannot be detected until reactivation of the disease occurs. Examples include infections caused by HSV-1 (fever blisters), HSV-2 (genital herpes), and VZV (chickenpox-shingles). In the case of chronic virus infections, the virus can be demonstrated in the body at all times and the disease may be present or absent for an extended period of time. Examples include hepatitis B (caused by HBV) and hepatitis C (caused by HCV). Slow infections are ones in which the infectious agents gradually increase in number over a very long period of time during which no significant symptoms are seen. Examples include AIDS (caused by HIV-1 and HIV-2) and certain lentiviruses that cause tumors in animals. Although not viruses, prions also cause slow infections.

Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.

  • Adenoviruses
  • Herpes Simplex
  • Varicella-Zoster Virus
  • Cytomegalovirus
  • Hepatitis B
  • Enteroviruses
  • Rhinoviruses
  • Rubella
  • Hepatitis C
  • Measles
  • Influenza
  • HIV Infection and AIDS

Summary

  1. Acute infections are of relatively short duration with rapid recovery.
  2. Persistent infections are where the viruses are continually present in the body.
  3. In a latent viral infection the virus remains in equilibrium with the host for long periods of time before symptoms again appear, but the actual viruses cannot be detected until reactivation of the disease occurs.
  4. In a chronic virus infection, the virus can be demonstrated in the body at all times and the disease may be present or absent for an extended period of time.
  5. Slow infections are ones in which the infectious agents gradually increase in number over a very long period of time during which no significant symptoms are seen.

Understanding the progress of viral infections

Even decades of research have only produced a few standardized vaccines and strategies for treatment to combat just a small number of viruses. Nor has there been much research into viral mechanisms of action &ndash which was a reason for Prof. Guiscard Seebohm and his team at the Institute for the Genetics of Heart Diseases of Münster University to focus their attention on precisely this topic. And the team has now succeeded in making a groundbreaking development: it has created a viral expression model which can be used to simulate and analyse a large number of viral infections &ndash including the one with SARS-CoV-2. The results can be read in the current issue of &ldquoScientific Reports&rdquo published by the &ldquoNature&rdquo journal.

One virus that is much less well-known than SARS-CoV-2, but which can be transmitted in the same way, is the Coxsackie virus B3 (CVB3). &ldquoIts symptoms,&rdquo explains Guiscard Seebohm, &ldquoare mostly similar to those for flu, as is the recovery time: after two or three weeks, any patients with a CVB3 infection are, as a rule, well again, and don&rsquot have any obvious long-term impairment.&rdquo But &ndash not always, says Guiscard Seebohm, who heads the Cellular Electrophysiology and Molecular Biology department. Apart from any acute infections, he explains, a viral infection also contains the risk of a chronic infection, with the consequence of continuous damage to certain internal organs &ndash which can lead to death. This means that months or even years later, an inflammation of the heart muscle, or type 1 diabetes, can occur in some patients who had CVB3 in the past. Histological examinations of patients showed, in some cases, serious damage to the tissue structure. Also: even years after the acute infection occurred, tissue analyses prove the existence of a virus in the genes.

So far there has been an insufficient amount of study devoted to the question of how a CVB3 infection becomes chronic and how exactly an acute infection progresses. In this respect, the Guiscard Seebohm team has succeeded in taking a great step forward. It developed an expression model for CVB3, based on stem cells, in order to get to the bottom of the mechanisms of action in this virus &ndash as a prototype for the effects of viruses in general. In a study, the model was tested for its controllability in heart muscle cells cultured from stem cells. In the process, the team of researchers was able to achieve a stable integration of the genetic information from a non-infectious variant of CVB3 into the genetic material in human stem cells. The latter can be converted into any kind of human tissue in the laboratory, making it possible to precisely investigate viral mechanisms. The CVB3 expression can be specifically activated by means of a chemical signal.

As a result of this unique human viral expression system based on stem cells, it will now be possible to simulate a large number of diseases in their progression and, for the first time, analyse them with the utmost precision

Guiscard Seebohm

Guiscard Seebohm is delighted at this success, because, as he says, &ldquoAs a result of this unique human viral expression system based on stem cells, it will now be possible to simulate a large number of diseases in their progression and, for the first time, analyse them with the utmost precision.&rdquo In Guiscard Seebohm&rsquos view there is something else that is just as important: the system is completely controllable. The team of researchers managed to steer the CVB3 expression in the expression model time-wise, both in stem cells and in differentiated heart muscle cells. At the same time, the researchers were able to vary the quantity of viral proteins produced as well as their localization. In other words, the extent of the viral infection, the infection pattern and the time progression can all be adapted to whatever topics researchers are working on.

The production of the first fully controllable viral expression model in human cells, its proven functionality, and the transferability to patients all open up numerous new approaches for research. It is not only that any infection with CVB3 and other viruses such as Corona and influenza can be examined with a very high resolution this new method also means that the borders of what can be researched can be extended. Follow-up studies on the controlled expression of CVB3 in hiPSC are already underway and showing promising results. Last but not least, Dr. Stefan Peischard, the lead author of the study now published, and his colleagues in the Seebohm team hope that their work will lead to significant benefits for patients.


4 Major Types of Infections

Acne is caused by Propionibacterium acnes, anaerobic diphtherias, coagulase negative staphylococci and micrococci. Toxin of certain organism and free fatty acids may be responsible for acne.

Staphylococcal Infection:

Staph aureus causes boils and carbuncles. These are suppurative lesion, with necrosis at the centre. They discharge a slough with pus. Carbuncle, a large abscess, usually occur in thick collagenous tissue such as back of the neck. Staph, aureus phage group I and II are main causes of boils, carbuncles, styes and sycosis barbae.

Streptococcal Infection:

1. Cellulitis-Superficial infection of subcutaneous tissue.

2. Erysipelas-Spreading infection of dermis.

3. Erythema and Impetigo-Streptococcal impetigo is less common.

4. Scarlet Fever-Usually associated with infection of throat.

Gram-negative infections are usually encountered in moist area of groin and axilla. Coliforms, Bacteroides sp and Proteus sp. May cause abscess between thigh and waist related to a preceding trauma:

Bacteriological Diagnosis:

Specimen: Pus, exudate, swabs from lesions

Culture: Inoculated blood agar is incubated — both aerobically and anaerobically. Identification is done by relevant tests.

Type # 2. Infection of Wounds:

Infection may occur in accidental or post-operative wound due to bacteria:

Pathogenesis, wound infection may be:

(a) Endogenous Infection:

Patient’s own bacterial flora i.e. Staph, aureus, from skin and anterior nares or coliforms.

(b) Exogenous Infection:

Many organisms are exogenous. Staphylococci from skin and anterior nares, organisms from hospital staff and visitors can be spread directly or indirectly by airborne route. Gram-negative enteric bacilli (60%) and Gram-positive cocci (30%) are responsible for hospital acquired infection.

Gram-negative bacilli may be patient’s own flora, or in damp environment. Antibiotic resistant organism (Ps. aeruginosa) from the environment may colonies the patient, traumatized skin (e.g. wound, burn, bedsore).

Laboratory Diagnosis:

Specimen: Pus, exudate and tissue from wound.

Microscopy: Direct Gram-stained smear will reveal the organism.

2. MacConkey agar for aerobic culture

3. Aminoglycoside agar for anaerobic culture.

4. Robertson s cooked meat medium

Isolates can be identified by biochemical and serological tests.

Type # 3. Infections of Burns:

Within 24 hours the large moist exposed surface of burns is colonised by bacteria (e.g. bacterial flora of skin, respiratory tract and intestine, streptococci and/ anaerobic spore bearing bacilli). Staph, aureus is the most common isolate from burn, followed by Ps. aeruginosa and various enterobacteria (e.g., Acinetobacter sp.) and Str. pyogenes group A, B, C and D.

The air-borne organisms reach the burnt area and invade directly the burnt tissue. Laboratory diagnosis is similar to that of wound infection.

Prophylaxis-Local application of antimicrobial agents (silver sulphadiazine cream, silver nitrate solution and chlorhexidine cream) reduce the colonisation of bacteria.

Type # 4. Infection of the Eyes:

Eyelid Infection:

Staph. aureus causes a small boil or abscess in one of the glands of the lash follicle. They would have originated from anterior nares or implanted from a septic lesion on the body.

2. Infection of lacrimal apparatus:

Infection of glands (dacryoadenitis) occurs from endogenous sources. Dacryoadenitis may be associated with mumps and gonorrhoea.

Because of flushing action of tears and presence of lysozyme that degrades peptidoglycan of the cell wall of Gram-negative bacilli the conjunctival sac is free from bacteria however, C. xerosis and Staph epidermidis colonies the conjunctival sac.

Conjunctivitis may occur due to conjunctival sac infection by pathogens.

Clinically, it may be acute type, mucopurulent type, non-purulent type and neonatal.

1. Staph, aureus causes acute conjunctivitis

In newborns, conjunctivitis caused by Staph, aureus is commonly observed in “Sticky eyes“. In older infants and children, infection due to H. influenza and Str. pneumonia is more common. C. Trachomatis (Serovars D-K) causes inclusion conjunctivitis and trachoma (A, B, C). Endemic area, trachoma is very common amongst children and is a very common cause of blindness in the world.

1. Adeno viruses (types 3, 7, 8, and 14)

Keratoconjunctivitis is caused by Adeno viruses type 7, 8 and Herpes virus (HSV1).

Conjunctivitis in neonates may be acquired either during birth from the mother’s genital tract or from some other external sources within 3 weeks of birth.

(a) From the genital tract-Infection of eye acquired by the newborn during birth is called “Ophthalmia neonatorum“:

2. C. trachomatis (D-K serovars)

3. Group B haemolytic streptococci.

(b) From the external source-Staph. aureus causes sticky eyes in the newborn.

It is inflammation of the cornea. The infective organisms of conjunctivitis can spread deeper into cornea leading to ulcers or a spreading keratitis.

2. Foreign body with secondary infection due to bacteria or fungi.

Corneal infection by fungus occur secondarily following injury. Saprophytic fungi, Aspergillus and Fusarium species are the common causes of infection by opportunistic fungi.

A. Bacterial infection except Chlamydial:

Exudate collected by a sterile platinum loop directly from the patient’s eye. Preparation of smear and culture should be done at the bedside. In case of delay in transport, swab of exudate should be placed in Stuart’s transport medium and despatched to the laboratory.

Stained smear reveal the presence of causative organisms.

Material is cultured on blood agar, and chocolate agar, incubated at 37°C in an atmosphere of 5-10% CO2.

Organism are identified by biochemical test and serotyping.

B. Chlamydial infection:

Scrapings from conjunctiva. Microscopy-Film is examined by immunofluorescence.

Tissue culture is performed in cycloheximide treated or irradiated McCoy cells for 2-3 days.

The organism produce intracytoplasmic inclusion which are identified by Giemsa stain or immunofluorescence.

Scrapings from the base or edge of corneal ulcer.

1. In 10% KOH mount, the fungi appear as branched, septate hyphae

2. Methylamine silver or PAS staining shows filamentous fungi with hyphae.

Scraping material is inoculated on Sabouraud’s dextrose agar.

It is serious Infection of the cellular tissue of the orbit. It may usually result from spread of infection from adjacent tissue (teeth and sinuses). Complications are panophthalmitis, brain abscess, meningitis. Painful swelling and protrusion of the eyeball may occur.

Staph, aureus and other pyogenic organisms can cause orbital cellulitis.

Specimen of pus collected by surgical incision can be subjected to the laboratory examination which is similar to that of conjunctivitis.

Panophthalmitis:

It is an inflammation of whole substance of the eye derived from infections of the outer eyes, foreign bodies and penetrating wounds (traumatic or surgical).

Gram negative enteric bacteria (Ps. aeruginosa, Proteus sp., Klebsiella sp., E. coli, Enterobacter sp.)

Diagnosis-Pus aspirated from the anterior chamber of the eye should be cultured.

Choroiditis and Chorioretinitis:

Aetiology: Bacterial, viral, fungal, protozoal infections may cause granulomatous lesion of choroid and retina.


Cellular Pathogenesis

Direct cell damage and death may result from disruption of cellular macromolecular synthesis by the infecting virus. Also, viruses cannot synthesize their genetic and structural components, and so they rely almost exclusively on the host cell for these functions. Their parasitic replication therefore robs the host cell of energy and macromolecular components, severely impairing the host's ability to function and often resulting in cell death and disease.

Pathogenesis at the cellular level can be viewed as a process that occurs in progressive stages leading to cellular disease. As noted above, an essential aspect of viral pathogenesis at the cellular level is the competition between the synthetic needs of the virus and those of the host cell. Since viruses must use the cell's machinery to synthesize their own nucleic acids and proteins, they have evolved various mechanisms to subvert the cell's normal functions to those required for production of viral macromolecules and eventually viral progeny. The function of some of the viral genetic elements associated with virulence may be related to providing conditions in which the synthetic needs of the virus compete effectively for a limited supply of cellular macromolecule components and synthetic machinery, such as ribosomes.

Damage of cells by replicating virus and damage by the immune response are considered further in Chapters 44 and 50, respectively.


Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.

Keywords: (ADP-ribosyl)hydrolase X domain alphavirus antiviral poly(ADP-ribosyl)polymerases coronavirus non-structural protein 3.

Conflict of interest statement

We declare we have no competing interests.

Figures

Model for the role of ADP-ribosylation following coronavirus infection. ( a ) Infection…

S2-MacroD reverses PARP14-derived ADP-ribosylation. (…

S2-MacroD reverses PARP14-derived ADP-ribosylation. ( a ) PARP14 can efficiently modify itself in…

Identification of key residues for…

Identification of key residues for ligand binding, catalysis and drug design. ( a…

Phylogenetic analysis of MacroD-like domains.…

Phylogenetic analysis of MacroD-like domains. ( a ) Evolutionary phylogenetic tree analysis of…

Evolutionary differences in the proximal…

Evolutionary differences in the proximal ribose and catalytic pocket. ( a ) Surface…

Loss of catalytic residues in…

Loss of catalytic residues in the macrodomain of insect-restricted alphaviruses. Non-structural polyprotein (precursors…

Assessment of similarities between viral,…

Assessment of similarities between viral, human and human-associated microbiome macrodomains. ( a )…

Two ADP-HPD ligands bound to…

Two ADP-HPD ligands bound to S2-MacroD mimic PAR dimer binding. ( a )…


Laboratory Diagnosis of Viral Infection

The choice of materials and methods for laboratory confirmation of viral infection depends on the stage of illness (Table 13.1).

The following methods are commonly employed:

(a) Microscopy: Cytopathic effect, inclusion bodies.

(b) Culture and isolation: Laboratory animals, chick embryo, tissue culture, cell culture.

(c) Serology: HI, CFT, EIA, Western blot test.

(d) Detection of viral proteins and genetic material: DNA probes, PCR.

Specimens to be collected early in the acute phase of the disease before the virus ceases to shed (Table 13.2).

Collection and transport of specimens:

Ideally all specimens for detection of virus should be processed by the laboratory immediately because many viruses are labile and the samples are also susceptible to bacterial and fungal overgrowth. Specimen should be placed in ice and transported in special media (Stuart’s viral transport media) containing proteins.

Blood for serological test is transported to laboratory in sterile test tube. Serum is separated as soon as possible. Blood for viral culture is to be transported in a sterile vial containing anticoa­gulant. Blood should be refrigerated at 4°C until processing, and can be stored for months at -20°C or below. Virus specific IgM should be tested before freezing since IgM may form insoluble aggregates upon thawing.

A. Microscopy (cytology):

Viruses produce cytopathic effect (CPE) which include: change in cell morphology, cell lysis, vacuolation, syncytia formation and inclusion bodies (Table 13.3).

1. Cytopathic effect (CPE):

Viruses cause cell degeneration or cell death which can be seen by microscopical examination of cultures. Each infectious virus particle gives rise to a localised focus of infected cells (a focus of cytopathology) that can be seen with the naked eye (Fig. 13.5). Such foci are called plaques and each plaque represents an infectious virus. Cell degeneration is manifested by certain changes.

(a) Syncytium formation (Fig. 13.1):

Syncytium or multinucleated giant cells result from fusion of con­tiguous cells in the monolayer as seen in measles virus.

(b) Cell necrosis and lysis:

Infected cells become pyknotic and granular, e.g. entero-viruses.

The cells do not fuse but produce large clumps resembling clusters of grapes, e.g. adenovirus.

Inclusion bodies are virus- specific intracellular globular masses produced during replication of virus in host cells and visible under the light microscope. They are far larger than a single mature virus particle (elementary body), size ranges from 20-25 μm and can be seen by light microscope.

Inclusion bodies formed by different viruses show distinct shape, size, location and staining properties and their presence in an infected cell is a presumptive histological evidence of viral infection.

These are generally acidophilic (eosino­philic) structures, appear pink when stained by Giemsa’s or eosin-methylene blue stain and exa­mined under light microscope. Some viruses produce basophilic inclusions (adenovirus). They may appear round, globular, oval or irregular.

(i) Intracytoplasmic Inclusion bodies may be formed in the nucleus, cytoplasm or both (Figs. 13.2 & 13.3):

(i) Intracytoplasmic bodies:

Small pox (Guarnieri bodies), rabies (Negri bodies), yellow fever (Councilman bodies). Inclusion bodies produced in fowl-pox virus infections are called Bollinger bodies which are larger than that formed in vaccinia virus infections. Intracytoplasmic molluscum bodies (20-30 μm) are large.

(ii) Intra-nuclear bodies:

These were classified by Cowdry (1934) into two types: Cowdry A in­cludes inclusion bodies of variable size and of granular appearance (herpes virus, yellow fe­ver virus) and type B of more circumscribed inclusion (adenovirus, poliovirus). Large intra­-nuclear inclusion is also seen in CMV infection of cell.

(iii) Inclusions formed in both nucleus and cytoplasm:

(e) Discrete focal degeneration:

It is found in her­pes virus infections.

Oncogenic virus transforms cells and causes loss of contact inhibition and the cell growth appears in a piled-up fashion producing “micro-tumours” (Fig. 13.1).

2. Immuno-electron microscopy:

When virus- specific antibody is added to a sample, the virus particles clump and thereby facilitate the detection and simultaneous identification of virus. This technique is useful in diagnosis of enteric and rota viruses which are found in abundance and have a characteristic morphology.

B. Culture and isolation:

Culture and isolation is the gold standard for prov­ing a viral aetiology of a syndrome (Table 13.4):

In the past, white mice and chimpanzees were inoculated with specimen for viral cultivation, but inoculation of laboratory animals has been largely replaced by the use of cell cultures.

Suckling mice (less than 48 hours old) are very susceptible to toga and coxsackie viruses, which are inoculated by intra-cerebral or intranasal routes.

2. Chick embryo (embryonated egg):

Eggs are kept in the incubator and embryos 7 to 12 days old are used. The embryonated egg is in­oculated by one of the several routes (Fig. 13.4).

After inoculation of the chick embryo, it is incubated and examined daily for virus growth:

(a) Chorioallantoic membrane (CAM):

CAM is inoculated mainly for growing poxvirus. Viral replication produces visible lesions (pocks), gray-white area in the transparent CAM. Each pock is derived from a single virion. Pocks produced by different viruses have different morphology.

Allantoic inoculation is done mainly for production of vaccine of influenza virus. Other allantoic vaccines include yellow fever (17D strain) and rabies (flury strain) vaccines. Duck’s egg, being larger, provide better yield of rabies virus than hen’s egg.

It is mainly inoculated for pri­mary isolation of influenza virus.

(iv) Yolk sac inoculation:

It is inoculated for cul­tivation of some viruses as well for some bac­teria (chlamydiae and rickettsiae).

3. Tissue culture:

There are three types of tissue culture — organ cul­ture, explant culture and cell culture. Small bits of organs (organ culture) from man and animal are maintained in tissue culture growth medium. Organ cultures are done mainly for highly specialised parasites of certain organ, e.g. tracheal ring culture for isolation of corona virus. Explant culture is rarely done nowadays.

The cell culture method is routinely employed nowadays for identification and cultivation of viruses. The growth medium for tissue culture is basically a balanced salt solution and contains 13 essential amino acids, glucose, salts, buffering system, protein supplement (lactalbumin hydro- lysate), calf serum (5%), antibiotics (penicillin, streptomycin) and phenol red (indicator).

Tissue fragments are trypsinised and the dissoci­ated cells are washed, counted and suspended in a growth medium. Most of the cell types undergo a very slow cell division once in 24 to 48 hours. Cell suspension is distributed in tubes, bottles or Petri dishes. Cells of fibroblastic or epithelial nature adhere and grow in the glass surface.

On incubation, the cells divide and spread out on the glass surface (wall of test tube) to form a confluent monolayer sheet of cells within a period of one week:

(a) Types of cell cultures:

Cell culture are classified into three types (primary cultures, diploid cultures, heteroploid or continuous cell line cultures) on the basis of their origin, chromo­somal characters and the number of genera­tions through which they can be maintained (Table 13.5).

Since all viruses do not grow in all cell lines, as a virus does not grow in a cell unless it carries receptor for the virus. Hence, specimen is to be inoculated in 3 to 4 cell lines with the hope that at least one of them will be permissive for the unknown virus.

(i) Primary cell culture:

These are normal cells obtained from fresh organs of animal or human being and cultured. Once the cells get attached to the vessel surface, they undergo mitosis until a confluent monolayer of cells covers the surface.

These cells are capable of only limited growth in culture and cannot be maintained in serial culture. They are commonly employed for primary isolation of viruses and in pre­paration of vaccine. The examples of primary culture include monkey kidney cell (Fig. 13.5), and human amnion cell culture.

(ii) Diploid cell cultures (Semi-continuous cell lines):

These cells of a single type, usually fibroblasts, contain the same number of chro­mosomes as the parent cells and are diploid. The diploid cell strains can be sub-cultured for lim­ited number of times. There is a rapid growth rate and after about 50 serial subcultures they undergo “senescence” and the cell strain is lost.

The diploid cell strains are susceptible to a wide range of human viruses. They are also used for isolation of some fastidious viruses and production of viral vaccines. The fibro­blasts are usually derived from embryo tissues (human embryo lung strains).

(iii) Heteroploid cultures (Continuous tumour cell lines) (Fig. 13.6):

These are cells of a single type capable of infinite growth in vitro. They are derived from immortalized cell lines (cancer cells), often of epithelial origin. These cells grow faster and their chromosomes are haploid. They are termed continuous cell lines as they can be serially cultivated indefinitely.

The standard continuous cell lines have been derived from human cancers, such as HeLa (derived from cervical cancer of a lady, Hela by name), HEp2 and KB cells.

Continuous cell lines are maintained either by serial subculture or by storing in deep freeze at -70°C so that these can be used when necessary. These are not used for preparation of viral vaccines, as vaccines prepared in cancer cells is considered unsafe for human use.

Primary cell cultures are generally best for virus isolation and rhesus monkey kidney cell cultures are widely used, which are sensitive to a wide range of viruses.

For precise identification of a vi­rus grown in cell cultures, several additional tests have to be performed:

1. Morphologic examination:

Morphological study of a virus can only be made by electron microscopy which is technically difficult and expensive. Moreover, this may not lead to success­ful identification of the virus.

2. Serological test:

Serologic tests help in most cases for precise identification of the virus:

It can be performed with any virus producing CPE. Standard known antiviral serum is mixed with the unknown virus recovered from the cell culture medium.

After incubating the antibody-virus mixture, it is inoculated into a fresh cell line. Specific antiserum neutralizes virus activity during incubation, and, consequently, the mixture fails to produce specific CPE. The haemadsorption viruses are typed by neutralisation test.

Cells infected with influenza virus, para-influenza virus, mumps virus, and toga virus express a viral glycoprotein (haemagglutinin) in the cell membrane that binds or adsorbs red blood cells of defined animal species to the infected cell surface (called haemadsorption) (Fig. 13.7).

(iii) Haemagglutination:

When the above-mentioned viruses-orthomyxo, paramyxo and toga viruses), carrying haemagglutinin in their envelope are shed into the culture medium, they can be detected by the agglutination of erythrocytes, a process called haemagglutination.

(iv) Haemadsorption inhibition test:

This is observed with haem agglutinating viruses (orthomyxo paramyxo and toga viruses). Medium from the cell culture is mixed with a specific antiserum, and the mixture is added to a fresh culture. After incubation, the second culture is tested for haemadsorption. A negative result indicates that the antiserum is specific and has reacted with the virus and, thereby, blocking haemad­sorption.

(v) Cytopathogenic effects interference inhibition test:

Specific antibody to the unknown interfering virus (e.g. rubella virus, when it is the suspe­cted identity of the unknown virus) is added to medium obtained from the cell culture. The mixture is then inoculated into a second culture.

After incubation for 2-3 days, the cytopathic virus (e.g. echovirus) is added to the second cell culture and incubated for 1-2 days. If cytopathogenic effects have occurred, it is concluded that the first culture was infected with the interfering virus against which the antiserum was employed.

(vi) Direct immunofluorescence:

A cell culture is inoculated with a specimen and incubated. Af­ter 24—48 hours the virus can be identified by adding a fluorescent monoclonal antibody to the suspected virus to a scraping obtained from the infected culture.

The scraping is then incubated for a short time, followed by washing to eliminate unreacted antiserum, the scraping is examined — under the fluorescent microscope. When the cells fluoresce, the vi­rus in question is responsible for the infection of the monolayer.

(vii) Immuno electron microscopy (IEM):

Although a virus can be visualised by electron microscope, but precise identification of a virus (e.g. herpes simplex, VZ, CMV and Epstein—Barr) is not possible on a mere morphological basis. IEM uses a specific antibody labelled with electron- dense tag to specifically identify the virus. The technique is very expensive and not useful for routine purpose.

Serology is useful and simpler in diagnosis in many cases. The course of the infection (acute or chronic) can also be determined by serology.

1. General considerations:

Total antibody assays are done, usually to ob­serve a significant rise of specific antibodies to a suspected virus on two paired samples, first when the patient is ill and second during convalescence (2 to 3 weeks later). There is usually a four-fold rise in antibody titre. The diagnosis is usually retrospec­tive and mainly useful in epidemiological study.

(a) Haemagglutination inhibition (HI) is used for the measurement of antibodies directed against haem agglutinating viruses, e.g. influenza viruses. Antibodies to the viral haemagglutinins in serum prevents a standardised amount of virus binding to and agglutinating erythrocytes.

(b)Complement fixation test (CFT) is the main­stay for some viral diseases. However, it can only detect CF-antibodies (i.e. IgG and IgM) and cannot differentiate between the two.

(c) Direct immunofluorescence test:

A fluorescein —Conjugated antiserum of the suspected virus is mixed with the infected tissue sample and examined under a fluorescent microscope for pale green fluorescent areas on the slide.

(d) Enzyme immunoassay (ELA):

(i) Measurement of antibody by EIA:

A known antigen is adsorbed to a solid phase-like test tube, micro-titre plate well to which proper dilution of patient’s serum is added and then incubated. If there is antibody in patient’s serum, it will attach to the antigen. The solid phase is washed off the un-reacted immunoglobulin’s.

After washing enzyme- labelled anti-human immunoglobulin (AHIG) is added to the solid phase. The solid phase is again washed off to remove unreacted antibody (AHIG). Then a subst­rate is added to the solid phase. The enzyme-labelled antibody (AHIG) binds to the patient’s antibody that has reacted with the immobilised antigen.

The enzyme splits the substrate to a colour com­pound. The intensity of the colour is proportional to the quantity of antibody present in the patient’s serum. The colour is measured by ELISA reader. These tests are rapid (takes 10-15 minutes) and easy.

(ii) Measurement of antigen:

A known specific antibody is adsorbed to the solid phase (e.g. well in micro-litre plate). Patient’s serum is added, and if viral particles or viral antigens are present in the circulation, they will be bound by the immobilised antibody. The solid phase is washed.

Then enzyme-la- belled antibody (e.g. AHG) of the same specificity to the solid phase is added. Af­ter incubating and washing off unbound reactants, a chromogenic substrate is added. A positive reaction is indicated by the de­velopment of colour.

(e) Western blot (immunoblot) test:

It is a most widely used confirmatory test for detection of positive HIV antibody immunoassay.

The test is done as follows:

(i) HIV antigens are separated by electro­phoresis on polyacrylamide gel which is blotted onto nitrocellulose paper strips.

(ii) The strip is incubated with patient’s anti­body.

(iii) After washing to remove the unbound antibody, an enzyme labelled anti-human globulin (AHG) antiserum is placed on the strip. The labelled antibody binds to any antibody captured by the viral antigens. When a chromogenic substrate is added subsequently, the antigen-antibody com­plexes are revealed as stained bands which represent the major antigenic proteins of HIV.

D. Detection of viral genetic material:

(1) Nucleic acid probes:

Nucleic acid probes are short segments of DNA complementary to specific regions of a viral genome. DNA probe analysis is especially useful to detect slowly replicating or non-productive viruses, e.g. CMV, human papilloma viruses and HIV (during window period).

(2) Polymerase chain reaction (PCR) and reverse transcriptase PCR:

PCR is a method that duplicates short DNA segments thousand to a million fold. When the viruses are present in low concentrations, they can be duplicated (amplified) using PCR.

1. Total particle count:

Total count of viruses irrespective of whether they are viable or nonviable is estimated by electron microscopy and haemagglutination:

(a) Electron microscopy:

The virus particles in a negatively stained suspension can be counted directly. The commonest method is to mix virus suspension with known concentration of latex particles and studied under the electron microscope. The number of virus particles in the suspension are calculated from the ratio of latex spheres to virus particles.

(b) Haemagglutination (HA):

Although it is not a very sensitive method, yet HA titre gives an approximate count. About 107 influenza viruses are necessary to bind erythrocytes (0.5 ml of 0.5% suspension) into a lattice-like aggregates of macroscopic size.

2. Infectious virions assay (assay of infectivity):

Quantitative infectivity assay:

The quantitative infectivity assay measures the number of viable infectious particles in a suspen­sion.

This can be measured by two methods:

Serial dilutions of a virus sus­pension are inoculated into confluent mono­layers of culture cells in a Petri dish or bottle. After allowing time for adsorption (1 to 4 hours), the monolayer is covered with agar gel which prevents diffuse spread of the viruses readily through the fluid medium.

Moreover, the agar gel layer restricts spread of the released progeny particles in such a way that only neighbouring cells are infected. Each infectious particle gives rise to a localised focus of infected cells, called a plaque, which can be seen with the naked eye (Fig. 13.5).

The titre of viruses is expressed in plaque forming units (PFU) per volume. Since each plaque represents an infectious virus, the plaque titre is the infectivity titre.

Since each pock in CAM arises from infection by a single virus particle, the number of pocks formed corresponds with the number of viruses present in the inoculum.

Antiviral Susceptibility Testing (AST):

It is done for defining mechanisms of antiviral resistance and to assess the frequency with which drug-resistant viral mutants emerge in clinical practice.

There are two types of AST:

These are in vitro suscepti­bility tests. Cell culture is incubated with several dilutions of the drug to which a constant amount of virus has been added. The test measures the reduction in the number of plaques, inhibition of viral DNA synthesis, and reduction in the yield of viral structural proteins (e.g. gp 24 of HIV).

These analyse viral nucleic acids to detect specific mutations respon­sible for drug resistance. CMV shows UL 97 (phosphotransferase) and UL 54 (polymerase) mutants with ganciclovir resistance. These are detected by PCR amplification and sequencing of entire UL 54 gene and fragment of UL 97 gene.


Bat tolerance to viral infections

Inflammatory molecules evolved partly to protect hosts from viruses, but increasing evidence suggests that they cause disease pathology and chronic conditions, and play a role in aging. By mitigating these effects, bats are able to both tolerate viral infections and live well beyond expectations.

What is interesting about bats? Bats effectively live everywhere that humans do, from the Alaskan Arctic to remote Hawaiian Islands. They are the only mammals that can truly fly and are the most abundant nocturnal insect predators. For epidemiologists and virologists, bats are important because they host a range of viruses, a handful of which cause severe disease in humans and other mammals. Questions remain regarding whether bats directly transmit these viruses to humans or whether they are disproportionate hosts for such viruses 1,2 . However, viruses causing rabies, Ebola virus disease, severe acute respiratory syndrome and Middle East respiratory syndrome (MERS) all have viral ancestors in bats. For mammalogists and other biologists, bats are of interest because they age remarkably well senescence, or ‘biological aging’, typically scales with body mass so that larger animals live longer. However, some bats with half the body mass of domestic mice have lifespans ten times that of the mice. Metabolic rates and other pace-of-life traits (Fig. 1a–f) are thought to drive this body mass–longevity relationship for most species however, during flight, normal bat metabolism can reach twice that of exhaustion compared to mice 3 . Now, growing numbers of studies are linking bats’ abilities to age with their role as viral hosts through genetic adaptations to their immune responses 4 .

af, Expected general relationships between mammals (diagonal lines). g, Bat viruses evolve in high IFN environments (green), so viral replication (yellow) continues when humans exhibit increased IFN expression following infection with bat viruses. Reduced inflammation (red) moderates disease pathology in bats and reduced B cell affinity maturation lowers antibody responses (blue).

In this issue of Nature Microbiology, Ahn et al. provide evidence that one mechanism bats have evolved to limit excessive inflammation during viral infection is through dampened transcriptional priming and lower functional capacity of the bat inflammasome sensor, NLR family pyrin domain containing 3 (NLRP3) (ref. 5 ). NLRP3 functions as a pathogen recognition receptor to activate inflammatory mediators. NLRP3-mediated inflammation has also been linked to aging and age-related chronic diseases. This study provides a mechanism for, and a provisional answer to, whether bats have evolved to tolerate or resist viral infections. This work suggests that bats have adapted to tolerate viral infection without development of pathology through inflammation (Fig. 1g). This finding helps explain another apparent feature of bats, which is why they seemingly don’t develop overt disease when infected, despite hosting a suite of viruses.

Previous studies have shown that parts of the innate immune system are ‘switched on’, even in unstimulated bat tissues, due to constitutive expression of three interferon (IFN)-α genes 6 . This constitutive IFN activation is hypothesized to have reduced the need for affinity maturation, a process during which B cells produce antibodies with increased antigen affinity during infection. These observations are relevant for epidemiologists because bats may limit viral replication without developing high antibody titres, thereby affecting serological assay interpretation through reduced antibody production (Fig. 1g). Further, the conditions bats provide for viruses are themselves hypothesized to facilitate high pathogenicity of bat viruses in other mammalian hosts, such as humans. Because of the constant high IFN activity, bat-borne viruses may be shed at low levels from bat cells without eliciting strong antibody responses 7 . Viral adaptation to these conditions through selection means that when other hosts (including humans) are infected by bat-derived viruses and produce similar immune responses, viral replication is not suppressed and pathology develops 7 .

Contrastingly, bats have been shown to have dampened IFN responses through substitutions in genes within the stimulator of IFN genes (STING) pathway 8 . In addition to reducing pathology during viral infection, this inhibitory IFN state, as well as the dampened NLRP3 inflammation 5 , may have evolved to reduce damage from the high metabolic demands of flight that cause DNA damage (Fig. 1e). These reductions in inflammation and adaptations for flight may have inadvertently increased bat lifespan 4 . This idea is supported by a recent field study which shows that telomeres, the protective caps of chromosomes, shorten more slowly within bat genera with the oldest living bat species 9 .

All these fascinating insights have been inspired through field observations but have relied on genomic, transcriptomic and in vitro studies. Despite these advances, there are numerous open questions in bat research. Ahn et al. showed that the bat-derived virus Pteropine orthoreovirus 3 and MERS coronavirus (MERS-CoV) cause NLRP3-mediated inflammation in mouse and human cell lines. Interestingly, the pathogenesis of these viruses (all RNA viruses) in humans can mainly be ascribed to aberrant innate immune responses. In vivo virus challenge studies with high doses of Ebola virus and MERS-CoV caused limited pathology in bats, despite high viral tissue titres 10,11 . However, questions remain as to whether bats are adapted to tolerate all viral infections or simply those they have co-evolved with. Rabies and related viruses persist in bat populations, but can kill bats. Are these viruses really the only exceptions? The study of MERS-CoV is valuable because, though sometimes thought of as a bat-derived virus, only MERS-CoV ancestors have been detected in bats, and MERS-CoV appears to be well established in camel populations with limited camel disease 12 . Therefore, bat NLRP3 responses to MERS-CoV hint that these responses may be general among bats. However, more cross-species studies using bat and non-bat viruses (including viruses distantly related to bat-derived viruses) in bat and non-bat cell lines are required 5,10,11 . Furthermore, while bats challenged with viruses may rarely develop disease, even in the presence of high viral loads, understanding disease in wild animals (in particular, small nocturnal flying bats) is difficult.

The dampened NLRP3-mediated inflammatory responses in bat tissues to three different types of RNA viruses 5 support the hypothesis that innate immune tolerance, rather than resistance, is a mechanism bats have evolved to host a diverse suite of viral infections with limited disease. Bat longevity and high viral diversity suggest that there are general patterns, but bats are diverse, and whether bats are overrepresented reservoir hosts of zoonotic viral infections is still arguable 1,2 . The fact that bats are extremely long-lived compared to other mammalian species given their size is not debatable, but how linked this is to their ability to host viruses remains to be seen. Recent work demonstrating temperature-independent filovirus (for example, Ebola virus) replication in bat cells 10 , in contrast to that predicted by the ‘flight as fever’ hypothesis 3 and the study by Ahn et al., are excellent examples of in vitro hypothesis testing. However, bats also use torpor, sometimes so deep that hibernating temperate bats may lower their body temperature to just above freezing for weeks, and even tropical bats have been shown to reduce their heart rates from over 1,000 beats per minute to less than 200 (ref. 13 ). What roles these physiological conditions have on bat longevity and viral dynamics within bats, and their consequences at the population level, have yet to be fully explored. Increasingly, however, hypotheses are being tested and each reveals fascinating insights into bats as animals and their role as viral reservoirs.


Discussion

Our quantitative analysis establishes estimates for the absolute number of virions present in an infected individual, as well as the number of virions produced during the infection and the total number of infected cells in the body. There are various ways in which one can leverage such quantitative estimates to produce insights regarding COVID-19. First, having absolute estimates allows us to compare them to other quantities in the human body and thus put the number of virions in context and even arrive at new insights. For example, a human body comprises 𢒃휐 13 cells ( 3 ). This means that even for our highest estimate, i.e. 10 11 virions per host, human cells outnumber the virions by more than 100 fold. We can also compare our estimate for the total number of infected cells with the total pool of cells expressing ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane protease, serine 2), the receptor and main protease SARS-CoV-2 relies on for infecting cells. Single-cell RNA-sequencing studies ( 35 – 37 ) indicate that a few percent of the cells in the lungs and airways express ACE2 and TMPRSS2. Most of the cells that have been found to express both are type 2 pneumocytes. While these results might be biased due to drop-out effects in measurements of only a few molecules ( 37 , 38 ), it is still reasonable that 1%�% of the lung and airway cells contain the necessary receptor to be infected by SARS-CoV-2, totaling

10 9 cells. This number is several orders of magnitude higher than our estimate for the total number of infected cells during peak infection (10 4 � 6 ). This suggests that out of the cells expressing both ACE2 and TMPRSS2, only a small fraction, e.g. 10 𢄥 � 𢄣 , are infected by the virus.

Because the immune system is the main line of defense against SARS-CoV-2, it is interesting to quantitatively examine the known immune response in comparison with the viral loads we estimated here. For example, we can compare the peak number of viral particles (10 9 � 11 ) to the number of antibodies the body produces to combat SARS-CoV-2 infection. Levels of SARS-CoV-2 specific IgG antibodies (CIgG) were measured 3 weeks after the onset of symptoms, showing a serum concentration of

10 μg/mL ( 39 ). Only 𢒅% of the total anti-spike (the viral protein responsible for allowing the attachment and fusion with the host cell) IgG antibodies has the capacity to neutralize the virus (fneutralizing) ( 40 ). Combining the concentration of neutralizing IgG antibodies with a mean IgG molecular weight (MWIgG) of 150 kDa ( 41 ) we estimate the number of neutralizing antibodies per mL of serum (Cneutralizing):

Combining this estimate with the measurement of viral concentration within the lung tissue and accounting for 30� spike trimers on each SARS-CoV-2 virion ( 42 , 43 ) we can estimate the ratio of neutralizing antibodies to viral spike proteins as

Previous work on other morphologically similar RNA viruses like influenza and flavivirus found that a ratio of 1 bound neutralizing antibody per 2𠄴 receptor-binding proteins was sufficient to neutralize binding of a virion to its cellular receptor in vitro ( 44 , 45 ). Taken at face value, our estimate seems to suggest an excess of neutralizing antibodies. There are several factors that will cause the effective concentration of antibodies the virus experiences to be lower. First, the antibody concentrations in the lung tissue tend to be lower than that of the blood. Second, many of the spike proteins are extensively glycosylated. These glycosylations shield many of the binding sites for neutralizing antibodies ( 43 ) and thus decrease the efficiency of neutralization ( 46 ). However, it is important to remember that the most relevant measure for the effectiveness of antibody neutralization, is the fraction of viral spike proteins that are bound by neutralizing antibodies. This fraction is determined by the strength of the binding of the neutralizing antibodies (nAb) to the viral particles, given by the dissociation constant Kd ( 45 ).

Following the first order relation:

As the dissociation constants for antibody-epitope binding are mostly in the range of 1� nM ( 47 , 48 ) we get:

Thus, even though the ratio between the number of neutralizing antibodies and viral particles is high, such a high number of antibodies is essential to ensure that enough of the epitopes are bound (even higher ratio is needed for some antiviral drugs, as shown in the SI).

Beyond the humoral arm of the immune response, T cells are also an integral part of the targeting of viral antigens. Although severe cases of COVID-19 tend to have lower concentration of T cells in the blood, they have a higher fraction of SARS-CoV-2–specific T cells than mild COVID-19 cases ( 49 ). Here SARS-CoV-2–specific T cells denotes T cells that showed markers for activation and proliferation after stimulation with SARS-CoV-2 peptide pools ( 49 ). We can use the concentrations of CD4+ and CD8+ cells in the blood in combination with their fraction of SARS-CoV-2–specific cells ( 49 ) to estimate 1𠄲 CD4+ cells/μL and 0.2𠄰.3 CD8+ cells/μL specific for SARS-CoV-2 in convalescent patients and severe cases. Assuming a patient’s blood volume is

5L and that 1𠄲% of lymphocytes reside in the blood ( 34 ), we estimate that there are up to 10 9 SARS-CoV-2 specific T cells in severe cases, with an unknown fraction found in the infected tissue, or 1 per 1� viral particles at the peak of infection, and 10 2 � 4 such T cells per infected cell.

In our comparisons, we usually rely on our estimates for the characteristic values for the peak viral load in infected individuals, which correspond to the center of the distribution of the measured values (specifically the interquartile range - between the quantiles 25%�%). However, it is important to note that there is a high degree of variability in viral loads, exceeding 6 orders of magnitude, as can be seen from samples taken from the upper respiratory system ( 50 ). This wide variation reflects the difference between people as well as differences in viral load through the progression of infection within an infected individual ( 51 ). Thus, extreme cases could exceed the interquartile range provided by an additional two orders of magnitude, reaching values of 10 13 viral particles in a single person at the peak of infection, while up to 10% of the cells expressing both ACE2 and TMPRSS2 are infected. The variation in the number of virions, as related to the severity of the disease and its outcome, is detailed in the SI. It is also important to note that viral load in different tissues in the host body changes throughout the infection, with some tissues likely infected early on and others later in the infection ( 52 ).

Another way in which we can use our estimates to produce new insights is by taking a global view and extrapolating from the numbers observed in a single infected individual to the entire population. For example, we can estimate the number of viral particles residing in all infected humans at a given time. The total number of viral particles at peak infection was shown above to be 10 9 � 11 viral particles (this range corresponds to the 25%�% percentile range). Because the viral loads of individuals are roughly log-normally distributed ( 53 ), the arithmetic average of the number of viral particles at peak infection would be on the high end of the range, even beyond the 75th percentile (10 11 � 12 particles). There is a rapid drop in viral loads after peak infection, thus the total number of viral particles is dominated by those infected individuals who are close to the infection peak (within 1𠄲 days). Assuming during most of the course of the pandemic there has been a total of 1� million infected people close to peak infection globally at any given time (including those undetected, see SI for details ( 54 )), we arrive at a total of 10 17 � 19 viral particles or 10 13 � 15 infectious units at any given time. Similarly, the arithmetic mean of the number of particles produced over the course of infection of an average individual is 10 12 𠄳휐 13 viral particles ( N ¯ viral particles produced per person ), or 10 8 𠄳휐 9 infectious units (see SI for the detailed derivation of the uncertainty range).

One can contextualize these estimates using an absolute mass perspective. Each virion has a mass of 𢒁 fg ( 5 ). Therefore even when the body carries 10 9 � 11 viral particles, these have a mass of only about 1� μg, i.e. 1� times less than the mass of a poppy seed. The total mass of virions residing in humanity at a given time is on the order of 0.1� kg. Furthermore, using the total number of viral particles produced throughout an infection we can derive the total mass of all the SARS-CoV-2 viral particles ever produced throughout this current pandemic (concentrating on humans which we find to currently dominate over animal reservoirs). We assume the total number of infected people will be in the range of 0.5𠄵 billion people, representing optimistic and pessimistic future scenarios for the pandemic (see SI for details). To calculate the total number of virions that will have been produced by the end of the pandemic, we multiply the total number of infected people by the total number of viral particles produced over an infection of an average person (which is the arithmetic mean of the distribution across people). We then multiply this number by the average mass of a single virion to find the total mass of viral particles produced globally for such widespread infection (see SI for details of the uncertainty estimate):

Finally, we use our estimates of the total number of viruses in an infected human to examine the evolution of SARS-CoV-2 and, specifically, estimate the rate of emergence of new variants. When studying the genetic diversity of SARS-CoV-2, we can define two different measures for diversity. The first is the diversity along a genetic lineage of virions - propagating from the ancestral strain in Wuhan until currently circulating virions. The second is the diversity among a population of virions - for example the population of virions present in the body of an infected individual. We start by calculating the average number of mutations accumulated along a specific lineage of ancestor virions leading from the beginning of viral replication in the host until the end of host infection. In these calculations we rely on estimates of the mutation rate per replication cycle per site (3휐 𢄦 nt 𢄡 cycle 𢄡 ) which have been measured for MHV, another betacoronavirus ( 5 ). We further assume that each human host is infected by a few infectious units ( 55 – 57 ), and use the estimated yield of

10� infectious units per cell. Each cycle of infection is therefore assumed to produce 10� infectious units that, in turn, go on to infect other cells. As estimated above, there are 3휐 5 𠄳휐 8 infectious units produced over the course of an infection. Assuming exponential growth, the entire course of infection will therefore take 3𠄷 viral replication cycles ( Figure 3A ). As the SARS-CoV-2 genome has a length of 30,000 nucleotides, we can compute the expected number of mutations accumulating in a virus that is the product of 3𠄷 replication cycles using the per cycle mutation rate:

We use our estimates for the total number of virions produced during an infection, along with other epidemiological and biochemical characteristics of SARS-CoV-2 to estimate the rate of mutation accumulation within an infected host (A) and within the population (B). We consider both the evolution along a specific genetic lineage of virions as well as the diversity among a population of virions - either within an infected host (A) or within the total population (B). In addition, we look at the de-novo mutation generated and transmitted to newly infected in comparison to all possible single base mutations (C).

Therefore, if we track a single lineage of virions from the time they started replicating in the body until the end of the infection, this lineage would accumulate in the range of 0.1𠄱 mutations on average across its entire genome ( Figure 3A ). Considering that the mean time between successive infections, known as the generation interval, is about 4𠄵 days, we can estimate an overall rate of 𢒃 mutations per month over the course of the epidemic ( Figure 3B ). This is consistent with empirical values observed during the pandemic for SARS-CoV-2 of about 10 𢄣 nt 𢄡 yr 𢄡 ( 58 , 59 ), also known as the evolution rate. The evolution rate is estimated from the observed rate of mutation accumulation across sequenced genomes from different time points over the course of the pandemic using reconstruction of phylogenetic trees ( 59 ). It therefore includes both the rate of accumulation of neutral mutations and the effects of natural selection. This estimated rate of evolution matches the number of mutations observed in variants present today, about a year after the onset of the pandemic, most of which contain about 20� mutations. The extreme examples in terms of number of mutations, of variants such as B.1.1.7, accumulated closer to 40 mutations compared to the first strains isolated.

We can use our estimates of the viral mutation rate to assess the expected rate of appearance of a specific single base mutation. Consider the example of a single nucleotide substitution resulting in the E484K mutation in which the Glutamate (E) in position 484 is replaced with Lysine (K). This mutation requires a specific substitution in a specific location: the first base of the codon must change from G to A. As each nucleotide can mutate to 3 others (e.g. G can become A, T or C) and the genome contains 30,000 nucleotides, there are �,000 possible single nucleotide substitutions to the SARS-CoV-2 genome. As concluded above, about 0.5 mutations are accumulated in every host infection cycle. Without accounting for the effects of selection (i.e. assuming the mutant virions are equally capable of infection and propagation), or the varying chances of mutation among nucleotides, we expect that such a specific mutation will be observed in one out of every

200,000 infections. Over the last months, hundreds of thousands of cases have been identified across the world every day and many additional cases have likely gone unidentified. Indeed, as shown in Figure 3C , the estimated number of mutations generated daily (10 5 � 6 mutations/day) likely exceeds the total number of possible single nucleotide substitutions to the SARS-CoV-2 genome (� 5 substitutions) assuming 0.3𠄳 million new cases a day worldwide. As such, our estimates imply that every single base mutation is being generated de-novo and transmitted to a new SARS-CoV-2 host, somewhere in the world, every day.

In addition to considering a specific lineage of SARS-CoV-2 viruses, we can also consider the genetic diversity at the population level and estimate the total variability across the entire repertoire of infectious units produced during a single course of infection. As we estimated that 3휐 5 𠄳휐 8 infectious units are produced during an infection, each one resulting from a lineage of ancestors and mutations, we expect overall to have about 10 5 � 8 mutations across all of the infectious units. Some of these mutations that occured in early cycles will appear in many later progeny within the host, while those generated in the most recent cycle will appear in only one viral genome. Because the SARS-CoV-2 genome is 30,000 nucleotides long, the 10 5 � 8 mutations across all of the virions produced over the course of a single infection probably cover every possible single nucleotide substitution ( Figure 3A ). They even cover a significant fraction of the possible pairs of single nucleotide substitutions. If we look globally at the entire number of infectious units of SARS-CoV-2 currently present within the infected human population, which we estimated above at 10 13 � 15 , we expect that every combination of two nucleotide substitutions and many, though not all, three nucleotide substitutions will be present in at least one infectious unit ( Figure 3B ).

This large genetic diversity might naively imply that advantageous mutations will rapidly take over the population due to natural selection, but there are several factors which slow down the rate of selection. These factors include epistasis, a phenomenon where a single mutation becomes advantageous only on the background of other specific mutations. Another key factor is the genetic bottleneck imposed during the transmission of virions between infected individuals. These bottlenecks are expected to slow selection as only a tiny fraction of the diversity generated in the host is passed onto future generations ( 55 – 57 ). This quantitative understanding brings to focus cases in which selection can occur for a significant amount of time with no bottlenecks, such as the case of long and persistent infections, for example in immunocompromised patients ( 60 – 62 ). We thus conclude that careful accounting of the number of virions can give insight into the process of viral evolution within and across hosts.

One of the strengths of a holistic quantitative analysis such as the one performed here is its ability to expose interesting “quirks” that are otherwise elusive. One such observation is the ratio of

10 4 between the RNA copies measured using RT-PCR and the number of infectious units measured in TCID50. Ratios on the order of 10 3 𠄰 4 between viral particles and PFUs were observed in animal viruses such as Poliovirus and Papillomavirus ( 63 ). Naively, such a ratio would suggest that only 0.01% of the virions produced are actually infectious. This ratio implies that SARS-CoV-2 is not very efficient in producing infectious progeny. While we do not have a clear explanation for this seeming low efficiency, there are several possible factors that will affect this ratio. First, measuring RNA copies may not correspond directly to actual virions, but also measures naked viral RNA. Second, while TCID50 is the most widely available assay for measuring infectious titer, it may not accurately reflect the actual number of infectious virions, for example, because conditions in the assay may not be optimal for SARS-CoV-2 infection. Another possibility is that many virions are non-infectious due to the neutralizing effect of binding antibodies, and thus the ratio may represent the effect of the immune response, and change over the period of infection.

Beyond exposing these quantitative aspects, a holistic analysis allows us to identify major knowledge gaps in the available literature. For example, the virion yield per infected cell is known only from a few studies on different kinds of betacoronavirus from over 40 years ago ( 21 , 22 ). Similarly, measurements of the mutation rate per nucleotide per cycle in SARS-CoV-2 are of much interest but missing. As discussed above, the quantitative relationship between viral RNA copies, viral particles and infectious units is not fully characterized for SARS-CoV-2, and thus further research could help better constrain and explain the differing values. In addition, a model describing the quantitative relationship between antibody production and infection metrics would help quantitatively test the estimates presented here.

Establishing estimates for the total number and mass of SARS-CoV-2 virions in infected individuals allows us to connect together various aspects of the pandemic, from immunology to evolution, and to highlight emerging patterns and relationships not obviously evident. Having better quantitative information on the process of infection at the cellular level, the intra-host level and the inter-host level will hopefully empower researchers with better tools to combat the spread of COVID-19 and to understand its evolution, including the rise of variants of concern.

Significance:

Knowing the absolute numbers of virions in an infection promotes better understanding of the disease dynamics and the response of the immune system. Here we use the best current knowledge on the concentrations of virions in infected individuals to estimate the total number and mass of SARS-CoV-2 virions in an infected person. Although each infected person carries an estimated 1� billion virions during peak infection, their total mass is no more than 0.1 mg. This curiously implies that all SARS-CoV-2 virions currently in all human hosts have a mass of between 100 gram and 10 kilogram. Combining the known mutation rate and our estimate of the number of infectious virions we quantify the formation rate of genetic variants.


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