Interim Risk Assessment and Biosafety Level Recommendations for Working With Influenza A(H7N9) Viruses

Prior to working with a pathogen in the laboratory, health and environmental related risks associated with its manipulation must be assessed. Biological risk assessment primarily focuses on the prevention of laboratory-acquired infections and unintended release of the pathogen into the environment. Due to the identification of human infections with a novel influenza A(H7N9) virus in China in March-April 2013, it is very important to conduct studies to better understand the extent of the outbreak; the properties of the wild type virus and its mode(s) of transmission; develop appropriate diagnostic tools and methods; and determine appropriate prevention and control strategies for veterinary and public health.

The preliminary data on the risks associated with this virus and type(s) of containment criteria to be applied when handling this agent are discussed herein. Some of the factors used to determine risk included the:

• pathogenicity of the agent,
• agent’s route of transmission,
• stability of the agent,
• infectious dose or concentration of the agent,
• origin of the agent,
• availability of effective prophylaxis against the agent (as per Biosafety in Microbiological and Biomedical Laboratories, 5^th Edition, 2009 available at https://www.cdc.gov/biosafety/publications/bmbl5/index.htm [BMBL]), and
• availability of the use of antiviral agents for treatment of human infections with influenza A(H7N9).

Additional information on these factors and interim guidance on the use of antiviral agents for treatment of human infections with influenza A(H7N9) is available. In the United States, biosafety and biocontainment best practice for the protection of the worker and public health is codified in the BMBL.

The recommendations provided herein from U.S. Department of Agriculture (USDA)’s Animal and Plant Health Inspection Service (APHIS) and U.S. Department of Health and Human Services (HHS)’s Centers for Disease Control and Prevention (CDC) are based on biosafety levels established in the BMBL with added enhancements to protect laboratory workers, the environment, and the public. Based on economic ramifications to agriculture and the source of the virus, additional APHIS containment requirements and personnel practices and/or restrictions may be required for those individuals working with wild type influenza A(H7N9) virus.

It should be noted that this risk assessment and its recommended laboratory requirement is for wild type influenza A(H7N9) viruses, and/or synthetic constructs containing any portion of the coding regions of all eight gene segments of the wild type virus. This guidance does not apply to work done with vaccine seed strains.

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Agent-Associated Risk Assessment Factors

Pathogenicity

The terms “low pathogenic avian influenza (LPAI)” viruses and “highly pathogenic avian influenza (HPAI)” viruses describe the severity of influenza disease in chickens. Novel influenza A(H7N9) is classified as a “low pathogenic avian influenza (LPAI)” since intravenous pathogenicity index test data indicates that infections in chickens are sub-clinical. However, novel influenza A(H7N9) viruses cause disease in human populations. It is important to note that the terms “low pathogenic avian influenza (LPAI)” viruses and “highly pathogenic avian influenza (HPAI)” do not necessarily correlate to the severity of disease in humans. Previous human infections with LPAI H7 subtype viruses have been rare (H7N2, H7N3 or H7N7) (Kurtz et al., 1996; Tweed et al., 2004; Nguyen-Van-Tam et al., 2006; Skowronski et al., 2006; Euro Surveillance 2007; Ostrowsky et al., 2012). These human infections resulted in symptoms of mild to moderate respiratory illness and/or conjunctivitis. As of May 10, 2013, 131 human cases of influenza A(H7N9) virus infection have been reported from multiple provinces in China (World Health Organization). The median age of the cases is approximately 60 years and most cases have experienced severe respiratory illness. There have been 24 reported deaths. Influenza A(H7N9) viruses have been detected in multiple avian species at live bird markets in Chinese provinces associated with human infections with influenza A(H7N9) virus. Many of the people infected with H7N9 are reported to have had contact with poultry. However, some cases reportedly have not had such contact and the exposure histories for many cases are unknown. Both animal-to-human and human-to-human routes of transmission are being actively investigated and currently there is no evidence of sustained human-to-human transmission of this influenza A(H7N9) virus. However, limited non-sustained human-to-human H7N9 virus transmission could not be excluded and remains a possibility in a few case clusters in family members (Li et al., 2013).

The influenza A(H7N9) viruses are novel reassortants that have surface glycoproteins [hemagglutinin (HA) and neuraminidase (NA)] that belong to the Eurasian avian influenza lineage and internal genes from the Eurasian H9N2 avian influenza lineage. These influenza A(H7N9) viruses do not contain a multi-basic cleavage site associated with HPAI viruses. Pathogenicity testing in chickens has confirmed that these viruses are LPAI. The presence of internal H9N2 genes suggests that these viruses are capable of replicating efficiently in poultry. However, the viruses also have several molecular features that suggest that some level of adaptation to mammalian species has occurred. At this time, the level of virulence for laboratory animals (mice and ferrets) is under evaluation.

Route of Transmission

There are different modes of transmission of influenza viruses among persons (Weber et al., 2008). Contact transmission includes direct contact and indirect contact with contaminated surfaces. Droplet transmission can occur either via large particles or small particles (also called droplet nuclei) dispersion through the air. Large or small particle aerosols of virus-laden respiratory secretions are expelled into the air from an infected individual during breathing, coughing, or talking (Milton et al., 2013; Lindsley et al., 2010; Stelzer-Braid et al., 2009). At this time, the transmissibility of H7N9 between mammals in the laboratory is under investigation.

Stability

Although influenza is regarded as an airborne infection, its ability to survive on surfaces is important in determining the appropriate level of containment. Infectious influenza virus has been maintained on surfaces for up to two weeks (Derrick and Edward, 1941; Bean et al., 1982; Wood et al., 2010) and in water for up to seven months (Stallknecht et al., 1990). Due to the potential for transmission of influenza A(H7N9) viruses by aerosol droplets, combined with its potential to remain viable in dry and liquid form for extended periods of time, proper safeguards should be in place to ensure protection of laboratory personnel and the environment.

Infectious Dose

The dose of a pathogen required to produce disease in a human or animal is an important factor in its biological risk assessment. Agents that produce disease at lower doses represent higher risks to individuals working with them. Influenza A(H7N9) viruses are known to grow to high titers in embryonating eggs and based on prior studies with other H7 subtype viruses, these viruses are expected to replicate to high titers in animals [plaque forming units (pfu) or 50% egg infectious dose (EID50) ≤108/ml]. The infectious dose for humans or poultry is unknown at this time.

Origin

The primary reservoir for influenza viruses classified as “H7” is waterfowl. Other hosts of the H7 subtype viruses can be the terrestrial, domesticated bird populations (chickens and turkeys). The animal reservoir of the H7N9 viruses and the mode of transmission that has occurred to infect humans in China remain unknown at this time, although poultry in live bird markets is highly suspected.

Availability and Efficacy of Vaccines

Currently, there are no licensed vaccines against H7 subtype viruses. Vaccination remains the optimal method for protection of individuals against influenza. Optimal efficacy is provided by influenza vaccines containing virus antigen well-matched to the viruses causing disease. Since the H7 HA is a novel glycoprotein not previously found in viruses that have circulated in humans, it is expected that there will be no neutralizing antibodies present in human populations. Several pre-pandemic H7 subtype vaccines have been developed and have undergone pre-clinical and phase I clinical testing. Two different inactivated vaccines based on Eurasian H7 viruses have been evaluated in small clinical trials. Both vaccines were poorly immunogenic in humans, inducing little or no serum antibodies to the H7 HA in adults that received two doses of vaccine even when high dose formualtions or an alum-adjuvanted vaccine was used (Cox et al., 2009; Couch et al., 2012). A live attenauted H7N3 influenza vaccine (LAIV) has also been assessed in adults and two doses were shown to elicit a neutralizing antibody response in approximately 50% of subjects (Talaat et al., 2009).

Availability and Efficacy of Therapeutic Agents

Laboratory testing conducted in China has shown that the influenza A(H7N9) viruses are sensitive to the anti-influenza drugs known as neuraminidase inhibitors (oseltamivir and zanamivir). When these drugs are given early in the course of illness due to other types of influenza A viruses (e.g., H1N1), they have been found to be effective in shortening the time to symptom improvement in otherwise healthy persons with typical acute uncomplicated influenza illness. Observational studies suggest that antiviral treatment of hospitalized patients with influenza may reduce complications associated with infection (e.g., pneumonia and ICU use) and mortality. However, at this time there is insufficient information to assess the effects of use of these drugs for the treatment of infection with influenza A(H7N9) virus. Interim guidance on the use of antiviral agents for treatment of human infections with influenza A(H7N9) is available.

Select Agent Status and Permitting Requirements

HHS has not determined if it will propose that the influenza A(H7N9) viruses should be regulated as a HHS select agent under 42 CFR Part 73. Based on the known sequence data and epidemiology, this influenza A(H7N9) virus will be handled as a LPAI and not subject to USDA select agent requirements. However, USDA and CDC permitting requirements for etiological agents for animals and humans, respectively, are in effect and specific applications to USDA and CDC must be made for importation or interstate movement of influenza A(H7N9) virus. Previous permits issued for general LPAI isolates are not applicable for influenza A(H7N9) viruses at this time. Issuance of a permit to work with this agent will be contingent upon adherence by the facility to the biosafety and biocontainment guidance contained in this document.

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Biosafety and Biocontainment Recommendations

Prerequisites for Staff Prior to Working in Laboratories with Influenza A (H7N9)

All staff working with influenza A(H7N9) viruses should be enrolled in a medical surveillance program that meets the requirements outlined below as well as the facility’s institutional policy. Serum samples may be collected in accordance with institutional policy and procedures for collection of serum before or immediately after a potential occupational exposure with convalescent serum samples three weeks after potential exposure. Unless an absolute medical contraindication exists, personnel are required to receive the current seasonal influenza vaccine. Immunization with seasonal influenza vaccine may reduce the chance of the possible reassortment of human influenza viruses with avian influenza viruses, which could result in a virus that more easily infects humans. Therefore, the facility’s institutional policy should be considered when an individual is unable to receive the vaccine.

Due to the potential for aerosol inhalation and the pathogenicity of influenza A(H7N9) viruses for humans observed to date, respiratory protection is mandatory for activities conducted at Biosafety Level 3 (BSL-3) or when formation of aerosols are anticipated. A primary prerequisite prior to wearing respiratory protection for activities conducted with influenza A(H7N9) viruses is enrollment in the institution’s Respiratory Protection Program followed by demonstrating competence in wearing a HEPA-filtered powered air-purifying respirator (PAPR), or a properly fit tested full face respirator with HEPA or N95 particulate protection. The selection of an appropriate respirator depends upon the type of activity anticipated in the laboratory. Training in the effective use of respirators is mandatory and requires yearly certification by your institution’s occupational health and safety program.

To prevent the potential spread of this virus to birds, personnel working in the BSL-3 laboratory with influenza A(H7N9) viruses must avoid contact with domestic or wild birds. All laboratorians working with influenza A(H7N9) viruses should comply with USDA policy of having no contact with any avian species or their housing when away from the workplace for at least five days after the last day of work on this virus in the laboratory. This includes home pets (e.g., canaries, parakeets, parrots) and poultry on farms and in backyards.

Laboratory Design and Containment Equipment

The primary factor in designing a biocontainment laboratory is protection of laboratory workers, the environment, and the public from accidental exposure to potentially biohazardous organisms. Until more comprehensive information is available on the transmissibility, mortality, and pathogenicity of influenza A(H7N9), primary and secondary barriers must be in place in order to prevent accidental personnel exposure to or environmental release of influenza A(H7N9) viruses. Primary barriers include laboratory equipment, such as biological safety cabinets and containment centrifuges, designed to protect the laboratory personnel. Secondary barriers include facility features such as specialized air handling and waste decontamination systems that protect the environment outside the laboratory. Primary barriers are explained below with special emphasis on proper laboratory procedure when handling influenza A(H7N9) virus. In a BSL-3 laboratory, biological safety cabinets (Class II or III) or other appropriate combinations of physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, and containment caging for animals) are used for all activities with infectious materials.

Laboratory Decontamination

1. Decontamination with an appropriate disinfectant is required:
   1. At the beginning and end of diagnostic or research activities with influenza A(H7N9).
   2. When the lab is decontaminated for annual maintenance.
   3. When personnel not authorized to work with infectious agents are assigned to the suite (e.g., for maintenance of equipment).
   4. When a major spill occurs outside of primary containment.
2. Individual biosafety cabinets (all interior surfaces) and containment centrifuges should be decontaminated with an Environmental Protection Agency approved disinfectant that has been validated for efficacy against avian influenza virus whenever diagnostic or research activities have been concluded.
3. To avoid inadvertent cross contamination (increasing the risk of unintended reassortment), work areas should be decontaminated between experiments. Supervisors should ensure that protocols contain strict measures for physical and/or temporal separation of virus strains.

Required Laboratory Biosafety Levels for Individuals Working with Influenza A(H7N9) Virus

Based on the available information regarding agent virulence in humans, and availability of antiviral agents for treatment of human infection, together with the potential of this exotic avian virus to spread in U.S. poultry, HHS/CDC and USDA/APHIS recommendations are that all in-vitro work (i.e., tissue culture) with the influenza A(H7N9) virus should be conducted within BSL-3 laboratories [BMBL]. Animal work involving this virus must be performed in Animal BSL-3 (ABSL-3) with enhanced practices [BMBL]. In addition to the standard practices, special practices, safety equipment, and facility requirements for BSL-3 and ABSL-3 laboratories provided in the BMBL, laboratories in which work with influenza A(H7N9) viruses is conducted must also maintain certain defined engineering enhancements, personal protective equipment (PPE) enhancements, and additional special practices. Specific recommendations are provided below:

• Engineering Enhancements
  • Air handling: Dedicated, single pass, directional, and pressure gradient ventilation systems must be used. Supply and exhaust air to and from the containment space is HEPA-filtered. The exhaust system should have a sealed ductwork system from the containment barrier to the filter. The supply and exhaust air handling systems must be interlocked in such a way as to prevent positive excursion. Air supply and exhaust systems must be independent.
  • Showers: A gown-in, shower-out procedure is used to enforce a change of street clothing. The value of forcing a change of clothing is to reduce the risk of fomite transmission of this highly contagious agent. Ideally, personal showers should be located at the containment/non-containment interface.
  • Decontamination of laboratory liquid effluents: Liquid effluents originating from laboratories should be collected locally and chemically disinfected or heat treated, or collected and processed in a central effluent decontamination system before being released into the local sanitary system. The decontamination of shower and toilet effluents is not a requirement, provided appropriate practices and procedures are in place for primary containment.
• PPE Enhancements
  • The use of adequate respiratory protection is required to guard against particulate challenge, even when work is conducted in a primary containment device (such as a Class II biological safety cabinet). Adequate respiratory protection should be selected based upon procedural risk assessment and must include a HEPA-filtered powered air pressure respirator (PAPR) or a properly fitted full face respirator with HEPA or N95particulate protection and head covering. PAPRs with shroud-type hoods are recommended when practical as these provide the highest levels of protection and the least amount of stress to the worker. Individuals using respiratory protection must be adequately trained and fit-tested in accordance with 29 CFR 1910.134. A disposable hood or head cover may be used depending upon the type of activity anticipated in the laboratory.
  • In addition to standard solid front BSL-3 protective clothing (tie back-or wrap around gowns, scrub suits, or coveralls), shoe covers and double gloves are worn and double gloves are used for all manipulations of virally infected materials.
• Additional Special Practices
  • A personnel isolation policy should be in place for all laboratory workers prohibiting all laboratory staff and visitors from having contact with susceptible avian species for a minimum of five days after the last possible exposure to influenza A(H7N9) virus. Laboratory workers should sign a written statement stating they understand and will abide by this policy.
  • An occupational health program should be in place for laboratory personnel. It is recommended that the occupational health plan include: 1) procedures for collection of serum before or immediately after a potential occupational exposure with convalescent serum samples three weeks after potential exposure; and 2) protocols for the medical evaluation, treatment and method of patient isolation (if necessary) of individuals with potential exposures.
  • Continued susceptibility of the influenza A(H7N9) virus to antiviral agents shall be established by sequence analysis or suitable biological assays. After manipulation of genes that influence sensitivity to antiviral agents, susceptibility to these agents shall be reconfirmed.
• ABSL-3 practices, procedures, and facilities are recommended for work involving animals plus specific precautions and procedures to include:
  • The use of primary containment caging systems.
  • Rigorous adherence to the use of negative pressure suites with HEPA-filtration treatment of exhaust air.
  • Specific decontamination methods for all infectious materials (solid and liquid waste).
  • Clothing change protocols and personal showers prior to exiting the vivarium, or movement to any animal room not used for research with avian influenza A(H7N9) virus.
  • The use of adequate respiratory protection to guard against particulate challenge even when work is conducted in a primary containment device (such as a Class II biological safety cabinet). Adequate respiratory protection must include a PAPR or a full face respirator with HEPA or N95 particulate protection and head covering. PAPRs with shroud-type hoods are recommended when practical as these provide the highest levels of protection and the least amount of stress to the worker. Individuals using respiratory protection must be adequately trained and fit-tested in accordance with 29 CFR 1910.134. A disposable hood or head cover may be used depending upon the type of activity anticipated in the laboratory.
  • An occupational health program should be in place for laboratory personnel. It is recommended that the occupational health plan include: 1) procedures for collection of serum before or immediately after a potential occupational exposure with convalescent serum samples three weeks after potential exposure; and 2) protocols for the medical evaluation, treatment and method of patient isolation (if necessary) of individuals with potential exposures.
  • A personnel isolation policy should be in place for all laboratory workers prohibiting all laboratory staff and visitors from having contact with susceptible avian species for a minimum of five days after the last possible exposure to influenza A(H7N9) virus. Laboratory workers should sign a written statement stating they understand and will abide by this policy.
• Specific ABSL-3 animal handling protocols must be developed by the facility. Prior to manipulation of animals, the animals must be adequately restrained (either physically or chemically) for the full duration of the procedure. All manipulations of the virus must be conducted inside a primary containment device such as a certified Class II biosafety cabinet. Laboratory animals should be housed within primary containment caging and, where possible, manipulated within Class II or Class III biosafety cabinets.
• In addition to procedures specified above, work with loose-housed or large animals (e.g., swine) must occur in BSL-3 Ag containment spaces designed for large animals and minimally have the following enhancements:
  • Rigorous adherence to the use of negative pressure suites with HEPA-filtration treatment of exhaust air (including vents).
  • Collection and treatment (heat or chemical) of all liquid effluents (except shower run-off and toilet discharge) originating from the containment area prior to discharge.
  • Heat treatment or equivalent decontamination process of all solid animal wastes and tissues prior to leaving the containment area.
  • Shower out for all personnel exiting the containment area.
• Proper training is the most important aspect of biosafety and is the responsibility of the Principal Investigator. A high degree of competence in handling human influenza viruses in BSL-2 conditions must be demonstrated prior to allowing an individual to work in BSL-3 or ABSL-3 conditions with these agents. Specific, documented training unique to the BSL-3 or ABSL-3 laboratory practices and procedures must be provided and coordinated through the institutional biosafety office.
• For work with this agent under ABSL-3 conditions individuals should have appropriate experience as determined by the laboratory supervisor and training in animal handling with the species used in the study.
• Access to a BSL-3 laboratory or animal area must be strictly controlled. Access is given only to staff members and individuals who have a work-related need to be in the laboratory.

Recommended Biosafety Level for Individuals Working with Diagnostic Specimens from Patients or Animals Suspected of Novel Influenza A(H7N9) Virus Infection

• Processing of samples and/or performing non-culture based diagnostic testing on clinical specimens from patients and animals with suspected influenza A(H7N9) virus infection may be performed in a Biosafety level (BSL)-2 laboratory as described in the BMBL with enhancements as follows: 1) laboratory personnel have specific training in handling pathogenic and potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents; and 2) the laboratory should have special engineering and design features to ensure directional airflow from clean to potentially contaminated areas. Supervisors are responsible for ensuring that laboratorians are properly trained to work safely in the laboratory. Laboratory safety procedures include all standard BSL-2 procedures and in addition require the following enhancements:
  • Special Microbiological Practices
    • All personnel must wear a disposable, wrap-around gown, shoe covers, an N95 respirator with eye protection or a face shield, or PAPR, and must double glove inside the room before beginning work. Individuals must be appropriately trained and those wearing N95 respirators fit-tested in accordance with 29 CFR 1910.134 (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=12716&p_table=standards).
    • Upon exiting the laboratory room, one must discard both pairs of gloves, shoe covers, gowns and respirator in a biohazard bag inside the room. Personal protective equipment (PPE) is not to be worn outside of the laboratory and must be removed prior to exiting the laboratory.
    • Eyeglasses worn while working in the BSL-2 laboratory without a face shield must be washed thoroughly with a germicidal soap before taking them out of the laboratory
    • All activities involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the laboratory. No work in open vessels is conducted on the open bench.
    • Work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious materials is finished.
• Laboratory materials and equipment to be removed from the laboratory must first be decontaminated with appropriate disinfectants following suggested contact times per institutional biosafety guidelines.
• Viral isolation
  • All growth of virus in cell culture or embryonating eggs from clinical specimens that are suspected cases of novel influenza A(H7N9) virus infection must be performed in a BSL-3 laboratory with BSL-3 practices as outlined above.

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Biosafety in Microbiological and Biomedical Laboratories 5th Edition. HHS Publication No. (CDC) 21-1112, Revised December 2009.