Evaluation of transmission risks associated with in vivo replication of several high containment pathogens in a biosafety level 4 laboratory

Abstract

Containment level 4 (CL4) laboratories studying biosafety level 4 viruses are under strict regulations to conduct nonhuman primate (NHP) studies in compliance of both animal welfare and biosafety requirements. NHPs housed in open-barred cages raise concerns about cross-contamination between animals, and accidental exposure of personnel to infectious materials. To address these concerns, two NHP experiments were performed. One examined the simultaneous infection of 6 groups of NHPs with 6 different viruses (Machupo, Junin, Rift Valley Fever, Crimean-Congo Hemorrhagic Fever, Nipah and Hendra viruses). Washing personnel between handling each NHP group, floor to ceiling biobubble with HEPA filter, and plexiglass between cages were employed for partial primary containment. The second experiment employed no primary containment around open barred cages with Ebola virus infected NHPs 0.3 meters from naïve NHPs. Viral antigen-specific ELISAs, qRT-PCR and TCID50 infectious assays were utilized to determine antibody levels and viral loads. No transmission of virus to neighbouring NHPs was observed suggesting limited containment protocols are sufficient for multi-viral CL4 experiments within one room. The results support the concept that Ebola virus infection is self-contained in NHPs infected intramuscularly, at least in the present experimental conditions, and is not transmitted to naïve NHPs via an airborne route.

Figure 1. Cage Arrangement for Experiment #1: Multi-virus Transmission.

Cages were setup in a manner that prevented any cross-contamination of viruses within the “biobubble”. The biobubble was a floor to ceiling plastic curtain (grey lines) with a HEPA filter in the top right hand corner (H). There was no curtain in front of the cage. Three mm plexiglass panels (red lines) were placed between cages within a quad to control the flow of air (arrows) towards the HEPA filter. Each quad housed one virus family (ie Arenaviridae) grouped such that the (A) top and bottom left side contained one virus (ie MACV) and the (B) top and bottom right side housed the other virus (ie JUNV) from the same family.

Figure 2. Cage arrangement for Experiment #2: EBOV Transmission.

The EBOV quad was approximately 0.3 m from the Cyno cages. A biobubble was not used therefore airflow was not directed with the use of Plexiglas panels and the use of the HEPA filtered exhaust. Subjects EBOV-1 and EBOV-2 were the EBOV infected rhesus macaques, and the cyno-1 and cyno-2 were the uninfected cynomolgus macaque controls.