BMBL-Section VII

The American Committee on Arthropod-Borne Viruses (ACAV) registered 535 arboviruses as of December 1991. In 1979, the ACAV's Subcommittee on Arbovirus Laboratory Safety (SALS) categorized each of the 424 viruses then registered in the Catalogue of Arboviruses and Certain other Viruses of Vertebrates (96) into 1 of 4 recommended practices, safety equipment, and facilities described in this publication as Biosafety Levels 1-4 (178). Since 1980, SALS has periodically updated the 1980 publication by providing a supplemental listing and recommended levels of practice and containment for arboviruses registered since 1979. SALS categorizations were based on risk assessments from information provided by a worldwide survey of 585 laboratories working with arboviruses. SALS recommended that work with the majority of these agents should be conducted at the equivalent of Biosafety Level 2, Table A. SALS also recognizes five commonly used vaccine strains, for which attenuation is firmly established, which may be handled safely at BSL 2, provided that personnel working with these vaccine strains are immunized, Table B. SALS has classified all registered viruses for which insufficient laboratory experience exists as BSL 3, Table C, and reevaluates the classification whenever additional experience is reported.

The viruses classified as BSL 2 are listed alphabetically in Table A and include the following agents which are reported to cause laboratory-associated infections (86, 151, 178).

The result of the SALS survey clearly indicate that the suspected source of the laboratory-associated infections listed above was other than exposure to infectious aerosols. Recommendations that work with the 341 arboviruses, listed in Table A, should be conducted at Biosafety Level 2 was based on the existence of adequate historical laboratory experience to assess risks for the virus which indicate that (a) no overt laboratory-associated infections are reported or (b) infections resulted from exposures other than to infectious aerosols or (c) if disease from aerosol exposure is documented, it is uncommon.

LABORATORY HAZARDS: agents listed in this group may be present in blood, CSF, central nervous system and other tissues, and infected arthropods, depending on the agent and the stage of infection. While the primary laboratory hazards are accidental parenteral inoculation, contact of the virus with broken skin or mucous membranes, and bites of infected laboratory rodents or arthropods, infectious aerosols may also be a potential source of infection.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 practices, safety equipment, and facilities are recommended for activities with potential infectious clinical materials and arthropods and for manipulations of infected tissue cultures, embryonated eggs, and rodents. Infection of newly hatched chickens with eastern and western equine encephalomyelitis viruses is especially hazardous and should be undertaken under Biosafety Level 3 conditions by immunized personnel. Investigational vaccines (IND) against eastern equine encephalomyelitis and western equine encephalomyelitis viruses are available through the Centers for Disease Control and Prevention and the U.S. Army Medical Research Institute for Infectious Diseases, (USAMRIID) Fort Detrick, Maryland. The use of these vaccines is recommended for personnel who work directly and regularly with these two agents in the laboratory. Western equine encephalomyelitis immune globulin (human) is also available from the Centers for Disease Control and Prevention. The efficacy of this product has not been established.

Prior to 1988, 12 laboratory-acquired dengue infections had been reported. However, from 1988 through 1991, four additional cases have been documented. In all four cases, proper protective gear (long sleeve lab gowns tying in back, gloves, masks, safety glasses) were not worn; and, in three instances, containment of potential aerosols in a laminar flow biosafety cabinet was ignored. These aerosols or infected fluids most likely produced contamination of broken, unprotected skin. An additional factor in these cases was work with highly concentrated amounts of virus. Safe manipulations of dengue viruses in the laboratory (particularly in concentrated preparations) requires strict adherence to Biosafety Level 2 recommendations.

VIRUS	CASES
Vesicular stomatitis	46
Colorado tick fever	16
Dengue	11
Pinchinde	17
Western equine encephalomyelitis	7 (2 deaths)
Rio Bravo	7
Kunjin	6
Catu	5
Caraparu	5
Ross River	5
Bunyamwera	4
Eastern equine encephalomyelitis	4
Zika	4
Apeu	2
Marituba	2
Tacaribe	2
Muructucu	1
O'nyong nyong	1
Modoc	1
Oriboca	1
Ossa	1
Keystone	1
Bebaru	1
Bluetongue	1

Arboviruses and Arenaviruses Assigned to Biosafety Level 2
Aabahoyo Abras Abu Hammad Acado Acara Aguacate Alfuy Almpiwar Amapari Ananindeua Anhanga Anhembi Anopheles A Anopheles B Apeu Apoi Aride Arkonam Aroa Aruac Arumowot Aura Avalon Bagaza Bahig Bakau Baku Bandia Bangoran Bangui Banzi Barmah Forest Barur Batai Batama Baulin Bebaru Belmont Benevides Benfica Bertioga Bimiti Birao Bluetongue Boraceia Botambi Boteke Bouboui Bujaru Bunyamwera Bunyip Burg E Arab Bushbush Bussuquara Buttonwillow Bwamba Cacao Cache Valley Caimito California enc. Calovo Candiru Cape Wrath Capim Caraparu Carey Island Catu Chaco Chagres Chandipura Changuinola Charleville Chenuda Chilibre Chobar gorge Clo Mor Colorado tick fever Corriparta Cotia Cowbone Ridge Csiro Village Cuiaba D'aguilar Dakar Bat Dengue-1 Dengue-2 Dengue-3 Dengue-4	Dera Ghazi Khan East equine. enc. (d) Edge Hill Entebbe Bat Ep. Hem. Disease Erve Eubenangee Eyach Flanders Fort Morgan Frijoles Gamboa Gan Gan Gomoka Gossas Grand Arbaud Great Island Guajara Guama Guaratuba Guaroa Gumbo Limbo Hart Park Hazara Highlands J Huacho Hughes Icoaraci Ieri Ilesha Ilheus Ingwavuma Inkoo Ippy Irituia Isfahan Itaporanga Itaqui Jamestown Canyon Japanaut Jerry Slough Johnston Atoll Joinjakaka Juan Diaz Jugra Jurona Jutiapa Kadam Kaeng Khoi Kaikalur Kaisodi Kamese Kammavan pettai Kannaman galam Kao Shuan Karimabad Karshi Kasba Kemerovo Kern Canyon Ketapang Keterah Keuraliba Keystone Kismayo Klamath Kokobera Kolongo Koongol Kotonkan Kowanyama Kunjin Kununurra Kwatta La Crosse La Joya Lagos Bat Landjia Langat Lanjan Las Maloyas Latino Le Dantec Lebombo Lednice Lipovnik	Lokern Lone Star Lukuni M'poko Madrid Maguari Mahogany Hammock Main Drain Malaka Manawa Manzanilla Mapputta Maprik Marco Marituba Marrakai Matariya Matruh Matucare Melao Mermet Minatitlan Minnal Mirim Mitchell River Modoc Moju Mono Lake Mont.myotis leuk. Moriche Mosqueiro Mossuril Mount Elgon Bat Murutucu Mykines Navarro Nepuyo Ngaingan Nique Nkolbisson Nola Ntaya Nugget Nyamanini Nyando O'nyong-nyong Okhotskiy Okola Olifantsvlei Oriboca Ossa Pacora Pacui Pahayokee Palyam Parana Pata Pathum Thani Patois Phnom-Penh Bat Pichinde Pixuna Pongola Ponteves Precarious Point Pretoria Prospect Hill Puchong Punta Salinas Punta Toro Qalyub Quaranfil Restan Rio Bravo Rio Grande Ross River Royal Farm Sabo Saboya Saint Floris Sakhalin Salehabad San angelo Sandfly f.(Naples) Sandfly f. (Sicilian) Sandjimba Sango	Sathuperi Sawgrass Sebokele Seletar Sembalam Serra do Navio Shamonda Shark River Shuni Silverwater Simbu Simian hem. fever Sindbis Sixgun City Snowshoe Hare Sokuluk Soldado Sororoca Stratford Sunday Canyon Tacaiuma Tacaribe Taggert Tahyna Tamiami Tanga Tanjong Rabo Tataguine Tehran Tembe Tembusu Tensaw Tete Tettnang Thimiri Thottapalayam Tibrogargan Timbo Timboteua Tindholmur Toscana Toure Tribec Triniti Trivittatus Trubanaman Tsuruse Turlock Tyuleniy Uganda S Umatilla Umbre Una Upolu Urucuri Usutu Uukuniemi Vellore Venkatapuram Vinces Virgin River VS-Indiana S-New Jersey Wad Medani Wallal Wanowrie Warrego West. equine enc.(d) Whataroa Witwatersrand Wonga Wongorr Wyeomyia Yaquinea Head Yata Yogue Zaliv Terpeniya Zegla Zika Zingilamo Zirqa
(d) A vaccine is available and is recommended for all persons working with this agent.

SALS has recommended that work with the 171 arboviruses included in the two alphabetical listings should be conducted at the equivalent of Biosafety Level 3 practices, safety equipment and facilities. These recommendations are based on the following criteria: for Table C , SALS considered the laboratory experience inadequate to assess risk, regardless of the available information regarding disease severity. For Table D , SALS recorded overt laboratory-associated infections with these agents which occurred by the aerosol route if protective vaccines were not used or were unavailable, and that the natural disease in humans is potentially severe, life threatening, or causes residual damage. Arboviruses were also classified BSL 3 if they cause diseases in domestic animals in countries outside the USA. Laboratory or laboratory animal-associated infections have been reported with the following BSL agents: (86, 151, 178).

Large quantities and high concentrations of Semliki Forest virus are commonly used or manipulated by molecular biologists under conditions of moderate or low containment. Although antibodies have been demonstrated in individuals working with this virus the first overt (and fatal) laboratory-associated infection with this virus was reported in 1979 (198). Because the outcome of this infection may have been influenced by a compromised host, an unusual route of exposure or high dosage, or a mutated strain of the virus, this case and its outcome are not typical. More recently, SFV was associated with an outbreak of febrile illness among European soldiers stationed in Bangui (118). The route of exposure was not determined in the fatal laboratory infection; for the natural infections, mosquitoes were the probable vector. SALS continues to classify SFV as a BL 3 virus, with the caveat that most activities with this virus can be safety conducted at Biosafety Level 2.

Some viruses (e.g., Akabane, Israel turkey meningoencepha-litis) are listed in Level 3, not because they pose a threat to human health, but because they are exotic diseases of domestic livestock or poultry.

LABORATORY HAZARDS: The agents listed in this group may be present in blood, cerebrospinal fluid, urine and exudates depending on the specific agent and stage of disease. The primary laboratory hazards are exposure to aerosols of infectious solutions and animal bedding, accidental parenteral inoculation, and broken skin contact. Some of these agents (e.g., VEE) may be relatively stable in dried blood or exudates. Attenuated vaccine strains for a number of these agents are listed in Table B.

RECOMMENDED PRECAUTIONS: Biosafety Level 3 practices, safety equipment, and facilities are recommended for activities using potentially infectious clinical materials and infected tissue cultures, animals, or arthropods.

A licensed attenuated live virus is available for immunization against yellow fever and is recommended for all personnel who work with this agent or with infected animals, and those who enter rooms where the agents or infected animals are present. An investigational vaccine (IND) available for immunization against Venezuelan equine encephalomyelitis is recommended for all personnel working with VEE (and the related Everglades, Mucambo, Tonate, and Cabassou viruses), infected animals, or entering rooms where these agents or infected animals are present. Likewise, investigational vaccines for Rift Valley fever and Junin viruses are available from USAMRIID. Work with Hantaan (Korean hemorrhagic fever) virus and related viruses (Puumala and Seoul) in rats, voles, and other laboratory rodents should be conducted with special caution, because of the extreme hazard of aerosol infection.

Table B. Vaccine Strains of BSL 3/4 Viruses Which May be Handled at BSL 2
Virus	Vaccine Strain
Chikungunya	131/25
Junin	Candid #1
Rift Valley fever (20)	MP-12
Venezuelan equine encephalomyelitis	TC-83
Yellow fever	17-D

VIRUS	CASES (SALS)
Venezuelan equine encephalomyelitis	150 (1 death)
Rift Valley fever	47 (1 death)
Chikungunya	39
Yellow fever	38 (8 deaths)
Japanese encephalitis	22
Louping ill	22
West Nile	18
Lymphocytic choriomeningitis	15
Orungo	13
Pery	13
Wesselsbron	13
Mucambo	10
Oropouche	7
Germiston	6
Bhanja	6
Hantaan	6
Mayaro	5
Spondweni	4
Murray Valley encephalitis	3
Semliki Forest	3 (1 death)
Powassan	2
DugbE	2
Issyk-kul	1
Koutango	1

Table C. Arboviruses and Certain Other Viruses Assigned to Biosafety Level 3 (on the basis of insufficient experience)
Adelaide River Agua Preta Alenquer Almeirim Altamira Andasibe Antequera Araguari Aransas Bay Arbia Arboledas Babanki Batken Belem Berrimah Bimbo Bobaya Bobia Bozo Buenaventura Cabassue(c,d) Cacipacore Calchaqui Cananeia Caninde Chim Coastal Plains Connecticut Corfou Dabakala Douglas Enseada Estero Real Fomede Forecariah Fort Sherman Gabek Forest Gadgets Gully Garba Gordil Gray Lodge Gurupi Iaco Ibaraki	Ife Ingangapi Inini Issyk-Kul Itaituba Itimirim Itupiranga Jacareacanga Jamanxi Jari Kedougou Khasan Kindia Kyzylagach Lake Clarendon Llano Seco Macaua Mapuera Mboke Meaban Mojui Dos Compos Monte Dourado Munguba Naranjal Nariva Nasoule Ndelle New Minto Ngari Ngoupe Nodamura Northway Odrenisrou Omo Oriximina Ouango Oubangui Oubi Ourem Palestina Para Paramushir Paroo River Perinet	Petevo Picola Playas Pueblo Viejo Purus Radi Razdan Resistencia Rochambeau Salanga San Juan Santa Rosa Santarem Saraca Saumarez Reef Sedlec Sena Madureira Sepik Shokwe Slovakia Somone Spipur Tai Tamdy Telok Forest Termeil Thiafora Tilligerry Tinaroo Tlacotalpan Tonate (c,d) Ttinga Xiburema Yacaaba Yaounde Yoka Yug Bogkanovac
(c.) SALS recommends that work with this agent should be conducted only in Biosafety Level 3 facilities which provide for HEPA filtration of all exhaust air prior to discharge from the laboratory. (d.) A vaccine is available and is recommended for all persons working with this agent.

Table D. Arboviruses and Certain Other Viruses Assigned to Biosafety Level 3
Aino Akabane Bhanja Chikungunya (c,d) Cocal Dhori Dugbe Everglades (c,d) Flexal Germiston (c) Getah Hantaan Israel Turkey mening. Japanese enc. Junin (c,d) Kairi Kimberley Koutango Louping Ill (a,c)	Mayaro Middelburg Mobala Mopeia (e) Mucambo (c,d) Murray Valley enc. Nairobi sheep disease(a) Ndumu Negishi Oropouche (c) Orungo Peaton Piry Powassan Puumala Rift Valley fever (a,b,c,d) Sagiyama	Sal Vieja San Perlita Semliki Forest Seoul Spondweni St. Louis enc. Thogoto Tocio (c) Turuna Venezuelan equine(c,d) encephalitis Vesicular Stomatitus (alagoas) Wesselsbron (a,c) West Nile Yellow fever (c,d) Zinga (b)
(a.) The importation, possession, or use of this agent is restricted by USDA regulation or administrative policy. See Appendix E. (b.) Zinga virus is now recognized as being identical to Rift Valley Fever virus. (c.) SALS recommends that work with this agent should be conducted only in Biosafety Level 3 facilities which provide for HEPA filtration of all exhaust air prior to discharge from the laboratory. (d.) A vaccine is available and is recommended for all persons working with this agent. (e.) This virus is presently being registered in the Catalogue of Arboviruses (96).

SALS has recommended that work with the 15 arboviruses, arenaviruses, or filoviruses (104) included in the alphabetical listing that follows should be conducted at the equivalent of Biosafety Level 4 practices, safety equipment, and facilities. These recommendations are based on documented cases of severe and frequently fatal naturally occurring human infections and aerosoltransmitted laboratory infections. SALS recommended that certain agents with a close antigenic relationship to the Biosafety Level 4 agents (e.g., Absettarov and Kumlinge viruses) also be handled at this level provisionally until sufficient laboratory experience was obtained to retain these agents at this level or to work with them at a lower level. Laboratory or laboratory animal-associated infections have been reported with the following agents: (65, 86, 90, 109, 151, 178, 194).

Rodents are natural reservoirs of Lassa fever virus (Mastomys natalensis), Junin and Machupo viruses (Calomys spp.) and perhaps other members of this group. Nonhuman primates were associated with the initial outbreaks of Kyasanur Forest disease (Presbytis spp.) and Marburg disease (Cercopithecus spp.); more recently, filoviruses related to Ebola were associated with Macaca spp. Arthropods are the natural vectors of the tick-borne encephalitis complex agents. Work with or exposure to rodents, nonhuman primates, or vectors naturally or experimentally infected with these agents represents a potential source of human infection.

LABORATORY HAZARDS: The infectious agents may be present in blood, urine, respiratory and throat secretions, semen and tissues from human or animal hosts, and in arthropods, rodents, and nonhuman primates. Respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and accidental parenteral inoculation are the primary hazards to laboratory or animal care personnel (109, 194).

RECOMMENDED PRECAUTIONS: Biosafety Level 4 practices and facilities are recommended for all activities utilizing known or potentially infectious materials of human, animal, or arthropod origin. A new, live attenuated investigational (IND) Junin virus vaccine (Candid #1) is available from the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) and is recommended for all laboratory and animal care personnel working with the agent or infected animals and for all personnel entering laboratories or animal rooms when the agent is in use. SALS has lowered the biohazard classification of Junin virus to BSL 3, provided all at risk personnel are immunized and the laboratory is equipped with HEPA filtered exhaust. Clinical specimens from persons suspected of being infected with one of the agents listed in this summary should be submitted to a laboratory with a Biosafety Level 4 maximum containment facility (23, 141).

VIRUS	CASES (SALS)
Junin	21 (1 death)
Marburg	25 (5 deaths)
Russian Spring-Summer encephalitis	8
Congo-Crimean hemorrhagic fever	8 (1 death)
Omsk hemorrhagic fever	5
Lassa	2 (1 death)
Machupo	1 (1 death)
Ebola	1

Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97
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Section VII