Biological Safety Manual – Chapter 08: Agent Summary Statements (Section IX: Viral Vectors)

Title

Biological Safety Manual – Chapter 08: Agent Summary Statements (Section IX: Viral Vectors)

Introduction

Viral vectors have become a staple of the molecular biology community. As such, it is important to understand the origins of these tools and potential implications of their use. The most commonly used viral vectors are outlined below. Included is information on virology, laboratory hazards, biological safety containment procedures, Personal Protective Equipment (PPE), disinfection, and animal use procedures.

Table of Contents

1. General Containment
2. Viral Vectors and Recombinant Nucleic Acids
3. Viral Vectors as Infectious Agents
4. Use of Cells Transfected by Viral Vectors in Animals
5. Adenovirus Vectors
6. Adeno-Associated Virus (AAV) Vectors
7. Epstein-Barr Virus Vectors
8. Herpesvirus Vectors
9. Retroviruses: Murine Leukemia Virus (MLV) Vectors
10. Retroviruses: Lentivirus Vectors
11. Poxviruses Vectors
12. Replication Competency Testing

General Containment

Suggested biological safety containment levels are provided for each vector system. In some cases, the use of a higher-containment level may be required, depending on the specific properties of the vector and/or insert. The biological safety containment level is ultimately determined by the Institutional Biosafety Committee (IBC) and/or Biological Safety Officer (BSO). Work with risk group 2 or 3 agents/viral vectors (such as Herpesvirus, Adenoviruses, Epstein Barr Virus, Retroviruses, Lentiviral vectors) require IBC review and approval prior to the start of work. Additionally, approval is required for research involving such vectors and animals.

Viral Vectors and Recombinant Nucleic Acids

All work involving recombinant nucleic acids must be registered with EHS via the online lab safety plan and approved by the IBC as required by UNC policy and the National Institutes of Health (NIH). Because UNC receives funding from NIH grants for research involving recombinant nucleic acids, ALL research conducted at UNC must comply with the NIH Guidelines for Research Involving Recombinant DNA Molecules and University policies.

The default biological safety containment level for recombinant viruses is BSL 2/ABSL 2; however, there are exceptions.

• A lower biological safety containment level may suffice for incomplete viruses cultured in vitro.
• A few animal and human viruses qualify for lower biological safety containment.
• Animals with recombinant viruses, which ordinarily require BSL 2 containment may be down-graded to ABSL 1 if and when animals are considered to be no longer shedding virus.

Contact EHS (919-962-5507) if you have any questions regarding containment requirements for viral vectors and/or recombinant nucleic acids.

Viral Vectors as Infectious Agents

Viral vectors, which are commercially acquired and 3rd generation or higher are not considered infectious agents by the UNC IBC, however all vectors still require IBC approval. Viral vectors which are not commercially acquired and/or which are 1st or 2nd generation are considered to be “potentially infectious”, and therefore must be registered as infectious agents.

Use of Cells Transfected by Viral Vectors in Animals

Human or animal cells transfected with 3rd generation (or greater) viral vectors should be washed three (3) times prior to administration, after which, animals injected with these cells may be housed as ABSL-1 containment.

Adenovirus Vectors

There are more than 50 immunologically distinct types of human adenovirus that can cause infection. Recombinant adenoviruses used for biomedical research are based on Adenovirus 5. These are medium-sized (90-100 nm), non-enveloped, icosohedral, double-stranded DNA viruses.

Virus packaged via transfection of HEK 293 cells with adenoviral-based vectors are capable of infecting human cells. The probability of producing replication competent adenovirus (RCA) increases with each successive amplification. RCA is produced when adenoviral DNA recombines with E1-containing genomic DNA in HEK 293 cells.

Potential Health Hazards

Adenoviruses often cause respiratory illness, including the common cold, bronchitis, and pneumonia. Additional clinical symptoms include conjunctivitis (“pink eye”), cystitis, gastroenteritis (stomach flu), tonsillitis, rash-associated illness, and rare cases of severe disease, especially in those with compromised immune systems.

NOTE: Adenoviral vectors do not have to be replication competent to cause corneal and conjunctival damage.

Laboratory Hazards

Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion. Adenovirus is unusually stable in the environment. Adenovirus can still be infectious following extraction with ether and/or chloroform.

Biosafety Containment

• BSL-2
• NO open-bench work
• Biological Safety Cabinet (BSC) required
• Eye protection, disposable gloves, laboratory coat required in addition to BSC.
• When centrifuging adenovirus, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC.

Animal Biosafety Containment

Adenoviral vector must be administered under ABSL-2 containment with the use of a BSC. Animals may shed/excrete adenovirus post-administration. Animals must be housed under ABSL-2 conditions for 72 hours during this period, after which animals may be moved to ABSL-1 housing.

Disinfection

Adenovirus susceptible to: 0.5% Sodium hypochlorite, 2% Glutaraldehyde, 5% Phenol, or Autoclave for 30 minutes at 121°C under 15 lbs. per square inch of steam pressure. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended. Alcohol/ethanol is NOT an effective disinfectant against adenovirus.

Additional Information

Public Health Agency of Canada-Adenovirus PSDS

Adeno-Associated Virus (AAV) Vectors

Adeno-Associated virus (AAV) is most often found in cells that are simultaneously infected with adenovirus. AAV are parvoviridae, icosahedral, 20-25 nm in diameter, single-stranded DNA viruses with a protein capsid. Wild type adenovirus or herpesvirus must be present in order for AAV to replicate Without these helper viruses, AAV will stably integrate into the host cell genome. Co-infection with helper virus triggers a lytic cycle.

Potential Health Hazards

AAV are infectious human viruses however, they are not known to cause direct disease in humans. However, AAV may be associated with insertional mutagenesis and cancer, thereby making AAV possibly not as safe as previously thought. The NIH Guidelines (Appendix B), state that “adeno-associated virus (AAV-all serotypes); and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus can in most cases, be handled safety at BSL 1. Work with other AAV serotypes will be at a minimum, BSL 2 facility. Using AAV in conjunction with Adenovirus or other helper viruses will be performed at BSL2 (and in some cases BSL2+).”

Laboratory Hazards

Inhalation of aerosolized droplets, mucous membrane contact, parenteral injection, or ingestion. There is no specific treatment for infection with AAV.

Biosafety Containment

Construction of AAV should be performed at BSL-2 within BSC. Once constructed, AAV may be manipulated at BSL-1. PPE required for manipulation at BSL-1 OR BSL-2: eye protection, lab coat, disposable gloves.

Animal Biosafety Containment

Animal housing must be maintained at ABSL 1. ABSL 2 if helper virus is present.

Disinfection

Adeno-Associated virus (AAV) susceptible to: 0.5% Sodium hypochlorite, 2% Glutaraldehyde, 0.25% sodium dodecyl sulfate, or Autoclave for 30 minutes at 121°C under 15 lbs. per square inch of steam pressure. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended. Alcohol/ethanol is NOT an effective disinfectant against AAV.

Additional Information

NIH Guidelines Appendix B-I: Risk Group 1 Agents

Epstein-Barr Virus Vectors

Epstein-Barr virus (EBV) is a member of the herpesvirus family and one of the most common human viruses. EBV is found worldwide and most people become infected with the virus sometime during their lives. In the United States, as many as 95% of adults between 35 and 40 years of age have been infected. It is an icosahedral, lipid envelope double-stranded linear DNA virus sized 120-150nm in diameter. EBV has been found in the tumor cells of a heterogeneous group of malignancies (i.e. 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 is often used to produce immortalized cell lines.

Potential Health Hazards

Infectious mononucleosis-acute viral syndrome with fever, sore throat, splenomegaly and lymphadenopathy; lasting one to several weeks; rarely fatal.

Burkitt’s lymphoma-monoclonal tumor of B cells; typically involving children; jaw involvement also common; hyperdemic in highly malarial areas.

Nasopharyngeal carcinoma-malignant tumor of epithelial cells of the nasopharynx; usually involving adults between 20 and 40 years of age.

Laboratory Hazards

Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion.

Biosafety Containment

• BSL-2
• NO open-bench work
• Biological Safety Cabinet (BSC) required
• Eye protection, disposable gloves, laboratory coat required in addition to BSC.
• When centrifuging EBV, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC.

Animal Biosafety Containment

EBV vector must be administered under BSL-2 containment. Animals must be housed as ABSL-2 containment.

Disinfection

Epstein-Barr Virus is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde, formaldehyde. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.

Additional Information

Public Health Agency of Canada-Epstein-Barr Virus PSDS

Herpesvirus Vectors

Herpes Simplex Virus (Types I and II) are icosahedral, lipid enveloped, double-stranded linear DNA viruses approximately 110-200nm in diameter. HSV types I and II can be differentiated immunologically. HSV-I is herpes gingivostomatitis; whereas HSV-II is herpes genitalis, or genital herpes. HSV-derived vectors are unique in that the vectors have a broad host cell range and cell tropism in dividing and non-dividing cells, able to infect almost every cell type in most vertebrates that have been examined.

Potential Health Hazards

Oral herpes-primary infection is typically mild and occurs 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; Possible CNS involvement (meningoencephalitis), 70% mortality rate if left untreated; causes approximately 2% of acute pharyngotonsillitis.

Genital herpes-sexually transmitted, associated with aseptic meningitis; vaginal delivery may pose risk to newborn (encephalitis and death).

Both HSV-I and HSV-II are capable of infecting the genital tract or oral mucosa.

Laboratory Hazards

Inhalation of aerosolized droplets, mucous membrane contact, parenteral inoculation, or ingestion. Only treatment available is anti-viral drug therapy for symptoms.

Biosafety Containment

• BSL-2
• NO open-bench work
• Biological Safety Cabinet (BSC) required
• Eye protection, disposable gloves, laboratory coat required in addition to BSC.
• When centrifuging HSV, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC.

Animal Biosafety Containment

HSV vector must be administered under BSL-2 containment. Animals must be housed as ABSL-2 containment.

Disinfection

HSV is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde, formaldehyde, iodine solutions containing ethanol. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.

Additional Information

Public Health Agency of Canada-Herpes Simplex Virus (HSV) PSDS

Retroviruses: Murine Leukemia Virus (MLV) Vectors

Murine Leukemia Virus (MLV) is an enveloped, icosahedral, single-stranded virus with a linear RNA genome, approximately 100nm in diameter. MLV integrates into the host genome and is present in infected cells as a DNA provirus. Cell division is required for infection.

The host range of recombinant MLV 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 both murine and non-murine cells, including human cells. VSV-G envelope allows infection in a wide range of mammalian and non-mammalian cells.

Potential Health Hazards

Recent data suggests a pathogenic mechanism by which chronic productive retroviral infection allowed insertional mutagenesis leading to cell transformation and tumor formation. The nature of the transgene or additional introduced genetic element(s) may pose additional risk.

Laboratory Hazards

In mice, virus is transmitted via blood from infected mother to offspring; may also occur via germ line infection. In vivo infection in humans appears to require direct parenteral injection with amphotropic or pseudo typed MLV. No recommended treatment.

Biosafety Containment

BSL-1 containment for ecotropic MLV demonstrated to be replication incompetent. PPE required for BSL-1 work: eye protection, lab coat, disposable gloves. BSL-2 containment for amphotropic or pseudotyped MLV. Biological Safety Cabinet (BSC) required. Eye protection, disposable gloves, laboratory coat required in addition to BSC. When centrifuging MLV, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC.

Animal Biosafety Containment

MLV vector must be administered under BSL-2 containment. Animals administered ecotropic MLV may be housed under ABSL-1 conditions. Animals administered amphotropic/pseudotyped MLV must be housed under ABSL-2 conditions for 72-hours post administration, after which animals may be moved to ABSL-1 housing.

Disinfection

MLV is susceptible to: 0.5% sodium hypochlorite, 70% ethanol, glutaraldehyde, formaldehyde, iodine solutions containing ethanol. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.

Additional Information

NIH Guidelines Appendix B-V: Animal Viral Etiologic Agents in Common Use

Retroviruses: Lentivirus Vectors

Lentiviruses are a subset of retroviruses that produce multi-organ diseases characterized by long incubation periods and persistent infection. Lentivirus vector systems can include viruses of non-human origin as well and simian and human viruses. There are five (5) serotypes recognized, based upon the mammalian hosts with which they are associated.

• Bovine lentiviruses: Bovine immunodeficiency virus, Jembrana disease virus
• Equine lentiviruses: Equine infectious anemia virus
• Feline lentiviruses: Feline immunodeficiency virus
• Ovine/caprine lentiviruses: Caprine arthritis-encephalitis virus, Ovine lentivirus, Visna virus
• Primate lentivirus group: Human immunodeficiency virus (HIV) types 1-3, Simian AIDS retrovirus (SRV- 1), Simian immunodeficiency virus (SIV)

Unlike simple retroviruses, lentiviruses, such as HIV, are capable of integrating into host chromosomes and infecting non-dividing cells. This feature was the major impetus for the development of various lentiviral vector-based gene delivery systems, which can deliver large genetic cargo to non-diving cells in vitro and in vivo. The more recent generation vectors have been designed to sufficiently diminish the possibility for recombination to occur resulting in wildtype-potentially infectious virus.

Lentiviral plasmids are based on the genomes of lentiviruses. In most cases, the plasmids have been derived from the HIV-1 genome. HIV lentiviral vectors however have been modified so they are safe to use in research labs. When producing lentiviral particles, three components are typically required:

1. A lentiviral vector or “transfer vector” containing the shRNA or transgene and the flanking LTRs
2. A packaging vector or set of packaging plasmids
3. An envelope vector

The second generation system has one packaging plasmid which includes all the important packaging components: Gag, Pol, Rev, and Tat. To produce virus, a single packaging plasmid, an envelope plasmid, and a transfer vector are required.

The main difference in the 3rd generation system is that there are 4 plasmids in total 2 packaging plasmids, an envelope plasmid, and a transfer plasmid, which are used to generate virus. The 3rd generation packaging system offers maximal biosafety but is more cumbersome to use, as it involves the transfection of four different plasmids in the producer cells.

In many cases, the NIH Guidelines and the Recombinant DNA Advisory Committee (RAC) guidance document on lentiviral vectors explicitly state the containment level.

• NIH Guidelines, Section III-D-3: Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems
• NIH Guidelines, Section III-D-4-a: Experiments Involving Whole Animals
• NIH Guidelines, Appendices B-II-D through B-IV-D: Risk Group Classification of Various Viruses

Potential Health Hazards

Lentiviruses are transmitted via direct exposure to infected bodily fluids. Lentiviruses persist lifelong – being both a function of their ability to integrate into the host chromosome and ability to evade host immunity. Lentiviruses replicate, mutate and undergo selection by host immune responses. The clinical manifestation of infection includes non-specific symptoms such as lymphadenopathy, anorexia, chronic diarrhea, weight loss, fever, and fatigue.

Laboratory Hazards

Direct contact with skin and mucous membranes, parenteral inoculation, ingestion.

Biosafety Containment

• BSL-2+ (for making lentivirus)
• BSL 2 for generating and manipulating lentivirus vectors
• NO open-bench work
• Biological Safety Cabinet (BSC) required
• Eye protection, disposable gloves, laboratory coat required in addition to BSC.
• When centrifuging lentivirus, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC.

Animal Biosafety Containment

Lentiviral vector(s) can be administered under BSL2 containment. Animals must be housed under ABSL-2 conditions for 72 hours during this period, after which animals may be moved to ABSL-1 housing.

Disinfection

Susceptible to: 0.5% sodium hypochlorite, 2% glutaraldehyde, formaldehyde, ethanol. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.

Additional Information

NIH Biosafety Considerations for Research with Lentiviral Vectors

Poxviruses Vectors

Poxviruses are the largest known DNA viruses and are distinguished from other viruses by their ability to replicate entirely in the cytoplasm of infected cells. The core contains a 200-kilobase (kb), double-stranded DNA genome and is surrounded by a lipoprotein core membrane. Poxviruses do not require nuclear factors for replication, and thus, can replicate with little hindrance in enucleated cells.

Recombinant vaccinia vectors-Vaccinia virus can accept as much as 25kb of foreign DNA, making it useful for expression large eukaryotic and prokaryotic genes. Foreign genes can be stably integrated into the viral genome, resulting in efficient replication and expression of biologically active molecules.

Vaccinia is used to generate live recombinant vaccine for the treatment of other illnesses.

Potential Health Hazards

Vaccinia virus normally has no serious health effects in humans, although it can cause disease of the skin when used to inoculate against the smallpox virus. Accidental infection with the virus can occur through contact between the vaccination lesion and broken skin (inadvertent inoculation). Serious complications such as ocular vaccinia, myopericarditis, eczema vaccinatum (a papular, vesicular and pustular rash that is very infectious, 38 cases per million doses), progressive vaccinia (progressive necrosis at the vaccination site, 3 cases per million doses), post-vaccinial CNS disease (headache, lethargy, seizures and coma, 12 cases per million doses), fetus malformations and abortion (very rare) sometimes occur after vaccination.

Laboratory Hazards

Direct contact with skin and mucous membranes, parenteral inoculation, ingestion, inhalation of aerosols.

Biosafety Containment

• BSL-2
• NO open-bench work
• Biological Safety Cabinet (BSC) required
• Eye protection, disposable gloves, laboratory coat required in addition to BSC
• When centrifuging poxvirus, rotors/buckets must be loaded/unloaded within the BSC and wiped down with appropriate disinfectant prior to being removed from BSC
• Consult University Employee Occupational Health Clinic (UEOHC) to determine if the Smallpox vaccine is appropriate for those working with vaccinia

Animal Biosafety Containment

Poxvirus vectors can be administered under BSL2 containment. Animals must be housed under ABSL-2 conditions for 72 hours following infection, after which animals may be moved to ABSL-1 housing.

Disinfection

Susceptible to: 0.5% sodium hypochlorite, 2% glutaraldehyde, formaldehyde, ethanol. Freshly prepared 10% household bleach (0.5% Sodium hypochlorite) recommended.

Additional Information

NIH Biosafety Considerations for Research with Lentiviral Vectors

Replication Competency Testing

A majority of viral vectors used today are disabled such that replication competent viruses are not readily formed by any biological process that might occur in normal hosts. EHS and the IBC encourages the use of such vectors in all relevant applications. In particularly sensitive applications, demonstrating that the viral stock used has no apparent contamination with replication competent vectors is essential. Of course, assays for replication competence will never be perfect or absolute, so the IBC asks that researchers use a current procedure of demonstrated sensitivity and specificity. Below is a summary guide of the current IBC recommendations for common classes of vectors.

If another procedure or reference method is used to accomplish the same conclusion, researchers are asked to submit that procedure and published article with their IBC registration. Even more rigorous testing may be required in some instances, such as a vector bearing a pathogenic gene or in human gene therapy, or in any materials that could be released in the environment.

When completing your IBC protocol, you should not check off that your vector has been tested for replication competency, unless you have documentation.