Can humans only "kill" viruses? Virologists: "Rebellion" is also possible

Most people's impression of biological research comes from science fiction movies: scientists get into trouble, humanity is in danger, and heroes save the earth. In 2020, the disaster really came, and the new coronavirus pandemic swept the world. What are virologists busy with? Do virologists have any unique tips to fight the epidemic? Viruses can cause diseases, but can viruses also cure diseases? Today's article will take you to understand the research and application of viruses.

Written by Li Qingchao (Shandong Normal University)

Viruses are an important class of pathogens that cause human diseases. Even before humans knew what viruses were, they had already tried to deal with them. In the Ming Dynasty, the Chinese began using smallpox to prevent smallpox. In 1798, British doctor Jenner invented the method of using cowpox as a vaccine to prevent smallpox.

In 1885, French scientist Pasteur invented the rabies vaccine.

In 1892, the first virus was discovered - tobacco mosaic virus, which is smaller than bacteria and can make plants sick.

The first animal virus was discovered in 1898 - the foot-and-mouth disease virus that infects cattle and sheep.

The first human virus was discovered in 1901 - the mosquito-borne yellow fever virus.

In 1911, the Rous sarcoma virus, which causes tumors in chickens, was discovered (Figure 1).

Figure 1. Some important nodes in the development of virology (source: Wikipedia, etc.)

Later, people's understanding of viruses became more and more profound, and virus research became a separate discipline: virology (Figure 2). Unlike what most people imagine, virology research not only answers basic scientific questions and prevents and treats viral diseases, but also involves the development and use of viruses. And these three functions are inseparable from the most important virus research tool: the reverse genetic operating system. This system can be said to be the "poison secret book" of virologists.

Figure 2. The main contents of virology research and related disciplines (drawn by the author). Including the structure, classification and evolution of viruses; the replication process of viruses; the interaction between viruses and hosts and their pathogenicity and carcinogenicity; virus research techniques (such as virus isolation and culture)

Step One: Virus Detection The first step in virology research is virus detection.

The detection of known viruses is mainly used in medical diagnosis and epidemiological surveys. The detection methods used mainly include symptom diagnosis, immunological testing, nucleic acid testing, and some coagulation experiments and other specific reaction detections.

For unknown viruses, they need to be discovered through the process of separation, cultivation, and identification, and finally the virus can be directly observed through an electron microscope. But ultimately, the genome sequence of the virus must be obtained through sequencing. This is because the genome sequence of the virus is its most core component. Grasping the genome sequence of the virus is equivalent to finding the virus.

Today's technology is developing rapidly. Researchers use deep sequencing technology (Figure 3) to easily discover a large number of new viruses. For patients suspected of being infected with unknown pathogens, we can take samples from appropriate parts, extract nucleic acids, build libraries, and perform deep sequencing. After sequencing, we use bioinformatics methods to find the pathogen sequence and give identification results.

Figure 3. Deep sequencing to detect new viruses, https://www.mgitech.cn/news/caseinfo/12/

After finding the virus, how do we conduct research? Next, we will introduce the most important research tool used by virologists: the reverse genetics operating system.

01 Genetics and reverse genetics

In order to understand reverse genetics, we need to first understand genetics. When we observe organisms, we can find some structural and functional characteristics, such as the color of hair, skin, and pupils. These are called phenotypes. The phenotype is determined by the genome of the organism, and the total combination of genes of the individual organism is called the genotype (Figure 4). The genotype determines the phenotype, and the phenotype reflects the genotype.

At the beginning, people did not know the nature of heredity, so they initially studied genetic issues from the perspective of phenotype. Mendel used peas and Morgan used fruit flies to study genetics. They used the phenotypes of red and white flowers in peas or red and white eyes in fruit flies to find genetic laws and locate genes related to phenotypes. Therefore, classical genetics studies genotypes from phenotypes, which is forward genetics.

Figure 4. Phenotype and genotype

https://www.sciencedirect.com/science/article/pii/S0042698901002620

https://www.niaid.nih.gov/diseases-conditions/pidds-genetics-inheritance

In forward genetics, it is necessary to screen mutants with specific phenotypes through natural mutation or induced mutagenesis, and then locate which gene determines this phenotype and study the function of the gene.

In reverse genetics, scientists get an unknown gene and can actively mutate it or change its expression level (overexpression, underexpression or knockout), and then observe what kind of phenotypic changes are caused by the gene mutation or the change in gene expression, and compare it with the phenotype of the wild type (normal genotype that has not been artificially changed) to infer the function of the gene. Therefore, reverse genetics studies the function of genes by changing the genotype and observing the results of phenotypic changes (Figure 5).

Figure 5. Forward genetics and reverse genetics

02 The prerequisite for reverse genetics is genetic engineering technology

Based on the concept, it is not difficult to see that in reverse genetics, specific nucleic acid sequences need to be modified, and this modification technology is actually only possible after we understand the structure and function of DNA, a genetic material, and develop a variety of genetic engineering tools, enzymes or genetic engineering methods. Genetic engineering is a technology that uses biotechnology to directly manipulate the genes of organisms and change the genetic composition of cells. Therefore, the idea of ​​reverse genetics appeared later than the idea of ​​classical genetics.

DNA is the genetic material of cellular organisms. It is more stable than RNA, and most of the tool enzymes in genetic engineering act on DNA, so the modification of genetic material is mainly carried out on DNA. We mainly carry out genetic material modification on plasmids (Figure 6), because plasmids are a kind of free DNA outside the genome that can replicate independently. It is relatively easy to amplify and can be carried out in bacteria or yeast. (For more information about plasmids, please go to Fang Pu to read "What is a plasmid? From biological weapons to genetically modified foods, it is related to it")

Figure 6. Plasmid

(Source: https://www.genome.gov/genetics-glossary/Plasmid)

03 Infectious clones

The viral genome is usually small, so we can assemble a double-linked DNA copy of the viral genome in a plasmid. Sending these plasmid DNAs with viral sequences, or RNA produced by transcription directed by the plasmid, into cells - a process called "transfection" - can instruct the cell factory to produce viral proteins, replicate new viral genomes, and assemble and release them to produce infectious virus particles.

The genome of a virus can be either DNA or RNA. The genome of a DNA virus can be amplified, cut, and then directly linked to a plasmid; while the genome of an RNA virus needs to be converted into DNA through the reverse transcription process of RNA-guided DNA synthesis before being linked to a plasmid. These plasmids that carry viral genome sequences and can produce infectious viral particles are called "infectious clones." By transfecting the constructed infectious clone into cells, or transfecting cells after transcription into RNA, new viruses can be produced (Figure 7).

Figure 7. Infectious clone construction and virus production, adapted from Wikipedia

04 Reverse genetic operating system

As mentioned earlier, the idea of ​​reverse genetics research is to change the gene sequence, study the phenotype from the genotype, and then study the gene function. The same is true for viruses: construct infectious clones, perform genetic engineering on them, and transfer the modified infectious clones into cells to produce viruses with mutations. These new viruses are then used to infect cells or hosts, and the phenotypes such as virus replication and host symptoms are observed to study the functions of virus-related genes (Figure 8). We call this system a reverse genetics operating system for viruses.

Figure 8. Reverse genetics system for virus research, adapted from Wikipedia

Take the recently released SARS-CoV-2 reverse genetics operating system as an example [1]: it uses yeast to obtain infectious clones. First, the SARS-CoV-2 genomic RNA is reverse transcribed and amplified to obtain DNA fragments, or the DNA version of the virus is directly synthesized. Then, the infectious clone plasmid is recombinantly synthesized in yeast. After the plasmid is extracted, it is transcribed and synthesized into RNA. After the RNA is transfected into cells, the SARS-CoV-2 can be produced (Figure 9).

Figure 9. Coronavirus infectious clone[1]

Whether it is for research, vaccines or treatment, to make a virus, we need to modify the plasmid. In the process of modification, we will use some genetic engineering tools such as enzyme cutting sites, screening markers, and necessary plasmid fragments. At the same time, our virus sequences are obtained from natural viruses. In the process of designing and manufacturing infectious clones, we will inevitably use existing theoretical models and design ideas. Even if a new virus is artificially synthesized from scratch, it will leave artificial traces in terms of codon usage frequency and so on. Therefore, it is easy to identify whether a virus is artificially synthesized.

Application of Virology Research The reverse genetics operating system of viruses is a very important tool in virology and related life sciences. It can be used for basic research, studying the functions of various genes in viruses, and for vaccine development. Viruses can also be used as vectors to load different gene sequences for other life science research. In addition, with the development of life sciences, the prospects for viral vectors to be used in cell therapy, gene therapy, and cancer treatment are becoming increasingly bright.

01 Basic research and application

The ideas and tools of virology are widely used in basic biological research. At present, viral vectors are widely used in various aspects such as gene overexpression, knockout, knockdown, and animal model modification. Take the application of pseudovirus in virology research as an example (Figure 10): We can use the basic method of reverse genetic operating system to make pseudovirus particles, so that the pseudovirus particle envelope contains the envelope protein of the virus under study, which can be used to simulate the antigenicity, neutralization and early infection process of virus particles; however, the particles only contain defective virus genomes or no genomes, so the pseudovirus cannot complete the virus replication cycle and will not cause the host to become ill. Due to its better safety, with the help of pseudovirus particles, we can study some dangerous viruses in laboratories with relatively low biosafety levels.

Figure 10. Pseudoviral particles used in coronavirus research (cited from GenScript)

02 Vaccine R&D and production

Each of us has been vaccinated. You can touch the scar on your arm, which is left by the BCG injection. Vaccines prevent diseases by stimulating the body to produce acquired immune protection. According to the components of the vaccine, it can be divided into inactivated vaccines, attenuated live vaccines, toxoids, subunit vaccines, recombinant protein or polypeptide vaccines, as well as viral vector vaccines, DNA or RNA vaccines, etc. Vaccines need to meet two conditions at the same time: they can stimulate the body to produce effective immune protection and not cause disease. The vaccination process is similar to a military training of the immune system: training the immune system without damaging the body. The so-called immunity is actually the ability of the body to quickly eliminate pathogens the next time it encounters pathogens, so that people (or animals) do not become ill.

Let's look at an example of a reverse genetic operating system for making vaccines (Figure 11). We know that influenza will mutate frequently, and the viral strains that are prevalent every year may be different. After changing their "vests", the immune system will not recognize them. Therefore, we must produce new influenza vaccines based on the viral strains that are prevalent that year. At this time, a reverse genetic operating system is needed: ① We detect the prevalent strong strains from the clinic and obtain its antigen coding sequence, ② Then, through genetic engineering, the coding sequence of the antigen part is recombined into the infectious clone of the weak vaccine strain; ③ Then we transfect these plasmids into the cells, and we can produce a new weak vaccine strain with strong strain antigens and no pathogenicity, which can be used to prevent the influenza that is prevalent that year.

Figure 11. Reverse genetics system to produce influenza vaccine (Source: Wikipedia, modified by the author)

The most effective way to prevent viral diseases is to develop vaccines. In order to prevent and control the COVID-19 pandemic, researchers around the world are working hard to develop vaccines for the novel coronavirus. The recombinant novel coronavirus vaccine (adenovirus vector) ("Ad5-nCoV") developed by the team of Academician Chen Wei in my country has entered Phase II clinical trials. As the name suggests, the virus vector used in this vaccine is adenovirus, which is a DNA virus without an envelope. We delete the disease-causing genes and some irrelevant genes in the adenovirus, and then recombinantly introduce the antigen protein expression gene of the novel coronavirus to complete the coronavirus vaccine based on the adenovirus vector. Adenovirus vectors are characterized by high efficiency, high titer (titer refers to the concentration of the virus), low pathogenicity, and will not integrate into the host cell chromosome. They are a commonly used viral vector. At present, researchers at home and abroad have also used strategies such as inactivated vaccines, subunit vaccines, pseudovirus particles, poxvirus vector vaccines, and nanoparticle vaccines to develop vaccines (Figure 12). Among them, nanoparticle vaccines are nanoparticles composed of viral antigens and self-assembled protein components.

Figure 12. COVID-19 vaccine development strategy

(Source: https://research.sinica.edu.tw/covid-19-vaccine-academia-sinica/)

03 Viruses can also cure diseases

Viruses or viral vectors can also be used in phage therapy, cell therapy, gene therapy, and cancer treatment and prevention.

Due to the discovery and application of various antibiotics, the harm of bacteria to human health has been greatly reduced. However, the abuse of antibiotics has brought about the problem of bacterial resistance. Some bacteria have multiple antibiotic resistance, which we call super bacteria. Super bacteria infection is very dangerous and a very difficult problem in medicine. Phages are viruses that can infect bacteria, so using phages to treat drug-resistant bacterial infections has become one of the ideas for treating bacterial infections. In 2015, a couple of scientists from the University of California were traveling in Egypt. The husband, Tom Patterson, was infected with super bacteria and was in critical condition. He was later cured by phage treatment (Figure 13).

Figure 13. Phage therapy for superbug infection

https://www.bbc.com/zhongwen/simp/world-50336647

Cancer is a type of disease that seriously threatens human health, and some viruses can cause tumor dissolution, which we call oncolytic viruses. Oncolytic viruses include adenovirus, poxvirus, alphavirus, Newcastle disease virus, herpes simplex virus-1, measles virus, etc. (Figure 14). After being modified, these viruses can be used to treat cancer: on the one hand, they do not cause disease, and on the other hand, they can kill tumor cells. There are many mechanisms for oncolytic viruses to treat tumors, such as destroying tumor blood vessels, cutting off tumor nutrition sources, directly killing tumor cells, or inducing cellular immune responses against tumors, etc.

Figure 14. Oncolytic viruses and their mechanisms of action [2]

The human immune system is like an army that can resist the invasion of foreign pathogens, and can also identify and eliminate abnormal "rebel" cells. However, some cells can evade this surveillance, are not recognized and eliminated by the immune system, and grow wantonly. These are cancer cells. Cell therapy methods can use lentiviral vectors to install receptors CAR that can recognize cancer cells on the soldiers of the immune system - T cells, helping the immune system to recognize and eliminate tumors (Figure 15). Lentiviral vectors are modified HIV viruses. They have high infection efficiency and can stably insert foreign genes into the cell genome. They are widely used in cell therapy.

Figure 15. CAR-T cell therapy

With the rapid development of gene editing technology, the prospects of gene therapy are becoming more and more promising. Gene therapy is to treat or prevent diseases by modifying genes. The main methods used are to replace mutant genes, knock out harmful genes, or introduce new genes. Gene therapy is particularly suitable for treating genetic diseases, and can also be used to treat viral diseases or cancer.

The recovery of the "butterfly boy" is a classic example of gene therapy. In 2017, Hassan, a boy with junctional epidermolysis bullosa, was hospitalized due to severe skin damage all over his body. His skin was extremely fragile and would break at the touch, and he was in danger of death. This serious genetic disease is caused by abnormal laminin genes in the skin, and people figuratively call this sick child the "butterfly boy." Scientists loaded the normal laminin gene into a retroviral vector, and then used the virus to infect skin cells cultured in vitro. These skin cells were introduced with normal genes by the viral vector, so that they could express normal proteins. After culturing the transgenic skin in vitro, the skin was transplanted onto the child's body, gradually replacing the original skin to achieve the purpose of treatment (Figure 16).

Figure 16. Genetically modified skin produced using viral vectors to treat junctional epidermolysis bullosa[3]

Spinal muscular atrophy is a fatal genetic disease. The motor neurons in the brainstem and spinal cord of children are gradually destroyed, and they slowly lose the ability to speak and walk. In the end, they can't even breathe or swallow, and eventually die. Studies have found that this disease is caused by abnormal SMN1 gene. The adenovirus-associated virus vector developed by Novartis in the United States can install the normal SMN1 gene into the patient's genome to treat this disease. This viral vector drug only needs to be injected once, and the cost of one injection is as high as 2 million US dollars (Figure 17). The AAV virus used here is a defective virus that relies on adenovirus for replication. It does not cause disease itself, can infect dividing and non-dividing cells (nerve cells usually do not divide), can be integrated into human chromosome 19, and is a commonly used viral vector.

Figure 17. The most expensive drug in history, AAV virus used to treat spinal muscular atrophy

Conclusion Viruses are important pathogens that cause human diseases. From ancient times to the present, various viruses have been nightmares for humans. Even in today's society, facing the COVID-19 pandemic, human health, economy, and society are still greatly impacted. As the boundary between man and nature becomes increasingly blurred, the climate and environment change dramatically, and international exchanges and transportation of humans or animals become frequent, emerging viral diseases will always be, and increasingly are, a serious threat to human society that is extremely likely to occur, has already occurred, or is occurring.

Virology research has enabled us to make great progress in early warning, treatment and prevention of emerging viral diseases. We are discovering new viruses faster and faster, making effective prevention measures and decisions faster and faster, and developing drugs and vaccines faster and faster. Compared with previous plagues in history, human society's ability to deal with infectious diseases is no longer what it used to be. At the same time, virology research has also brought many remarkable results to basic research in life sciences and medical research. We often say that the 21st century is the century of life sciences, and many experts also regard life science technologies represented by gene editing as the main component of the fourth industrial revolution, and all of this is inseparable from virology tools. Therefore, virology research is very important both in terms of the development of life sciences and human health.

References

[1] https://www.biorxiv.org/content/10.1101/2020.02.21.959817v1.full.pdf

[2] Ungerechts, G., Bossow, S., Leuchs, B., Holm, PS, Rommelaere, J., Coffey, M., Coffin, R., Bell, J., and Nettelbeck, DM (2016). Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses. Molecular Therapy - Methods & Clinical Development 3, 16018–13.

[3] https://www.nature.com/articles/nature24753

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