Team:HZAU-China/Safety/Security Evaluatio

• -Safety forms
• -Security Evaluation
• -Social Approval Degree
• -The Safety of Flea
• -Helpful Ideas

The reason why we take Rabies G protein as our topic

Rabies is caused by the rabies virus infection in the central nervous system characterized as fatal zoonotic diseases. Once infected by the disease, the mortality rate is almost 100%. Studies have shown that rabies virus can cross the human blood-brain barrier and then invade the brain. But in the human body it can not be cleaned mainly because of its ability to evade the host immune clearance. Escape mechanism is the key factor for the rabies virus inhibiting the inflammatory response, as well as inhibiting the body's resistance to innate immunity and acquired immunity, then clear up the viral infection caused by the T-lymphocytes. It is effective to use rabies virus vaccine vaccination to prevent and control the incidence of rabies. Currently, people widely employ the primary cell culture vaccines and refine passage cells to purify vaccine. Although these vaccines have better immune effect, people still cannot receive the high cost of production. To solve the problem, transportation and storage must rely on the low-temperature "cold chain". What is more, the mode of administration and other factors will limit the application to store rabies virus. It is inconvenient to inoculate the reservoir hosts, for example, dogs, foxes and other wild or domestic animal. Inoculating the source of rabies virus effectively may cut off the route of transmission, which still depends on developing a new generation of genetically engineered recombinant vaccines. The most successful experience in Europe and other developed countries for rabies control is the use of attenuated vaccine and recombinant live vector vaccine to immune the wildlife. In recent years, with the increasing number of dogs, cats and other pets, rabies is becoming more and more serious. In order to respond the policy of our government to increase the force of compulsory vaccination to animals, we need to develop a safe and effective rabies weak poison vaccine to finish the target. So we choose the Rabies G protein as our title, and we hope one day we can use the flea carried with our vaccine to end up the rabies virus disease.

I. The level of Laboratory Biosafety

The biological methods used in our laboratory are just the normal operation such as PCR, digestion, ligation, electric conversion and so on. Well protective measures have been taken to the use and storage of toxic reagents. Besides, we do not try any dangerous experiment operation. Therefore, according to《Laboratory Biosafety Manual》 - Third Edition(Edited by WTO), the level of security for the genetic manipulation is Class I .

II .The safety Evaluation of rabies G protein

1 The source of the G protein

We got it from the State Key Laboratory of Agricultural Microbiology.

2 Is there long-term security applications record?

Yes. Our school has the exact record of the poison to extend and the main biological characteristics of monitoring. Besides, we have the special staff to record and store them.

3 Did it cause any adverse impact on human health or the environment?

Rabies virus strains ERA glycoprotein (G) gene ... The glycoprotein can induce the body to produce neutralizing antibody. Besides, the structure of the rabies virus and the cell receptors are under the influence of the G protein. Currently, No related stories have proved that rabies G protein caused any adverse impact on human health or the environment.

So from the website https://2013.igem.org/Safety/Risk_Group_Table., we determine the security level of rabies G protein type II.

III. The safety Evaluation of vector

1 Vector we used

Shuttle vector, namely pht304, phy300, and pma5 plasmid.

2 The stability of plasmid and the degree of the potential risk level

Plasmid we used is provided by State Key Laboratory, and its stability has a lot of data to be proved, and there is no potentially dangerous.

According https://2013.igem.org/Safety/Risk_Group_Table, the security level of plasmid is type I.

IV .The safety evaluation of receptor microorganisms

1 The type of receptor microorganisms

Bacillus subtilis 168. (Protease defect strains）

2 Natural wild species or artificial cultivation strains ?

We choose the artificial cultivation strains.

3 Genetic variation of receptor microorganisms:

We use LB medium to cultivate the microorganism, then extract plasmid and PCR. The DNA sequencing results further confirmed that PCR products were truly target gene, and it showed that chassis microorganism’s inheritance is stable.

4 Looking over the history, is there the possibility that the receptor organisms will evolve into harmful ones?

The possibility is so low that we can just leave it alone.

5 Risk groups of the species:

We used Bacillus subtilis as chassis organism which have many advantages, so it fell into Risk group I.

V .The safety evaluation of recombinant organism

1 Introduction

The recombinant plasmid contains rabies virus glycoprotein, signal peptide gene and antimicrobial peptide gene; the organism expresses G-protein effectively, meanwhile, it can secrete antimicrobial peptides, which can kill pathogenic bacteria in the fleas forestomach; Bacillus subtilis can survive in the forestomach and generate biofilm. Then fleas’ forestomach will be blocked. By this way the strains will injected into stray dogs’ blood with the gastric disorder causing nausea of flea.

2 Dose it produce toxic substance to animal?

G protein rabies recombinant strains are not pathogenic to the host. If animals are vaccinated high dose(107 PFU）, there is even no adverse reaction; The organism won’t produce poisonous substance.

3 Potential dangerousness to human ?

The recombinant organism doesn’t have pathogenicity to human ，and research has shown that powerful immune system of human will clear it up soon.

4 The survive ability and ways to spread

First, we mix recombinant Bacillus subtilis into blood and feed fleas with them ,and the bacteria will survive in the forestomach, and fleas vomit them into target animals. In addition, the infected fleas couldn’t suck blood because of biofilm, and they won’t live for a long time; Bacillus subtilis might be passed to offspring by flea droppings, or uninfected flea suck infected dogs’ blood, which contain recombinant organism; there are just speculations; Due to limitation of experimental time, the survival in the environment is not clear; Transmission capacity will be simulated through the mathematics modeling.

5 The probability of genetic material exchange with other microorganism (especially for pathogene )

Genome exposure is scarcely possible under natural conditions.

6 The probability of genetic material exchange with animals:

Genetic material exchange is impossible because of a low homology with mammal.

So according to the Website http://en.wikipedia.org/wiki/Risk_management , the level of recombinant organism is type II

1 When it comes to unusual breeding animal species in domestic, you should describe the nature environment of the animal and other concerned information in detail.

The recombinant plasmid does not involve unusual breeding animal or cultivate plant spices.

2 The ecological correlationship with other microorganism in ecosystem; The infection to human and animal causative agent such as virus.

Rabies virus G protein does not exchange with other microorganism on genetic material, because it cannot meet the condition (such as high dose, two kinds of microbes into the cell at the same time) of homologous recombination. In addition, the synthetic bacterial strain will eliminate multiplication of other harmful microorganism, but doesn’t have negative influence on the health of animals and human as well as ecologic environment.

3 In the geographical distribution of domestic and natural habitats, Will natural distribution change because of certain conditions change?

Domestic and foreign evidence so far suggests that rabies virus mainly infects stray dogs, cats and wild animal, because it is a blood borne, because the distribution of fleas and receptor animal change and have a certain impact on the natural ecological environment.

4 Whether does it have the ecological specificity, such as in the environment adaptability?

No ecological specificity.

5 The impact on the ecological environment and its potential hazard degree;

It can be predicted, and it will cause a certain impact on the ecological environment in a short period of time. The flea population will increase. But because of the shorter life expectancy, we use dedicated host fleas and harmful microorganisms are cleared up, so its harmfulness won’t be too serious.

6 If is there a possibility of genetic variation? Or does it have an adverse effect on animal health, human health or the environment?

So far, it has not been reported.

7 The safety to animal and the mechanism of genetically modified microorganisms.

After using synthetic biology vaccine immune animal subjects, by stimulating the body's immune system, the humoral immunity, cellular immunity and mucosal immunity, the immune animal acquired the ability of resistance to wild virus infection.

8 The survival prospects of vivo of target and non target animal.

Recombinant vaccine virulence is weak; it can cause short-term infection to the target animal. What’s more, the recombinant strain has the narrow host range, so there is little possibility to do harm to the non target animal.

9 The effects of target and non target animal.

It has not been proven, but it can be predicted with high dose vaccination of animal subjects, does not appear abnormal symptoms.

10 The drift of host and vector.

This virus has been reported that the parental virus host range is narrow, mainly in dog and cat. The recombinant gene column of the vaccine has mutated, and the virulence of the vaccine strain is greatly reduced. The drift problem of vector has not yet been proved.

As we descried before, the level of Ecological security is type II.