Division of Research Safety

The University of Illinois
at Urbana-Champaign

Biological Safety

Research Utilizing Viral Vectors

Introduction

More researchers, many of whom are not virologists by training, are choosing to utilize viral vectors. The following information was developed which defines terms, identifies research that requires IBC registration, and provides information about the virus/viral vectors, practices and Biosafety approaches that may be used as a starting point for a risk assessment.

Bacterial viruses (bacteriophage or phage): Recombinant bacterial viruses require registration with the IBC. However, these viruses are not generally considered harmful to humans, animals, plants, or the environment. Thus, wild type bacterial viruses do not as a rule require registration as biohazards. If a non-recombinant bacterial virus is found or suspected to cause harm, then registration would be required.

Eukaryotic viruses: All wild type and recombinant eukaryotic viruses will require registration as a biohazard. Note that field release or field sampling of viruses, although not involved with containment, do require IBC oversight and registration. The biosafety containment level may be inferred by consulting primarily the current edition of the BMBL http://www.cdc.gov/biosafety/publications/bmbl5/index.htm and secondarily the American Biological Safety Association site for Risk Group Classification for Infectious Agents (http://www.absa.org/riskgroups/) and the Material Data Safety Sheets compiled by the Public Health agency of Canada (http://www.phac-aspc.gc.ca/msds-ftss/index.html). Unfortunately many viruses, especially plant and non-zoonotic animal viruses, do not have documented formal risk analyses. In these cases, the PI will do the risk assessment based on experience or the literature, and the IBC will either concur or require modifications.

Recombinant DNA or RNA Viruses

All recombinant DNA and RNA must be registered. These include recombinant viruses from all sources, even if the PI was not involved in the manipulations, but obtained the materials from an external source. Ultimately the identity and portion of the viral backbone present, the inserted or modified recombinant nucleic acid, the host cell, and the experimental context determine the containment level. These rules also hold for cases when recombinant viruses are prepared by a collaborator or made from a commercial kit. The NIH Guidelines (http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_lnk_2002z.pdf) and the Recombinant DNA Advisory Committee (RAC) guidance document on lentiviral vectors (http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti_Containment_Guidance.pdf) explicitly dictate the containment level in most instances. The three common ones are Section III-D-3 for recombinant viruses in tissue culture systems, Section III-D-4-a for recombinant viruses in animals, and Appendices B-II-D to B-IV-D for Risk Group Classification of Various Viruses. Other NIH sections are relevant to viruses as well as to other agents. In common practice, the default biosafety level for recombinant viruses is BL-2 and ABL-2. The following are exceptions to that generality:

1. A lower level may suffice if an incomplete virus is cultured in vitro (NIH Guidelines).
2. A few animal and/or human viruses qualify for a lower level (NIH Guidelines).
3. Certain recombinant viruses that are infectious to humans and carry a gene that will confer a selective growth advantage or lead to immune dysfunction require BL-2 containment and the demonstration of replication incompetency prior to the actual research (University of Illinois at Urbana-Champaign IBC and NIH Guidelines).
4. Animals with recombinant viruses that ordinarily require ABL-2 containment can be down-graded to ABL-1 if and when the animals are considered to no longer be shedding.

A frequently encountered situation with viral vectors is to take advantage of their high specific infectivity rates, while still using them as plasmids. The replication functions are first genetically dissected from the packaging (infectivity) functions. Both functions are then transiently combined on separate molecules (in trans) to package an infectious but non-replicating recombinant molecule. The recombinant virus can be eventually lost or persist in the target cell either integrated into, or independent of, the chromosomes. However, as long as the packaging and replicating functions are kept from recombining into a single molecule, no further viral-driven replication or viral life cycles will ensue. However, if an endogenous virus is present in the host, the deficient viral vector may be rescued by this endogenous virus in trans producing a fully competent viral particle. The successful execution of this dictum has implications from both a biosafety and research standpoint. A number of strategies have been developed to minimize the occurrence of recombining the two functions.

1. Ensure that no homologous recombination occurs by neatly dissecting the replication from the packaging functions.
2. Minimize the probability of homologous and end-joining recombination by separating the individual packaging functions into several different plasmid molecules.
3. Utilize a heterologous (from a different virus) packaging system to minimize the probability of homologous recombination. In this case, the host range and tissue tropism are determined by the packaging system, and the virus is said to be “pseudotyped”.
4. Utilize a SIN (self-inactivating) derivative, which lacks the native viral 3’ enhancer/promoter. Upon integration, only the recombinant gene without the rest of the virus is transcribed and thus virus should not be able to undergo another round of replication.

However, two main risks are still in play: the virus is highly infectious, and integration into the genome can have severe consequences.

Each of the following virus information fact sheets should be considered a starting point for conducting a risk assessment. Utilizing each viral vector system brings along the biology of the virus and must be considered.

• Retroviruses/Moloney Murine Leukemia Virus (MoMuLV)
• Retroviruses/Lentiviruses
• Adenovirus
• Adeno-Associated virus (AAV)
• Herpesvirus/Gammaherpesvirus/Epstein Barr (EB)
• Herpesvirus/Type A/Herpes simplex virus type-1 and type-2
• Poxviridae/Vaccinia

Retroviruses/Moloney Murine Leukemia Virus (MoMuLV)

Risks of MMLV vectors: Data suggests a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis leading to cell transformation and tumor formation. Pseudotyping also increases host range and risk of infection.

• General criteria for a risk assessment:
  • Host Range: The host range of recombinant MoMuLV vectors is dependent on the specificity of the viral envelope. The ecotropic env gene produces particles that infect only rodent cells. Amphotropic env gene allows infection of murine and non-murine cells, including human.
  • Zoonosis: None, unless pseudotyped
  • Clinical Manifestation: In vivo infection in humans appears to require direct injection with amphotropic or pseudotyped virus. Cell transformation and tumor formation.
  • Infectious Dose: Unknown
  • Cell types able to be infected transduced: Cell division is required for infection.
  • Transgene considerations: Is the transgene oncogenic or biotoxic? If so, increased risk.
• Host-vector systems:
  • Vector information: MoMuLV integrates into the host genome and is present in infected cells as a DNA provirus.
  • Packaging cell line: Certain cell lines provide the packaging signal in trans. A helper virus can increase the risk due to recombination and reconstitution of helper virus resulting in contamination.
  • What is the potential for generation of replication competent virus from vectors by recombination? Recombination may occur (1) between endogenous murine retroviral sequences in the packaging cells line and (2) between vector coding sequences and the helper packaging sequences to generate infectious viral particles.
  • Envelope protein in the packaging system: The packaging cell line determines if the vector is ecotropic, xenotropic or amphotropic.
• General containment considerations:
  • BL-1 is appropriate when ecotropic or the native envelope is used and it has been shown to be replication incompetent. BL-2 is appropriate for many constructs, while higher levels may be required depending upon the construct. During production phase of the vector, BL-2 is appropriate until replication incompetence is demonstrated.
• Animal Research Considerations:
  • Housing and husbandry: ABL-1 Contact with feces or urine from infected animals for 72 hours post infection. Contact with tissues and body fluids of infected animals.
  • Vector Administration: ABL-1 or ABL-2 depending on the construct/pseudotyping
• Disinfection:
  • 10% bleach (recommended)

Retroviruses/Lentiviruses

Human: HIV; Animals: EIV, FIV, SIV, etc.

Risks of Lentiviral vectors: HIV-AIDS in humans.

• General Criteria for a risk assessment:
  • Host Range: Humans and animals
  • Zoonosis: Suspected in HIV
  • Clinical Manifestation: Insidious onset with non-specific symptoms such as lymphadenopathy, anorexia, chronic diarrhea, weight loss, fever, and fatigue; opportunistic infections and malignant diseases without a known cause for immune deficiency
  • Infectious Dose: Unknown
  • Cell types able to be infected transduced: dividing and non-dividing cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, muscle and liver cells
  • Transgene Considerations: Is the transgene oncogenic? If so, increased risk.
• Host-vector systems:
  • Vector information: Lentiviral vectors integrate into the host genome and may cause insertional mutagenesis as lentivirus tends to insert in transcriptionally active sites. Commercial lentiviral vector systems that include specific genes (gag, pol, env) for virulence or pathogenicity on 2, 3 or 4 plasmids lower the risk for recombination and the production of replication-competent HIV thereby providing greater safety to the researcher.
  • Is the vector on multiple plasmids separating vector and packaging functions?
  • How many plasmids? (e.g. 2 plasmid system (increased risk) vs. 4 plasmid (less risk) Commercial systems are available with 2, 3, 4 plasmid systems.
  • What is the potential for generation of replication competent virus from vectors by recombination? 4 plasmid systems are less risky
  • Are certain viral genes deleted thereby increasing the safety of the system? Some commercial systems have essential viral genes deleted, e.g. Tat (required for replication)
  • Envelope Protein in the packaging system: The lentiviral particles are often pseudotyped with the envelope of the vesicular stomatitis virus (VSV), which extends the host range to introduce genes into a broad range of tissues and can be used in vivo.
• General containment considerations:
  • BL-2 with a 4 plasmid system
• Animal research considerations:
  • Housing and husbandry: ABL-2 for animals xenografted with human cells or animal cells permissive for HIV-1.
  • Is the animal a permissive host or non-permissive host for the viral vector? Wild type mice are not permissive and therefore, are a system of containment themselves.
  • Animal engrafted with permissive cells: ABL-2
  • Vector Administration: ABL-2
• Disinfection:
  • 10% bleach (recommended), 2% glutaraldehyde, 4% formaldehyde, 70% ethanol

Adenovirus

Risks of Adenovirus vectors: Infections and, in rare cases, severe disease.

• General criteria for a risk assessment:
  • Host Range: Humans and animals
  • Zoonosis: Yes, for specific strains
  • Clinical Manifestation: Infections of the upper respiratory tract, conjunctivitis, tonsillitis and gastroenteritis (stomach flu). Rare cases of severe disease can occur.
  • Infectious Dose: >150 plaque forming units when given intranasally
  • Cell types able to be infected/transduced: Ad vectors can infect a variety of non-dividing cells and elicit high levels of transgene expression. Ad attaches to the coxsackievirus and adenovirus receptor (CAR) present in many human cells.
  • Transgene Considerations: Is the transgene oncogenic or biotoxic? If so, risk is increased.
• Host-vector systems:
  • Vector information: Adenovirus does not normally involve integration into the host genome, rather Ad replicates as episomal elements in the nucleus of the host cell consequently there is minimal risk of insertional mutagenesis. Newer commercial vectors have many built-in safety features in Ad systems, such as deletion of viral genes to limit homologous recombination or infectivity. However, transfected Ad vectors can be expressed in cells, cause lysis and produce infection.
  • Are the vector and packaging functions separate? Newer Ad vectors are “gutless” with only the long terminal repeats and a packaging sequence of the virus. All the necessary viral genes provided in trans either by a helper virus, plasmid or integrated into a helper cell genome (human embryonic kidney cells, line HEK 293).
  • What is the potential for generation of replication competent virus from vectors by recombination? Recombination can occur between (1) the viral sequences found in cotransfected plasmids and (2) the DNA of the packaging cell line and the vector to reconstitute infectious Ad particles. Adenovirus is recombinogenic and the use of 293 cells in conjunction with E1-delected adenoviral vector leads to a systematic rescue of wild type adenovirus by a process that involves only a single recombination event.
• Are certain viral genes deleted thereby increasing the safety of the system? e.g.
  • E1 or E3 essential genes for replication and, if deleted, decrease risk but are not sufficient to drop the containment level to BL-1.
• General containment considerations:
  • BL-2 containment; Adenovirus is quite stable in the environment. No open bench work. Face protection (goggles/face shield) must be worn when working with the agent/vector. Adenovirus does not have to be replication-competent to cause corneal and conjunctival damage.
• Animal research considerations:
  • Housing and husbandry: ABL-2 housing for 48-72 hours post injection/exposure. Adenovirus is excreted by infected animals.
  • Is the animal a permissive host or non-permissive host for the viral vector? In general, Ad5 vectors transduce human cells (with CAR receptors) quite well, whereas mouse cells are mostly refractory to Ad5.
  • Animal host or animals engrafted with cells permissive for adenovirus: ABL-2
  • Vector Administration: ABL-2 during injection
• Disinfection:
  • 10% bleach (recommended), 5% phenol Note: Alcohol is not an effective disinfectant against adenovirus species.

Adeno-Associated virus (AAV)

Risks of AAV vectors: Recent events suggest that AAV vectors may be associated with insertional mutagenesis and cancer, and therefore, may not be as safe as previously thought. AAV can preferentially insert at a specific site on human chromosome 19 and remain latent. Potentially at a later time when a helper virus is present, AAV can be reactivated and produce infection. Some characteristics of AAV may increase risk, including the following: detection in embryonic tissue, as association with male infertility, and the ability to replicate in some cases without a helper virus.

• General criteria for a risk assessment:
  • Host Range: Humans and animals
  • Zoonosis: AAV is not known to cause direct disease in humans.
  • Clinical Manifestation: None
  • Infective Dose: Unknown but can be aerosol transmitted
  • Cell types able to be infected/transduced: A wide range of host of human and non-human cell lines cells which can be dividing and non-dividing.
  • Transgene Considerations:Is the transgene oncogenic or biotoxic? If so, increased risk.
• Host-vector systems
  • Vector information: The long terminal repeats flank the transgene, and the AAV Rep and Cap and helper virus genes are provided in trans. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome so expression of the transgene is long term and stable. In humans AAV inserts preferentially at a specific site on chromosome 19.
  • Helper virus: Adenovirus, herpesvirus, vaccinia virus or CMV, for replication
  • Deletion of specific genes: Allows AAV to insert specifically in other chromosomal sites
• General containment considerations:
  • BL-1and BL-2, strain specific
• Animal Research Considerations:
  • Housing and husbandry: ABL-1. When a known helper virus is present or the host animal may potentially contain virus that could act as a helper (e.g. mice replete with retroviruses) the work must be done under ABL-2 containment.
  • Is the animal a permissive or non-permissive host for the viral vector? Mice are permissive.
  • Vector Administration: ABL-2 if a helper virus is present.
• Disinfection:
  • 10% bleach (recommended)

Herpesvirus/Gammaherpesvirus/Epstein Barr (EB)

Risks of EB vectors: Human cancer

• General Criteria for a risk assessment:
  • Host Range: Humans or animals
  • Zoonosis: None
  • Clinical Manifestation: Epstein-Barr virus (EBV), a ubiquitous B-lymphotrophic herpesvirus, causes mononucleosis and is a common childhood disease worldwide; Burkitt's tumour is worldwide but hyperendemic in highly malarial areas such as tropical Africa; carcinoma is worldwide but highest in Southeast Asia and China, especially in males; the tumor cells of a heterogeneous group of malignancies (Burkitt's lymphoma, lymphomas associated with immunosuppression, other non-Hodgkin's lymphomas, Hodgkin's disease, nasopharyngeal carcinoma, gastric adenocarcinoma, lymphoepithelioma-like carcinomas, and immunodeficiency-related leiomyosarcoma). EBV is a transforming virus and can immortalize B-cells and cause lymphoma in various animal models
  • Infectious Dose: Unknown
  • Cell types able to be infected transduced: Human B cells and nasopharyngeal cells
  • Transgene Considerations: Is the transgene oncogenic or biotoxic? If so, increased risk.
• Host-vector systems:
  • Vector information: The genes of EB are used to immortalize episomal plasmids that can express genes of interest in human cells. Several hybrid vector systems, including Ad-EBV, HSV-EBV, and retrovirus-EBV, have been constructed.
• General containment considerations:
  • EB is often used to produce immortalized cell lines and should be worked with under BL-2 containment.
• Animal Research Considerations:
  • Housing and husbandry: ABL-2
  • Is the animal a permissive or non-permissive host for the viral vector? EBV replicates poorly in rodents unless a mammalian origin of replication is inserted.
  • Animal host or animals engrafted with permissive cells: ABL-2
  • Vector Administration: ABL-2
• Disinfection:
  • 10% bleach (recommended), 70% ethanol

Herpesvirus/Type A/Herpes simplex virus type-1 and type-2

Risks of HSV vectors: Infections in humans, mild to severe. Therapeutic medications are available.

• General Criteria for a risk assessment:
  • Host Range: Human and animals
  • Zoonosis: None
  • Clinical Manifestation: Classic presentation of primary HSV-1 is herpes gingivostomatitis - oral mucosa, HSV 1 - primary infection usually mild (10% of cases can be severe) and in early childhood; reactivation of latent infection results in fever blisters or cold sores, usually on the face and lips which crust and heal within a few days, may be CNS involvement (meningoencephalitis), 70% mortality rate if left untreated; causes about 2% of acute pharyngotonsillitis; Classic presentation of a primary HSV-2 infection is herpes genitalis, HSV 2 - genital herpes, sexually transmitted, associated with aseptic meningitis, vaginal delivery can cause risk to newborn, encephalitis and death; HSV-1 and HSV-2, either may infect the genital tract or oral mucosa.
  • Infectious Dose: Not known
  • Cell types able to be infected transduced: The vectors have a wide host range and cell tropism in dividing and non-dividing cells (e.g. neurons), infecting almost every cell type in most vertebrates that have been examined.
  • Transgene Considerations: Is the transgene oncogenic or biotoxic? If so, increased risk.
• Host-vector systems:
  • Vector information: Herpes simplex virus (HSV) vectors have some unique features that categorizes them as either replicating, non-replicating or “gutless”. In addition, the natural property of the virus to infect and establish latent infection indefinitely in post-mitotic neurons has generated substantial interest in using it to deliver therapeutic genes to the nervous system.
  • What is the potential for generation of replication competent virus from vectors by recombination? Recombination can rescue mutations or deletions producing infectious virus
  • Are certain viral genes deleted thereby increasing the safety of the system? Commercial vector systems have specific constructs with many viral genes deleted so much so, that a helper virus is required for packaging to produce replication competent virus.
• General containment considerations:
  • BL-2 containment
• Animal Research Considerations:
  • Housing and husbandry: ABL-2
  • Animal host or animals engrafted with permissive cells: ABL-2
  • Vector Administration: ABL-2
• Disinfection:
  • 10% bleach (recommended), 70% ethanol

Poxviridae/Vaccinia

Risks of VV vectors: A specific strain of vaccinia has been used as the vaccine for Smallpox, a disease that has been eradicated from the world. There are many strains with varying virulence in animals as well as humans. The potential for human infection from a localized skin infection to severe systemic infection exists. Prophylactic vaccination is available and recommended (but not required) if working with certain strains but not recommended for highly attenuated strains. Certain individuals are more likely to experience severe side effects. Please consult your personal physician and the following website for advice: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1.htm

• General Criteria for a risk assessment:
  • Host Range: Humans
  • Zoonosis: Anthropozoonotic; no animal reservoir.
  • Clinical manifestation: Virus disease of skin induced by inoculation for the prevention of smallpox - vesicular or pustular lesion, area of induration or erythema surrounding a scab or ulcer at inoculation site. A systemic infection can cause major complications such as encephalitis, progressive vaccinia (immunocompromised susceptible), eczema vaccinatum - a localized or systemic dissemination of vaccinia virus, fetal vaccinia; minor complications - generalized vaccinia with multiple lesions; auto-inoculation of mucous membranes or abraded skin, benign rash, secondary infections; complications are serious for those with eczema or who are immunocompromised; death is most often the result of postvaccinial encephalitis or progressive vaccinia
  • Infectious Dose: Vaccines have potency of 108 pock-forming units/ml; infectious dose unknown
  • Cell types able to be infected transduced: All mammalian cell types
  • Transgene considerations: The containment for work with this vector may be changed due to the expressed protein being carried by the vector. Particular consideration should be given to developing a recombinant-host system that is more virulent than wild type.
• Host-vector systems:
  • Vector information: No helper system is used for this viral system and VV does not integrate into the host genome.
• General containment considerations:
  • BL-2 All procedures should be conducted in a certified biosafety cabinet.
• Animal Research Considerations:
  • Housing and husbandry: ABL2
  • Animal work: If a procedure can not be performed in the biosafety cabinet (i.e. surgery) the following PPE is required: Lab coat, gloves, full face shield or the equivalent.
  • Serious ocular infections can occur even in vaccinated individuals
  • Is the animal a permissive host or non-permissive host for the viral vector? Naturally or experimentally infected laboratory animals are a potential source of infection to exposed unvaccinated laboratory personnel. Serious ocular infections can occur even in vaccinated individuals.
  • Vector Administration: ABL-2
• Disinfection:
  • 10% bleach (recommended), 70% ethanol

Viral Vector Table

Viral Vector	Risk Group	Hazard	Biosafety Level	Animal Biosafety Level	Disinfection
Murine Retrovirus (Ecotropic)	1	Injection, splash to face	1	ABL-1	10% bleach (recommended)
Murine Retrovirus (amphotropic or pseudotyped)	2	Injection, splash to face	2	ABL-2 (with special consideration for transgenes, toxins, oncogenes, elements that alter host range, etc.)	10% bleach (recommended)
Lentivirus	2	Injection, splash to face	2	ABL-2 (if animals or cells are permissive)	10% bleach (recommended), 2% glutaraldehyde, 4% formaldehyde, 70% ethanol
Adenovirus	2	Droplets, aerosol, injection	2	ABL-2 (49-72 hours post injection/exposure)	10% bleach (recommended), 5% phenol Note: Alcohol is not an effective disinfectant against adenovirus species.
Adeno-Associated virus (AAV)	2		2 (helper virus), 1 no helper virus	ABL-1 if no helper virus, ABL-2 with a helper virus or other special considerations	10% bleach (recommended)
Herpesvirus I and II	2	Aerosols, injection	2	ABL-2	10% bleach (recommended), 70% ethanol
Epstein Barr	2	Aerosols, injection	2	ABL-2	10% bleach (recommended), 70% ethanol
Vaccinia	2	Aerosols, Splash to face (Severe ocular infection), droplets, injection	2	ABL-2	10% bleach (recommended), 70% ethanol

Questions?

Contact the Division of Research Safety, Biological Safety Section (333-2755 or via e-mail) or visit our website: http://www.drs.illinois.edu/bss/.

Other Biosafety Facts Sheets are available from the Biological Safety Section at our website: http://www.drs.illinois.edu/bss/factsheets/.

Quick Links

• NIH Guidelines for Recombinant DNA and Gene Transfer
• Biosafety in Microbiological and Biomedical Laboratories
• ABSA