Biological Safety Manual – Chapter 08: Agent Summary Statements (Section VI: Arboviruses and Related Zoonotic Viruses)

Title

Biological Safety Manual – Chapter 08: Agent Summary Statements (Section VI: Arboviruses and Related Zoonotic Viruses)

Introduction

In 1979, the American Committee on Arthropod-Borne Viruses (ACAV) Subcommittee on Arbovirus Laboratory Safety (SALS) first provided biosafety recommendations for each of the 424 viruses then registered in the International Catalogue of Arboviruses, including Certain Other Viruses of Vertebrates.¹ Working together, SALS, the CDC and the NIH have periodically updated the catalogue by providing recommended biosafety practices and containment for arboviruses registered since 1979. These recommendations are based, in part, on risk assessments derived from information provided by a worldwide survey of laboratories working with arboviruses, new published reports on the viruses, as well as discussions with scientists working with each virus.

Table 3, located at the end of this chapter, provides an alphabetical listing of 597 viruses and includes common name, virus family or genus, acronym, BSL recommendation, the basis for the rating, the antigenic group 2 (if known), HEPA filtration requirements, and regulatory requirements (i.e. import/export permits from either the CDC or the USDA). In addition, many of the organisms are classified as Select Agents and require special security measures to possess, use, or transport (See Chapter 14). Table 1 provides a key for the SALS basis for assignment of viruses listed in Table 3.

Agent summary statements have been included for certain arboviruses. They were submitted by a panel of experts for more detailed consideration due to one or more of the following factors:

• at the time of writing this edition the organism represented an emerging public health threat in the United States
• the organism presented unique biocontainment challenge(s) that required further detail
• the organism presented a significant risk of laboratory-acquired infection

These recommendations were made in August 2005; requirements for biosafety, shipping, and select agent registration can change. Please be sure to confirm the requirements with the appropriate Federal agency. If the pathogen of interest is one listed in Chapter 12, contact the USDA for additional biosafety requirements. USDA guidance may supersede the information found in this chapter.

Recommendations for the containment of infected arthropod vectors were drafted by a subcommittee of the American Committee on Medical Entomology (ACME), and circulated widely among medical entomology professionals (See Chapter 13).

Some commonly used vaccine strains for which attenuation has been firmly established are recognized by SALS. These vaccine strains may be handled safely at BSL-2 (Table 2). The agents in these Tables may require permits from USDA/DOC/DHHS.

Table of Contents

1. Table 1: Explanation of Symbols Used in Table 3 to Define Basis for Assignment of Viruses to Biosafety Levels
2. Table 2: Vaccine Strains of BSL-3 and -4 Viruses That May Be Handled As BSL-2
3. Viruses With BSL-2 Containment Recommended
4. Viruses With BSL-3 Containment Recommended
5. Viruses With BSL-4 Containment Recommendations
6. Dealing With Unknown Arboviruses
7. Agent: West Nile Virus (WNV)
8. Agent: Eastern Equine Encephalitis (EEE) Virus, Venezuelan Equine Encephalitis (VEE) Virus, and Western Equine Encephalitis (WEE) Virus
9. Agent: Rift Valley Fever Virus (RVFV)
10. References
11. Table 3: Alphabetic Listing of 597 Arboviruses and Hemorrhagic Fever Viruses

Table 1

Explanation of Symbols Used in Table 3 to Define Basis for Assignment of Viruses to Biosafety Levels

Symbol	Definition
S	Results of SALS survey and information from the Catalog.1
IE	Insufficient experience with virus in laboratory facilities with low biocontainment.
A	Additional criteria.
A1	Disease in sheep, cattle or horses.
A2	Fatal human laboratory infection – probably aerosol.
A3	Extensive laboratory experience and mild nature of aerosol laboratory infections justifies BSL-2.
A4	Placed in BSL-4 based on the close antigenic relationship with a known BSL-4 agent plus insufficient experience.
A5	BSL-2 arenaviruses are not known to cause serious acute disease in humans and are not acutely pathogenic for laboratory animals including primates. In view of reported high frequency of laboratory aerosol infection in workers manipulating high concentrations of Pichinde virus, it is strongly recommended that work with high concentrations of BSL-2 arenaviruses be done at BSL-3.
A6	Level assigned to prototype or wild-type virus. A lower level may be recommended for variants with well-defined reduced virulence characteristics.
A7	Placed at this biosafety level based on close antigenic or genetic relationship to other viruses in a group of 3 or more viruses, all of which are classified at this level.
A8	BSL-2 hantaviruses are not known to cause laboratory infections, overt disease in humans, or severe disease in experimental primates. Because of antigenic and biologic relationships to highly pathogenic hantaviruses and the likelihood that experimentally infected rodents may shed large amounts of virus, it is recommended that work with high concentrations or experimentally infected rodents be conducted at BSL-3.

Table 2

Vaccine Strains of BSL-3 and -4 Viruses That May Be Handled As BSL-2

Virus	Vaccine Strain
Chikungunya	181/25
Junin	Candid #1
Rift Valley fever	MP-12
Venezuelan equine encephalomyelitis	TC83
Yellow fever	17-D
Japanese encephalitis	14-14-2

Based on the recommendations listed within tables, the following guidelines should be adhered to where applicable.

Viruses With BSL-2 Containment Recommended

The recommendation for conducting work with the viruses listed in Table 3 at BSL-2 was based on the existence of adequate historical laboratory experience to assess the risks when working with this group of viruses. This indicates:

1. no overt laboratory-associated infections are reported
2. infections resulted from exposures other than by infectious aerosols
3. if disease from aerosol exposure is documented, it is uncommon

Laboratory Hazards

Agents listed in this group may be present in blood, CSF, various tissues, and/or infected arthropods, depending on the agent and the stage of infection. The primary laboratory hazards comprise accidental parenteral inoculation, contact of the virus with broken skin or mucous membranes, and bites of infected laboratory rodents or arthropods. Properly maintained BSCs, preferable Class II, or other appropriate personal protective equipment or physical containment devices are used whenever procedure with a potential for creating infectious aerosols or splashes are conducted.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for activities with potentially infectious clinical materials and arthropods and for manipulations of infected tissue cultures, embryonate hen’s eggs, and rodents.

Large quantities and/or high concentrations of any virus have the potential to overwhelm both innate immune mechanisms and vaccine-induced immunity. When a BSL-2 virus is being produced in large quantities or in high concentrations, additional risk assessment is required. This might indicate BSL-3 practices, including additional respiratory protection, based on the risk assessment of the proposed experiment.

Viruses With BSL-3 Containment Recommended

The recommendations for viruses listed in Table 3 that require BSL-3 containment are based on multiple criteria. SALS considered the laboratory experience for some viruses to be inadequate to assess risk, regardless of the available information regarding disease severity. In some cases, SALS recorded overt LAI transmitted by the aerosol route in the absence or non-use of protective vaccines, and considered that the natural disease in humans is potentially severe, life threatening, or causes residual damage.¹ Arboviruses also were classified as requiring BSL-3 containment if they caused diseases in domestic animals in countries outside of the United States.

Laboratory Hazards

The agents listed in this group may be present in blood, CSF, 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 contact with broken skin. Some of these agents (e.g., VEE virus) may be relatively stable in dried blood or exudates.

Containment Recommendations

BSL-3 practices, containment 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. It is recommended for all personnel who work with this agent or with infected animals, and those entering rooms where the agents or infected animals are present. Junin virus has been reclassified to BSL-3, provided that all at-risk personnel are immunized and the laboratory is equipped with HEPA-filtered exhaust. SALS also has reclassified Central European tick-borne encephalitis (CETBE) viruses to BSL-3, provided all at-risk personnel are immunized. CETBE is not a registered name in The International Catalogue of Arboviruses(1985). Until the registration issue is resolved taxonomically, CETBE refers to the following group of very closely related, if not essentially identical, tick-borne flaviviruses isolated from Czechoslovakia, Finland and Russia: Absettarov, Hanzalova, Hypr, and Kumlinge viruses. While there is a vaccine available that confers immunity to the CETBE group of genetically (>98%) homogeneous viruses, the efficacy of this vaccine against Russian spring-summer encephalitis (RSSE) virus infections has not been established. Thus, the CETBE group of viruses has been reclassified as BSL-3 when personnel are immunized with CETBE vaccine, while RSSE remains classified as BSL-4. It should be noted that CETBE viruses are currently listed as Select Agents and require special security and permitting considerations (See Chapter 14).

Investigational vaccines for eastern equine encephalomyelitis (EEE) virus, Venezuelan Equine Encephalitis (VEE), western equine encephalomyelitis (WEE) virus, and Rift Valley fever viruses (RVFV), may be available in limited quantities and administered onsite at the Special Immunization Program, USAMRIID. Details are available at the end of this chapter.

The use of investigational vaccines for laboratory personnel should be considered if the vaccine is available, initial studies have shown the vaccine to be effective in producing an appropriate immunologic response, and the adverse effects of vaccination are within acceptable parameters. The decision to recommend vaccines for laboratory personnel must be carefully considered and based on an risk assessment which includes a review of the characteristics of the agent and the disease, benefits versus the risk of vaccination, the experience of the laboratory personnel, laboratory procedures to be used with the agent, and the contraindications for vaccination including the health status of the employee.

If the investigational vaccine is contraindicated, does not provide acceptable reliability for producing an immune response, or laboratory personnel refuse vaccination, the use of appropriate personal protective equipment may provide an alternative. Respiratory protection, such as use of a PAPR, should be considered in areas using organisms with a well-established risk of aerosol infections in the laboratory, such as VEE viruses. Any respiratory protection equipment must be provided in accordance with the institution’s respiratory protection program. Other degrees of respiratory protection may be warranted based on an assessment of risk as defined in Chapter 3 of this manual. All personnel in a laboratory with the infectious agent must use comparable personal protective equipment that meets or exceeds the requirements, even if they are not working with the organism. Sharps precautions as described under BSL-2 and BSL-3 requirements must be continually and strictly reinforced, regardless of whether investigational vaccines are used.

Non-licensed vaccines are available in limited quantities and administered on-site at the Special Immunization Program of USAMRIID, located at Ft. Detrick, Frederick MD. As IND vaccines are administered under a cooperative agreement between the U.S. Army and the individual’s requesting organization. Contact the Special Immunization Program by telephone at 301-619-4653.

Enhanced BSL-3 Containment

Situations may arise for which enhancements to BSL-3 practices and equipment are required. An example would be when a BSL-3 laboratory performs diagnostic testing on specimens from patients with hemorrhagic fevers thought to be due to dengue or yellow fever viruses. When the origin of these specimens is Africa, the Middle East, or South America, such specimens might contain etiologic agents, such as arenaviruses, filoviruses, or other viruses that are usually manipulated in a BSL-4 laboratory. Examples of enhancements to BSL-3 laboratories might include:

1. enhanced respiratory protection of personnel against aerosols;
2. HEPA filtration of dedicated exhaust air from the laboratory;
3. personal body shower.

Additional appropriate training for all animal care personnel should be considered.

Viruses With BSL-4 Containment Recommendations

The recommendations for viruses assigned to BSL-4 containment are based on documented cases of severe and frequently fatal naturally occurring human infections and aerosol- transmitted laboratory infections. SALS recommends that certain agents with a close antigenic relationship to agents assigned to BSL-4 also be provisionally handled at this level until sufficient laboratory data indicates their retention at this level or movement to work at a lower level.

Laboratory Hazards

The infectious agents may be present in blood, urine, respiratory and throat secretions, semen, and other fluids and tissues from human or animal hosts, and in arthropods, rodents, and NHP. 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.^3,4

Containment Recommendations

BSL-4 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious materials of human, animal, or arthropod origin. 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 BSL- 4 maximum containment facility.⁵

Dealing With Unknown Arboviruses

The ACAV has published reports documenting laboratory workers who acquired arbovirus infections during the course of their duties.⁶ In the first such document, it was recognized that these laboratory infections typically occurred by unnatural routes such as percutaneous or aerosol exposure, that “lab adapted” strains were still pathogenic for humans, and that as more laboratories worked with newly identified agents, the frequency of laboratory-acquired infections was increasing. Therefore, to assess the risk of these viruses and provide safety guidelines to those working with them, ACAV appointed SALS to evaluate the hazards of working with arboviruses in the laboratory setting.^7,8 The SALS committee made a series of recommendations, published in 1980, describing four levels of laboratory practices and containment guidelines that were progressively more restrictive. These levels were determined after widely-distributed surveys evaluated numerous criteria for each particular virus including:

1. past occurrence of laboratory acquired infections correlated with facilities and practices used
2. volume of work performed as a measure of potential exposure risk
3. immune status of laboratory personnel
4. incidence and severity of naturally-acquired infections in adults
5. incidence of disease in animals outside the United States (to assess import risk)

While these criteria are still important factors to consider in any risk assessment for manipulating arboviruses in the laboratory, it is important to note that there have been many modifications to personal laboratory practices (e.g., working in BSC while wearing extensive personal protective equipment in contrast to working with viruses on an open bench top) and significant changes in laboratory equipment and facilities (e.g. BSC, PAPR) available since the initial SALS evaluation. Clearly, when dealing with a newly recognized arbovirus, there is insufficient previous experience with it; thus, the virus should be assigned a higher biosafety level. However, with increased ability to safely characterize viruses, the relationship to other disease-causing arboviruses can be established with reduced exposure to the investigators. Therefore, in addition to those established by SALS, additional assessment criteria should be considered.

One criterion for a newly identified arbovirus is a thorough description of how the virus will be handled and investigated. For example, experiments involving pure genetic analysis could be handled differently than those where the virus will be put into animals or arthropods.⁹ Additionally, an individual risk assessment should consider the fact that not all strains of a particular virus exhibit the same degree of pathogenicity or transmissibility. While variable pathogenicity occurs frequently with naturally identified strains, it is of particular note for strains that are modified in the laboratory. It may be tempting to assign biosafety levels to hybrid or chimeric strains based on the parental types but due to possible altered biohazard potential, assignment to a different biosafety level may be justified.¹⁰ A clear description of the strains involved should accompany any risk assessment.

Most of the identified arboviruses have been assigned biosafety levels; however, a number of those that are infrequently studied, newly identified, or have only single isolation events may not have been evaluated by SALS, ACAV, CDC, or the NIH (See Table 3). Thorough risk assessment is important for all arboviral research and it is of particular importance for work involving unclassified viruses. A careful assessment by the laboratory director, institutional biosafety officer and safety committee, and as necessary, outside experts is necessary to minimize the risk of human, animal, and environmental exposure while allowing research to progress.

Chimeric Viruses

The ability to construct cDNA clones encoding a complete RNA viral genome has led to the generation of recombinant viruses containing a mixture of genes from two or more different viruses. Chimeric, full-length viruses and truncated replicons have been constructed from numerous alphaviruses and flaviviruses. For example, alphavirus replicons encoding foreign genes have been used widely as immunogens against bunyavirus, filovirus, arenavirus, and other antigens. These replicons have been safe and usually immunogenic in rodent hosts leading to their development as candidate human vaccines against several virus groups including retroviruses.^11-14

Because chimeric viruses contain portions of multiple viruses, the IBC, in conjunction with the biosafety officer and the researchers, must conduct a risk assessment that, in addition to standard criteria, includes specific elements that need to be considered before assigning appropriate biosafety levels and containment practices. These elements include:

1. the ability of the chimeric virus to replicate in cell culture and animal model systems in comparison with its parental strains;¹⁵
2. altered virulence characteristics or attenuation compared with the parental viruses in animal models;¹⁶
3. virulence or attenuation patterns by intracranial routes using large doses for agents affecting the CNS;^17,18 and
4. demonstration of lack of reversion to virulence or parental phenotype.

Many patterns of attenuation have been observed with chimeric flaviviruses and alphaviruses using the criteria described above. Additionally, some of these chimeras are in phase II testing as human vaccines.¹⁹

Chimeric viruses may have some safety features not associated with parental viruses. For example, they are generated from genetically stable cDNA clones without the need for animal or cell culture passage. This minimizes the possibility of mutations that could alter virulence properties. Because some chimeric strains incorporate genomic segments lacking gene regions or genetic elements critical for virulence, there may be limited possibility of laboratory recombination to generate strains exhibiting wild-type virulence.

Ongoing surveillance and laboratory studies suggest that many arboviruses continue to be a risk to human and animal populations. The attenuation of all chimeric strains should be verified using the most rigorous containment requirements of the parental strains. The local IBC should evaluate containment recommendations for each chimeric virus on a case-by-case basis, using virulence data from an appropriate animal model. Additional guidance from the NIH Office of Biotechnology Activities Recombinant DNA Advisory Committee may be necessary.

Agent: West Nile Virus (WNV)

WNV has emerged in recent years in temperate regions of Europe and North America, presenting a threat to public and animal health. This virus belongs to the family Flaviviridaeand the genus Flavivirus, Japanese encephalitis virus antigenic complex. The complex currently includes Alfuy, Cacipacore, Japanese encephalitis, Koutango, Kunjin, Murray Valley encephalitis, St. Louis encephalitis, Rocio, Stratford, Usutu, West Nile, and Yaounde viruses. Flaviviruses share a common size (40-60nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense, single stranded RNA approximately 10,000-11,000 bases) and virus morphology. The virus was first isolated from a febrile adult woman in the West Nile District of Uganda in 1937.²⁰ The ecology was characterized in Egypt in the 1950s; equine disease was first noted in Egypt and France in the early 1960s.^21,22 It first appeared in North America in 1999 as encephalitis reported in humans and horses.²³ The virus has been detected in Africa, Europe, the Middle East, west and central Asia, Oceania (subtype Kunjin virus), and most recently, North America.

Occupational Infections

LAI with WNV have been reported in the literature. SALS reported 15 human infections from laboratory accidents in 1980. One of these infections was attributed to aerosol exposure. Two parenteral inoculations have been reported recently during work with animals.²⁴

Natural Modes of Infection

In the United States, infected mosquitoes, primarily members of the Culexgenus, transmit WNV. Virus amplification occurs during periods of adult mosquito bloodfeeding by continuous transmission between mosquito vectors and bird reservoir hosts.

People, horses, and most other mammals are not known to develop infectious viremias very often, and thus are probably “dead-end” or incidental hosts.

Laboratory Safety

WNV may be present in blood, serum, tissues, and CSF of infected humans, birds, mammals, and reptiles. The virus has been found in oral fluids and feces of birds. Parenteral inoculation with contaminated materials poses the greatest hazard; contact exposure of broken skin is a possible risk. Sharps precautions should be strictly adhered to when handling potentially infectious materials. Workers performing necropsies on infected animals may be at higher risk of infection.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for activities with human diagnostic specimens, although it is unusual to recover virus from specimens obtained from clinically ill patients. BSL-2 is recommended for processing field collected mosquito pools whereas BSL-3 and ABSL-3 practices, containment equipment, and facilities are recommended, for all manipulations of WNV cultures and for experimental animal and vector studies, respectively.

Dissection of field collected dead birds for histopathology and culture is recommended at BSL-3 containment due to the potentially high levels of virus found in such samples. Non-invasive procedures performed on dead birds (such as oropharyngeal or cloacal swabs) can be conducted at BSL-2.

Special Issues

Transfer of Agent

Importation of this agent may require CDC and/or USDA importation permits. Domestic transport of this agent may require a permit from USDA/APHIS/VS. A DoC permit may be required for the export of this agent to another country. See Chapter 11 for additional information.

Agent: Eastern Equine Encephalitis (EEE) Virus, Venezuelan Equine Encephalitis (VEE) Virus, And Western Equine Encephalitis (WEE) Virus

VEE, EEE, and WEE viruses are members of the genus Alphavirus in the family Togaviridae. They are small, enveloped viruses with a genome consisting of a single strand of positive-sense RNA. All three viruses can cause encephalitis often accompanied by long-term neurological sequelae. Incubation period ranges from 1-10 days and the duration of acute illness is typically days to weeks depending upon severity of illness. Although not the natural route of transmission, the viruses are highly infectious by the aerosol route; laboratory acquired infections have been documented.²⁵

Occupational Infections

These alphaviruses, especially VEE virus, are infectious by aerosol in laboratory studies and more than 160 EEE virus, VEE virus, or WEE virus laboratory-acquired infections have been documented. Many infections were due to procedures involving high virus concentrations and aerosol-generating activities such as centrifugation and mouth pipetting. Procedures involving animals (e.g. infection of newly hatched chicks with EEE virus and WEE virus) and mosquitoes also are particularly hazardous.

Natural Modes of Infection

Alphaviruses are zoonoses maintained and amplified in natural transmission cycles involving a variety of mosquito species and either small rodents or birds. Humans and equines are accidental hosts with naturally acquired alphavirus infections resulting from the bites of infected mosquitoes.

EEE virus occurs in focal locations along the eastern seaboard, the Gulf Coast and some inland Midwestern locations of the United States, in Canada, some Caribbean Islands, and Central and South America.²⁶ Small outbreaks of human disease have occurred in the United States, the Dominican Republic, Cuba, and Jamaica. In the United States, equine epizootics are common occurrences during the summer in coastal regions bordering the Atlantic and Gulf of Mexico, in other eastern and Midwestern states, and as far north as Quebec, Ontario, and Alberta in Canada.

In Central and South America, focal outbreaks due to VEE virus occur periodically with rare large regional epizootics involving thousands of equine cases and deaths in predominantly rural settings. These epizootic/epidemic viruses are theorized to emerge periodically from mutations occurring in the continuously circulating enzootic VEE viruses in northern South America. The classical epizootic varieties of the virus are not present in the U.S. An enzootic subtype, Everglades virus (VEE antigenic complex subtype II virus), exists naturally in southern Florida, while endemic foci of Bijou Bridge virus (VEE antigenic complex subtype III-B virus), have been described in the western United States.²⁷

The WEE virus is found mainly in western parts of the United States and Canada. Sporadic infections also occur in Central and South America.

Laboratory Safety

Alphaviruses may be present in blood, CSF, other tissues (e.g., brain), or throat washings. The primary laboratory hazards are parenteral inoculation, contact of the virus with broken skin or mucus membranes, bites of infected animals or arthropods, or aerosol inhalation.

Containment Recommendations

Diagnostic and research activities involving clinical material, infectious cultures, and infected animals or arthropods should be performed under BSL-3 practices, containment equipment, and facilities. Due to the high risk of aerosol infection, additional personal protective equipment, including respiratory protection, should be considered for nonimmune personnel. Animal work with VEE virus, EEE virus and WEE virus should be performed under ABSL-3 conditions. HEPA filtration is required on the exhaust system of laboratory and animal facilities using VEE virus.

Special Issues

Vaccines

Two strains of VEE virus (TC-83 and V3526) are highly attenuated in vertebrate studies and have been either exempted (strain TC-83) or excluded (strain V3526) from Select Agent regulations. Because of the low level of pathogenicity, these strains maybe safely handled under BSL-2 conditions without vaccination or additional personal protective equipment.

Investigational vaccine protocols have been developed to immunize at-risk laboratory or field personnel against these alphaviruses, however, the vaccines are available only on a limited basis and may be contraindicated for some personnel. Therefore, additional personal protective equipment may be warranted in lieu of vaccination. For personnel who have no neutralizing antibody titer (either by previous vaccination or natural infection), additional respiratory protection is recommended for all procedures.

Select Agent

VEE virus and EEE virus are Select Agents requiring registration with CDC and/or USDA for possession, use, storage and/or transfer. See Chapter 14 for additional information.

Transfer of Agent

Importation of this agent may require CDC and/or USDA importation permits. Domestic transport of this agent may require a permit from USDA/APHIS/VS.

Agent: Rift Valley Fever Virus (RVFV)

RVFV was first isolated in Kenya in 1936 and subsequently shown to be endemically present in almost all areas of sub-Saharan Africa.²⁸ In periods of heavy rainfall, large epizootics occur involving primarily sheep, cattle, and human disease, although many other species are infected. The primordial vertebrate reservoir is unknown, but the introduction of large herds of highly susceptible domestic breeds in the last few decades has provided a substrate for massive virus amplification. The virus has been introduced into Egypt, Saudi Arabia, and Yemen and caused epizootics and epidemics in those countries. The largest of these was in 1977 to 1979 in Egypt with many thousands of human cases and 610 reported deaths.²⁹

Most human infections are symptomatic and the most common syndrome consists of fever, myalgia, malaise, anorexia, and other non-specific symptoms. Recovery within one to two weeks is usual but hemorrhagic fever, encephalitis, or retinitis also occurs. Hemorrhagic fever develops as the primary illness proceeds and is characterized by disseminated intravascular coagulation and hepatitis. Perhaps 2% of cases will develop this complication and the mortality is high. Encephalitis follows an apparent recovery in <1% of cases and results in a substantial mortality and sequelae. Retinal vasculitis occurs in convalescence of a substantial but not precisely known proportion of cases. The retinal lesions are often macular and permanent, leading to substantial loss of visual acuity. Infected sheep and cattle suffer a mortality rate of 10-35%, and spontaneous abortion occurs virtually in all pregnant females. Other animals studied have lower viremia and lesser mortality but may abort. This virus is an OIE List A disease and triggers export sanctions.

Occupational Infections

The potential for infection of humans by routes other than arthropod transmission was first recognized in veterinarians performing necropsies. Subsequently, it became apparent that contact with infected animal tissues and infectious aerosols were dangerous; many infections were documented in herders, slaughterhouse workers, and veterinarians. Most of these infections resulted from exposure to blood, other tissues including aborted fetal tissues of sick animals.

There have been 47 reported laboratory infections; before modern containment and vaccination became available virtually every laboratory that began work with the virus suffered infections suggestive of aerosol transmission.^30,31

Natural Modes of Infection

Field studies show RVFV to be transmitted predominantly by mosquitoes, although other arthropods may be infected and transmit. Mechanical transmission also has been documented in the laboratory. Floodwater Aedes species are the primary vector and transovarial transmission is an important part of the maintenance cycle.³² However, many different mosquito species are implicated in horizontal transmission in field studies, and laboratory studies have shown a large number of mosquito species worldwide to be competent vectors, including North American mosquitoes.

It is currently believed that the virus passes dry seasons in the ova of flood-water Aedes mosquitoes. Rain allows infectious mosquitoes to emerge and feed on vertebrates. Several mosquito species can be responsible for horizontal spread, particularly in epizootic/epidemic situations. The vertebrate amplifiers are usually sheep and cattle, with two caveats; as yet undefined native African vertebrate amplifier is thought to exist and very high viremias in humans are thought to play some role in viral amplifications.³³Transmission of diseases occurs between infected animals but is of low efficiency and virus titers in throat swabs are low. Nosocomial infection rarely if ever occurs. There are no examples of latency with RVFV, although virus may be isolated from lymphoid organs of mice and sheep for four to six weeks post-infection.

Laboratory Safety

Concentrations of RVFV in blood and tissues of sick animals are often very high. Placenta, amniotic fluid, and fetuses from aborted domestic animals are highly infectious. Large numbers of infectious virus also are generated in cell cultures and laboratory animals.

Containment Recommendations

BSL-3 practices, containment equipment and facilities are recommended for processing human or animal material in endemic zones or in non-endemic areas in emergency circumstances. Particular care should be given to stringent aerosol containment practices, autoclaving waste, decontamination of work areas, and control of egress of material from the laboratory. Other cultures, cells, or similar biological material that could potentially harbor RVFV should not be used in a RVFV laboratory and subsequently removed. Diagnostic or research studies outside endemic areas should be performed in a BSL-3 laboratory. Personnel also must have additional respiratory protection (such as a PAPR) or be vaccinated for RVFV. In addition, for research conducted in non-endemic areas, the USDA may require full BSL-3Ag containment (See Chapter 12).

Special Issues

Vaccines

Two apparently effective vaccines have been developed by Department of Defense (DOD) and have been used in volunteers, laboratory staff, and field workers under investigational protocols, but neither vaccine is available at this time.

Select Agent

RVFV is a Select Agent requiring registration with CDC and/or USDA for possession, use, storage and/or transfer. See Chapter 14 for additional information. The live- attenuated MP-12 vaccine strain is specifically exempted from the Select Agent rules. In general, BSL-2 containment is recommended for working with this strain. The USDA may require enhanced ABSL-3 or ABSL-3 facilities and practices for working with RVFV in the United States (See Chapter 12). Investigators should contact the USDA for further guidance before initiating research.

Transfer of Agent

Importation of this agent may require CDC and/or USDA importation permits. Domestic transport of this agent may require a permit from USDA/APHIS/VS.

References

1. American Committee on Arthropod-borne Viruses. Subcommittee on Information Exchange International catalogue of arboviruses including certain other viruses of vertebrates. 3rd ed. San Antonio (TX): American Society of Tropical Medicine and Hygiene; 1985.
2. Casals J, Brown LV. Hemagglutinations with arthropod-borne viruses. J Exp Med. 1954;99:429-49.
3. Leifer E, Gocke DJ, Bourne H. Lassa fever, a new virus disease of man from West Africa. II. Report of a laboratory acquired infection treated with plasma from a person recently recovered from the disease. Am J Trop Med Hyg. 1970;19:667-9.
4. Weissenbacher MC, Grela ME, Sabattini MS, et al. Inapparent infections with Junin virus among laboratory workers. J Infect Dis. 1978;137:309-13.
5. Centers for Disease Control and Prevention, Office of Biosafety. Classification of etiologic agents on the basis of hazard. 4th edition. US Department of Health, Education and Welfare; US Public Health Service. 1979.
6. Hanson RP, Sulkin SE, Beuscher EL, et al. Arbovirus infections of laboratory workers. Extent of problem emphasizes the need for more effective measures to reduce hazards. Science. 1967;158:1283-86.
7. Karabatsos N. Supplement to international catalogue of arboviruses, including certain other viruses of vertebrates. Am J Trop Med Hyg. 1978;27:372-440.
8. Department of Health, Education, and Welfare. International catalogue of arboviruses including certain other viruses of vertebrates. Berge TO, editor. Washington, DC. 1975. Pub No. (CDC) 75-8301.
9. Hunt GJ, Tabachnick WJ. Handling small arbovirus vectors safely during biosafety level 3 containment: Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) and exotic bluetongue viruses. J Med Entomol. 1996;33:271-7.
10. Warne SR. The safety of work with genetically modified viruses. In: Ring CJA, Blair ED, editors. Genetically engineered viruses: development and applications. Oxford: BIOS Scientific Publishers; 2001. p. 255-73.
11. Berglund P, Quesada-Rolander M, Putkonen P, et al. Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein and challenge with a high dose of SHIV-4 virus. AIDS Res Hum Retroviruses. 1997;13:1487-95.
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Table 3

* Federal regulations, import/export requirements, and taxonomic status are subject to changes. Check with the appropriate federal agency to confirm regulations.

Notes

1. Containment requirements will vary based on virus concentration, animal species, or virus type. See the Hantavirus agent summary statement in the viral agent chapter.
2. Tentative placement in the genus.
3. These organisms are considered pathogens of significant agricultural importance by the USDA (See Chapter 12) and may require additional containment (up to and including BSL-3-Ag containment). Not all strains of each organism are necessarily of concern to the USDA. Contact USDA for more information regarding exact containment/permit requirements before initiating work.
4. Alternate name for Ganjam virus.
5. Garissa virus is considered an isolate of this virus, so same containment requirements apply.
6. Antigenic groups designated A, B, and C refer to the original comprehensive and unifying serogroups established by Casals, Brown, and Whitman based on cross-reactivity among known arboviruses (2,21). Group A viruses are members of the genus Alphavirus, group B belong to the family Flaviviridae, and Group C viruses are members of the family Bunyaviridae.