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Thursday, 11 July, 2002, 23:28 GMT 00:28 UK
First synthetic virus created

By Dr David Whitehouse
BBC News Online science editor

Scientists have assembled the first synthetic virus.

The US researchers built the infectious agent from scratch using the genome sequence for polio.

It was just a matter
of time before it was done

Dr CJ Peters,
University of Texas
Scientists are divided about whether a virus is alive. For those that think it is, then this synthetic artefact would constitute a simple form of life.

Responding to criticisms that such research could lead to bioterrorists engineering new lethal viruses, the scientists behind the experiment said that only a few people had the knowledge to make it happen.

'New reality'

To construct the virus, the researchers say they followed a recipe they downloaded from the internet and used gene sequences from a mail-order supplier.

The polio virus: Now made by man
Having constructed the
virus, which appears to be identical to its natural counterpart, the researchers, from the University of New York at Stony Brook, injected it into mice to demonstrate that it was active.

The animals were paralysed and then died.

"The reason we did it was to prove that it can be done and it now is a reality," said Dr Eckard Wimmer, leader of the biomedical research team and co-author of the study published in the journal Science.

"This approach has been talked about, but people didn't take it seriously," said Dr Wimmer.

"Now people have to take it seriously. Progress in biomedical research has its benefits and it has its down side. There is a danger inherent to progress in sciences. This is a new reality, a new consideration."

'Very easy to do'

According to researcher Jeronimo Cello, the polio virus assembled in the laboratory is one of the simplest known viruses. "It was very easy to do," he said.

Dr Eckard Wimmer: "The world had better be prepared"
The more dangerous
smallpox virus would be complex and difficult to assemble, but Cello says, "it would probably in the future be possible".

Dr Wimmer added: "The world had better be prepared."

Smallpox has been eradicated in the wild, but specimens are stored in the United States and the Soviet Union.

Dr Wimmer said assembling the polio virus showed that eradicating a virus in the wild might not mean it was gone forever because biochemists could now reconstruct those viruses from blueprints.

Following last year's terrorist and anthrax-by-mail attacks, US officials became concerned about the threat of smallpox and arranged for the manufacture of enough vaccine to protect the US population.

Matter of time

Dr CJ Peters, director for the Center for Biodefense at the University of Texas Medical Center at Galveston, said experts had known for years that it was theoretically possible to assemble a virus in the lab.

This is a new reality

Dr Eckard Wimmer
"We've known this could be

done. We've known it was just a matter of time before it was done," he said.

Dr Peters said he was concerned that publicity about a synthesized virus might lead some people to believe "that there is nothing that can be done about bioterrorism - which is not the case".

He added that it was possible that viruses like Ebola could be assembled in laboratories, but there were only a few people in the world with that skill.

Polio is on the brink of being eradicated worldwide and there are plans to stop inoculations against the disease after it disappears from nature.

Dr Wimmer said that this policy should be reconsidered. Stopping vaccination could lead to a generation of people highly susceptible to polio, enhancing its appeal as a weapon.

The World Health Organization is planning to stockpile vaccines against a return of polio and Dr Wimmer said that policy should be followed everywhere.

Responding to criticisms that such research could lead to bioterrorists engineering new lethal viruses, the scientists behind the experiment said that only a few people had the knowledge to make it happen.

To construct the virus, the researchers say they followed a recipe they downloaded from the internet and used gene sequences from a mail- order supplier.

According to researcher Jeronimo Cello, the polio virus assembled in the laboratory is one of the simplest known viruses. "It was very easy to do," he said.

So, it doesn't matter how few people know how to do this. Apparently the instructions are on the internet (why not - everything else is), and its easy, and money has a way of motivating people who otherwise wouldn't have attempted making something more dangerous.

Just because it was possible doesn't mean it should have been done.

Dr Wimmer said assembling the polio virus showed that eradicating a virus in the wild might not mean it was gone forever because biochemists could now reconstruct those viruses from blueprints.

You could build the smallpox virus genome from scratch and then put it into another viral capsid (1), one that would be easily internalized, such as an adenovirus (2), as used for gene therapy and one of the five major virus families that cause the common cold; once internalized by the host, perhaps as easily as being picked up from a doorknob like a cold is at the office during cold and flu season, the ensuing viral replication would then result in the production of a lot of native smallpox in the native smallpox capsid and convert into a normal mode of transmission. Such an agent could be spread intially throughout large office buildings by cleaning personnel at night. Over the course of a week or so, before people caught on to the fact that the disease was smallpox and got an inkling of its initial mode of infection, large numbers of public places (armrests on airside chairs at airports, computer keyboards in public schools and office buildings, desktops, even kindergarten crayons, etc.) could be contaminated, and many first generation cases started. If the initial infection is widespread, sudden, and unanticipated, the resulting numbers of secondary cases could be enough to cause major problems. Various terrorist scenarios have been proposed for such modes of transmission as 3 aerosol bombs infecting 1000 people in each of 3 shopping malls or 5 al Qaeda terrorists (Science Magazine: BIOTERRORISM: How Devastating Would a Smallpox Attack Really Be? by Martin Enserink). The authors conclude that the resulting infection would be small. But a recombinant smallpox genome in an adenovirus or rhinovirus capsid spread quietly could be a different story entirely. Imagine, instead of 5 al Qaeda terrorists, 100 janitors at large junior and senior high schools and another 20 or 30 housekeeping staff at major airports.

1.

J Virol 1998 Dec;72(12):10298-300
In vitro construction of pseudovirions of human papillomavirus type 16: incorporation of plasmid DNA into reassembled L1/L2 capsids.

Kawana K, Yoshikawa H, Taketani Y, Yoshiike K, Kanda T.

Division of Molecular Genetics, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan.

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a beta-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced beta-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.

PMID: 9811779 [PubMed - indexed for MEDLINE]

J Gen Virol 1999 Jan;80 ( Pt 1):39-46
The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

Ou WC, Wang M, Fung CY, Tsai RT, Chao PC, Hseu TH, Chang D.

Department of Microbiology, Chung Shan Medical and Dental College, Taichung, Taiwan, Republic of China.

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.

PMID: 9934681 [PubMed - indexed for MEDLINE]

2.

Adenoviruses develop within the nuclei of infected cells, where they often can be observed packed in an apparently crystalline arrangement. In humans, adenoviruses cause acute mucous-membrane infections of the upper respiratory tract, the eyes, and frequently the regional lymph nodes, bearing considerable resemblance to the common cold. Like the cold viruses, adenoviruses are often found in latent infections in clinically healthy persons. There are more than 45 different members of the adenovirus group, but only a few commonly cause illness in humans; it is thus possible to prepare a vaccine against these viruses. In contrast, there are more than 100 cold viruses, all of which are fairly commonly found as disease agents; this great number makes the development of a vaccine for the common cold virtually impossible.

J Neurosci Methods 2002 Feb 15;114(1):99-106
High-efficiency adenovirus-mediated in vivo gene transfer into neonatal and adult rodent skeletal muscle.

Sapru MK, McCormick KM, Thimmapaya B.

Molecular Neuroscience Laboratory, Departments of Kinesiology and Psychiatry, University of Illinois at Chicago, MC 194, 901 West Roosevelt Road, Chicago, IL 60608, USA. msapru@uic.edu

Several methodological limitations have emerged in the use of viral gene transfer into skeletal muscle. First, because the nuclei of mature muscle fibers do not undergo division, the use of strategies involving replicative integration of exogenous DNA is greatly limited. Another important limitation concerns the maturation-dependent loss in muscle fiber infectivity with adenoviral vectors. In this study, we investigated the possibility that high-titer infections with recombinant adenovirus, expressing a foreign marker gene under the control of a strong viral promoter, can significantly improve the efficiency of gene transfer in vivo into neonatal and adult rat skeletal muscle. High-titer (2 x 10(10) plaque forming units) intramuscular injection of replication-defective adenovirus vector, expressing green fluorescent protein (GFP) under the control of cytomegalovirus promoter, resulted in GFP expression in 99 +/- 0.34% of fibers in the adult soleus muscle and in approximately 85 +/- 1.44% of fibers in the adult tibialis anterior muscle. Interestingly, reduction in injected adenoviral dose significantly reduced the number of GFP-positive fibers in the adult tibialis anterior muscle, but not in the soleus muscle. However, in neonates, adenoviral infection resulted in GFP expression in 96-99% of the fibers in the tibialis anterior and the gastrocnemius muscles regardless of administered adenoviral dose.

PMID: 11850044 [PubMed - indexed for MEDLINE]

"The reason we did it was to prove that it can be done and it now is a reality," said Dr Eckard Wimmer

And like the four-minute mile, no one could do it until someone did it, then everyone could do it.

This is bad Voodoo for sure.

He added that it was possible that viruses like Ebola could be assembled in laboratories, but there were only a few people in the world with that skill.

Okay, so why is it that statement doesn't exactly give me that "warm and fuzzy" feeling inside?