Biological Safety Manual - Chapter 08: Agent Summary Statements (Section V: Viral Agents)

University-Policy EHS---Biological-Safety-Manual

Title

Agent: Hantaviruses
Agent: Hendra Virus (Formerly Known as Equine Morbillivirus) and Nipah Virus
Agent: Hepatitis A Virus, Hepatitis E Virus
Agent: Hepatitis B Virus, Hepatitis C Virus (Formerly Known as nonA nonB Virus), Hepatitis D Virus
Agent: Herpesvirus simiae (Cerocopithecine herpesvirus I, Herpes B Virus)
Agent: Human Herpes Virus
Agent: Influenza
Agent: Lymphocytic Choriomeningitis Virus
Agent: Poliovirus
Agent: Poxviruses
Agent: Rabies Virus (and Related Lyssaviruses)
Agent: Retroviruses, Including Human and Simian Immunodeficiency Viruses (HIV and SIV)
Agent: Severe Acute Respiratory Syndrome (SARS) Coronavirus
References
Section Va: Reassortant Influenza A Viruses

Agent: Hantaviruses

Hantaviruses are negative sense RNA viruses belonging to the genus Hantavirus within the family Bunyaviridae. The natural hosts of hantaviruses are rodent species and they occur worldwide. Hantavirus pulmonary syndrome (HPS) is a severe disease caused by hantaviruses such as Sin Nombre virus or Andes virus whose hosts are rodents in the sub familySigmodontinae. This subfamily only occurs in the New World, so HPS is not seen outside North and South America. Hantaviruses in Europe and Asia frequently cause kidney disease, called nephropathica epidemica in Europe, and hemorrhagic fever with renal syndrome (HFRS) in Asia.

Occupational Infections

Documented laboratory-acquired infections have occurred in individuals working with hantaviruses.^1-4 Extreme caution must be used in performing any laboratory operation that may create aerosols (centrifugation, vortex-mixing, etc.). Rats, voles, and other laboratory rodents, should be conducted with special caution because of the extreme hazard of aerosol infection, especially from infected rodent urine.

Natural Modes of Infection

HPS is a severe, often fatal disease that is caused by Sin Nombre and Andes or related viruses.^5,6 Most cases of human illness have resulted from exposures to naturally infected wild rodents or to their excreta. Person-to-person transmission does not occur, with the exception of a few rare instances documented for Andes virus.⁷ Arthropod vectors are not known to transmit hantaviruses.

Laboratory Safety

Laboratory transmission of hantaviruses from rodents to humans via the aerosol route is well documented.^4-7 Exposures to rodent excreta, especially aerosolized infectious urine, fresh necropsy material, and animal bedding are presumed to be associated with risk. Other potential routes of laboratory infection include ingestion, contact of infectious materials with mucous membranes or broken skin and, in particular, animal bites. Viral RNA has been detected in necropsy specimens and in patient blood and plasma obtained early in the course of HPS;^8,9 however, the infectivity of blood or tissues is unknown.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for laboratory handling of sera from persons potentially infected with hantaviruses. The use of a certified BSC is recommended for all handling of human body fluids when potential exists for splatter or aerosol.

Potentially infected tissue samples should be handled in BSL-2 facilities following BSL-3 practices and procedures. Cell-culture virus propagation and purification should be carried out in a BSL-3 facility using BSL-3 practices, containment equipment and procedures.

Experimentally infected rodent species known not to excrete the virus can be housed in ABSL-2 facilities using ABSL-2 practices and procedures. Primary physical containment devices including BSCs should be used whenever procedures with potential for generating aerosols are conducted. Serum or tissue samples from potentially infected rodents should be handled at BSL-2 using BSL-3 practices, containment equipment and procedures. All work involving inoculation of virus-containing samples into rodent species permissive for chronic infection should be conducted at ABSL-4.

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.

Agent: Hendra Virus (Formerly Known as Equine Morbillivirus) and Nipah Virus

Hendra virus and Nipah virus are members of a newly recognized genus called Henipavirus, within the family Paramyxoviridae. Outbreaks of a previously unrecognized paramyxovirus, at first called equine morbillivirus, later named Hendra virus, occurred in horses in Australia in 1994 and 1995. During 1998-1999 an outbreak of illness caused by a similar but distinct virus, now known as Nipah virus, occurred in Malaysia and Singapore. Human illness, characterized by fever, severe headache, myalgia and signs of encephalitis occurred in individuals in close contact with pigs (i.e., pig farmers and abattoir workers).^10-14 A few patients developed a respiratory disease. Approximately 40% of patients with encephalitis died. Recently, cases of Nipah virus infection were described in Bangladesh, apparently the result of close contact with infected fruit bats without an intermediate (e.g., pig) host.

Occupational Infections

No laboratory-acquired infections are known to have occurred as a result of Hendra or Nipah virus exposure; however, three people in close contact with ill horses developed encephalitis or respiratory disease and two died.^15-20

Natural Modes of Infection

The natural reservoir hosts for the Hendra and Nipah viruses appear to be fruit bats of the genus Pteropus.^21-23 Studies suggest that a locally occurring member of the genus, Pteropusgiganteus, is the reservoir for the virus in Bangladesh.²⁴ Individuals who had regular contact with bats had no evidence of infection (antibody) in one study in Australia.²⁵

Laboratory Safety

The exact mode of transmission of these viruses has not been established. Most clinical cases to date have been associated with close contact with horses, their blood or body fluids (Australia) or pigs (Malaysia/Singapore) but presumed direct transmission from Pteropus bats has been recorded in Bangladesh. Hendra and Nipah viruses have been isolated from tissues of infected animals. In the outbreaks in Malaysia and Singapore, viral antigen was found in central nervous system, kidney and lung tissues of fatal human cases²⁶ and virus was present in secretions of patients, albeit at low levels.²⁷ Active surveillance for infection of healthcare workers in Malaysia has not detected evidence of occupationally-acquired infections in this setting.²⁸

Containment Recommendations

Because of the unknown risks to laboratory workers and the potential impact on indigenous livestock should the virus escape a diagnostic or research laboratory, health officials and laboratory managers should evaluate the need to work with the virus and the containment capability of the facility before undertaking any work with Hendra, Nipah or suspected related viruses. BSL-4 is required for all work with these viruses. Once a diagnosis of Nipah or Hendra virus is suspected, all diagnostic specimens also must be handled at BSL-4. ABSL-4 is required for any work with infected animals.

Special Issues

Select Agent

Hendra and Nipah virus are Select Agents requiring registration with CDC and/or USDA for possession, use, storage and/or transfer. See Chapter 15 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. A DoC permit may be required for the export of this agent to another country. See Chapter 12 for additional information.

Agent: Hepatitis A Virus, Hepatitis E Virus

Hepatitis A virus is a positive single-stranded RNA virus, the type species of the Hepatovirus genus in the family Picornaviridae. Hepatitis E virus is a positive single stranded RNA virus, the type species of the genus Hepevirus, a floating genus not assigned to any family.

Occupational Infections

Laboratory-associated infections with hepatitis A or E viruses do not appear to be an important occupational risk among laboratory personnel. However, hepatitis A is a documented hazard in animal handlers and others working with naturally or experimentally infected chimpanzees and other nonhuman primates.²⁹ Workers handling other recently captured, susceptible primates (owl monkeys, marmosets) also may be at risk for hepatitis A infection. Hepatitis E virus appears to be less of a risk to personnel than hepatitis A virus, except during pregnancy, when infection can result in severe or fatal disease.

Natural Modes of Infection

Most infections with hepatitis A are foodborne and occasionally water-borne. The virus is present in feces during the prodromal phase of the disease and usually disappears once jaundice occurs. Hepatitis E virus causes acute enterically-transmitted cases of hepatitis, mostly waterborne. In Asia, epidemics involving thousands of cases have occurred.

Laboratory Safety

The agents may be present in feces and blood of infected humans and nonhuman primates. Feces, stool suspensions, and other contaminated materials are the primary hazards to laboratory personnel. Care should be taken to avoid puncture wounds when handling contaminated blood from humans or nonhuman primates. There is no evidence that aerosol exposure results in infection.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for the manipulation of hepatitis A and E virus, infected feces, blood or other tissues. ABSL-2 practices and facilities are recommended for activities using naturally or experimentally infected nonhuman primates or other animal models that may shed the virus.

Special Issues

Vaccines

A licensed inactivated vaccine against hepatitis A is available. Vaccines against hepatitis E are not currently available.

Transfer of Agent

Agent: Hepatitis B Virus, Hepatitis C Virus (Formerly Known as nonA nonB Virus), Hepatitis D Virus

Hepatitis B virus (HBV) is the type species of the Orthohepadnavirus genus in the family Hepadnaviridae. Hepatitis C virus (HCV) is the type species of the Hepacivirus genus in the family Flaviviridae. Hepatitis D virus (HDV) is the only member of the genus Deltavirus.

These viruses are naturally acquired from a carrier during blood transfusion, vaccination, tattooing, or ear piercing with inadequately sterilized instruments. Non-parenteral routes are also important, and cases may result from domestic and sexual contact, especially homosexual practices.

Individuals who are infected with the HBV are at risk of infection with HDV, a defective RNA virus that requires the presence of HBV virus for replication. Infection with HDV usually exacerbates the symptoms caused by HBV infection.

Occupational Infections

Hepatitis B has been one of the most frequently occurring laboratory-associated infections, and laboratory workers are recognized as a high-risk group for acquiring such infections.³⁰

Hepatitis C virus infection can occur in the laboratory situation as well.³¹ The prevalence of antibody to hepatitis C (anti-HCV) is slightly higher in medical care workers than in the general population. Epidemiologic evidence indicates that HCV is spread predominantly by the parenteral route.³²

Laboratory Safety

HBV may be present in blood and blood products of human origin, in urine, semen, CSF and saliva. Parenteral inoculation, droplet exposure of mucous membranes, and contact exposure of broken skin are the primary laboratory hazards.³³ The virus may be stable in dried blood or blood components for several days. Attenuated or avirulent strains have not been identified.

HCV has been detected primarily in blood and serum, less frequently in saliva and rarely or not at all in urine or semen. It appears to be relatively unstable to storage at room temperature and repeated freezing and thawing.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious body fluids and tissues. Additional primary containment and personnel precautions, such as those described for BSL-3, may be indicated for activities with potential for droplet or aerosol production and for activities involving production quantities or concentrations of infectious materials. ABSL-2 practices, containment equipment and facilities are recommended for activities utilizing naturally or experimentally infected chimpanzees or other NHP. Gloves should be worn when working with infected animals and when there is the likelihood of skin contact with infectious materials. In addition to these recommended precautions, persons working with HBV, HCV, or other bloodborne pathogens should consult the OSHA Bloodborne Pathogen Standard.³⁴ Questions related to interpretation of this Standard should be directed to federal, regional or state OSHA offices.

Special Issues

Vaccines

Licensed recombinant vaccines against hepatitis B are available and are highly recommended for and offered to laboratory personnel.³⁵ Vaccines against hepatitis C and D are not yet available for use in humans, but vaccination against HBV will also prevent HDV infection.

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: Herpesvirus simiae (Cerocopithecine herpesvirus I, Herpes B Virus)

B virus is a member of the alphaherpesvirus genus (simplexvirus) in the family Herpesviridae. It occurs naturally in macaque monkeys, of which there are nine distinct species. Macaques may have primary, recurrent, or latent infections often with no apparent symptoms or lesions. B virus is the only member of the family of simplex herpesviruses that can cause zoonotic infections. Human infections have been identified in at least 50 instances, with approximately 80% mortality when untreated. There remains an approximate 20% mortality in the absence of timely treatment with antiviral agents.³⁶

There have been no reported cases in situations where prompt first aid with wound or exposure site cleansing was performed, and no cases where cleaning and post exposure prophylaxis were done. Cases prior to 1970 were not treated with antiviral agents, since at this time they were unavailable. Morbidity and mortality associated with zoonotic infection results from invasion of the central nervous system, resulting in ascending paralysis ultimately with loss of ability to sustain respiration in the absence of mechanical ventilation. From 1987-2004, five additional fatal infections bring the number of lethal infections to 29 since the discovery of B virus in 1933.

Occupational Infections

B virus is a hazard in facilities where macaque monkeys are present. Mucosal secretions (saliva, genital secretions, and conjunctival secretions) are the primary body fluids associated with risk of B virus transmission. However, it is possible for other materials to become contaminated. For instance, a research assistant at the Yerkes Primate Center who died following mucosal splash without injury in 1997 was splashed with something in the eye while transporting a caged macaque. In part on this basis, the eye splash was considered low risk. However, feces, urine or other fluids may be contaminated with virus shed from mucosal fluids. Zoonoses have been reported following virus transmission through a bite, scratch, or splash accident. Cases of B virus have also been reported after exposure to monkey cell cultures and to central nervous system tissue.

There is often no apparent evidence of B virus infection in the animals or their cells and tissues, making it imperative that all suspect exposures be treated according to recommended standards.³⁶The risks associated with this hazard are, however, readily reduced by practicing barrier precautions and by rapid and thorough cleansing immediately following a possible site contamination. Precautions should be followed when work requires the use of any macaque species, even antibody negative animals. In most documented cases of B virus zoonosis, virus was not recovered from potential sources except in four cases, making speculations that some macaque species may be safer than others unfounded. The loss of five lives in the past two decades underscores that B virus infections have a low probability of occurrence, but when they do occur it is with high consequences.

Specific, regular training in risk assessments for B virus hazards including understanding the modes of exposure and transmission should be provided to individuals encountering B virus hazards. This training should include proper use of personal protective equipment, which is essential to prevention. Immediate and thorough cleansing following bites, scratches, splashes, or contact with potential fomites in high-risk areas appears to be helpful in prevention of B virus infections.³⁷ First aid and emergency medical assistance procedures are most effective when institutions set the standard to be practiced by all individuals encountering B virus hazards.

Natural Modes of Infection

B virus occurs as a natural infection of Asiatic macaque monkeys, and some 10% of newly caught rhesus monkeys have antibodies against the virus, which is frequently present in kidney cell cultures of this animal. Reservoir species include Macaca mulatta, M. fascicularis, M. fusata, M. arctoides, M. cyclopsis and M. radiata. In these species the virus causes vesicular lesions on the tongue and lips, and sometimes of the skin. B virus is not present in blood or serum in infected macaques. Transmission of B virus appears to increase when macaques reach sexual maturity.

Laboratory Safety

The National Academies Press has recently published ILAR's guidelines for workings with nonhuman primates.³⁸ Additional resources are provided in the references following this agent summary. Asymptomatic B virus shedding accounts for most transmission among monkeys and human workers, but those working in the laboratory with potentially infected cells or tissues from macaques are also at risk. Exposure of mucous membranes or through skin breaks provides this agent access to a new host, whether the virus is being shed from a macaque or human, or present in or on contaminated cells, tissues, or surfaces.³⁶ B virus is not generally found in serum or blood, but these products obtained through venipuncture should be handled carefully because contamination of needles via skin can occur. When working with macaques directly, virus can be transmitted through bites, scratches, or splashes only when the animal is shedding virus from mucosal sites.

Fomites, or contaminated surfaces, e.g., cages, surgical equipment, tables, etc. should always be considered sources of B virus unless verified as decontaminated or sterilized. Zoonotically infected humans should be cautioned about autoinoculation of other susceptible sites when shedding virus during acute infection.

Containment Recommendations

BSL-2 practices and facilities are suitable for all activities involving the use or manipulation of tissues, cells, blood, or serum from macaques with appropriate personal protective equipment. BSL-3 practices are recommended for handling materials from which B virus is being cultured using appropriate personal protective equipment, and BSL-4 facilities are recommended for propagation of virus obtained from diagnostic samples or stocks. Experimental infections of macaques as well as small animal models with B virus are recommended to be restricted to BSL-4 containment.

All macaques regardless of their origin should be considered potentially infected. Animals with no detectable antibody are not necessarily B virus-free. Macaques should be handled with strict barrier precaution protocols and injuries should be tended immediately according to the recommendations of the B Virus Working Group led by NIH and CDC.³⁶ Barrier precautions and appropriate first aid are the keys to prevention of severe morbidity and mortality often associated with B virus zoonoses. These prevention tools were not implemented in each of the five of B virus fatalities during the past two decades. Guidelines are available for safely working with macaques and should be consulted.^36,39 The correct use of gloves, masks, and protective coats, gowns, aprons, or overalls is recommended for all personnel while working with non- human primates, especially macaques and other Old World species, including for all persons entering animal rooms where non-human primates are housed. To minimize the potential for mucous membrane exposure, some form of barrier is required to prevent droplet splashes to eyes, mouth, and nasal passages. Types and use of personal protective equipment, e.g., goggles or glasses with solid side shields and masks, or wrap-around face shields should be determined with reference to the institutional risk assessment. Specifications of protective equipment must be balanced with the work to be performed so that the barriers selected do not increase work place risk by obscuring vision and contributing to increased risk of bites, needle sticks,scratches, or splashes.

Special Issues

Post-exposure prophylaxis with oral acyclovir or valacyclovir should be considered for significant exposures to B virus. Therapy with intravenous acyclovir and/or ganciclovir in documented B virus infections is also important in reduction of morbidity following B virus zoonotic infection.³⁶ In selected cases, IND permission has been granted for therapy with experimental antiviral drugs. Because of the seriousness of B virus infection, experienced medical and laboratory personnel should be consulted to develop individual case management. Barrier precautions should be observed with confirmed cases. B virus infection, as with all alphaherpes viruses, is lifelong in macaques⁴⁰ and there are no effective vaccines available at present.

Select Agent

B virus is a Select Agent requiring registration with CDC and/or USDA for possession, use, storage and/or transfer. See Chapter 15 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: Human Herpes Virus

The herpesviruses are ubiquitous human pathogens and are commonly present in a variety of clinical materials submitted for virus isolation. Thus far, nine herpesviruses have been isolated from humans: herpes simplex virus-1(HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella- zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesviruses (HHV) 6A, 6B, 7, and 8.⁴¹

HSV infection is characterized by a localized primary lesion. Primary infection with HSV-1 may be mild and unapparent occurring in early childhood. In approximately 10% of infections overt illness marked by fever and malaise occurs. HSV-1 is a common cause of meningoencephalitis. Genital infections, usually caused by HSV-2, generally occur in adults and are sexually transmissible. Neonatal infections are most frequently caused by HSV-2 but HSV-1 infections are also common. In the neonate, disseminated disease and encephalitis are often fatal. EBV is the cause of infectious mononucleosis. It is also associated the pathogenesis of several lymphomas and nasopharyngeal cancer.⁴² EBV is serologically distinct from the other herpesviruses; it infects and transforms Blymphocytes. HCMV infection is common and often undiagnosed presenting as a nonspecific febrile illness. HCMV causes up to 10% of all cases of mononucleosis in young adults. The most severe form of the disease is seen in infants infected in utero. Children surviving infection may evidence mental retardation, microencephaly, motor disabilities and chronic liver disease.⁴² HCMV is one of the most common congenital diseases.

VZV is the causative agent of chickenpox and herpes zoster. Chickenpox usually occurs in childhood and zoster occurs more commonly in adults. HHV-6 is the causative agent of exanthema subitum (roseola), a common childhood exanthem.⁴³ Nonspecific febrile illness and febrile seizures are also clinical manifestations of disease. HHV-6 may reactivate in immunocompetent individuals during pregnancy or during critical illness. Two distinct variants, HHV-6A and HHV-6B, exist; the latter causing roseola. HHV-7 is a constitutive inhabitant of adult human saliva.⁴⁴ Clinical manifestations are less well understood but the virus has also been associated with roseola. HHV-8, also known as Kaposi's sarcoma-associated virus, was first identified by Chang and co-workers in 1994.⁴² HHV-8 is believed to be the causative agent of Kaposi's sarcoma and has been associated with primary effusion lymphoma.⁴⁵The natural history of HHV-8 has not been completely elucidated. High risk groups for HHV-8 include HIV- infected men who have sex with men and individuals from areas of high endemicity such as Africa or the Mediterranean.⁴⁵ The prevalence of HHV-8 is also higher among intravenous drug users than in the general population.⁴⁵ At least one report has provided evidence that, in African children, HHV-8 infection may be transmitted from mother to child.⁴⁶ While few of the human herpesviruses have been demonstrated to cause laboratory-acquired infections, they are both primary and opportunistic pathogens, especially in immunocompromised hosts. Herpesvirus simiae (B-virus, Monkey B virus) is discussed separately in another agent summary statement in this chapter.

Occupational Infections

Few of the human herpesviruses have been documented as sources of laboratory acquired infections. In a limited study, Gartner and co-workers have investigated the HHV-8 immunoglobulin G (IgG) seroprevalence rates for healthcare workers caring for patients with a high risk for HHV-8 infection in a non-endemic area. Healthcare workers in contact with risk group patients were infected more frequently than healthcare workers without contact with risk groups. Workers without contact with risk group patients were infected no more frequently than the control group.⁵³

Although this diverse group of indigenous viral agents has not demonstrated a high potential hazard for laboratory-associated infection, frequent presence in clinical materials and common use in research warrant the application of appropriate laboratory containment and safe practices.

Natural Modes of Infection

Given the wide array of viruses included in this family, the natural modes of infection vary greatly, as does the pathogenesis of the various viruses. Some have wide host ranges, multiply effectively, and rapidly destroy the cells they infect (HSV-1, HSV-2). Others have restricted host ranges or long replicative cycles (HHV-6).⁴¹ Transmission of human herpesviruses in nature are, in general, associated with contact close, intimate contact with a person excreting the virus in their saliva, urine, or other bodily fluids.⁴⁷ VZV is transmitted person-to-person through direct contact, through aerosolized vesicular fluids and respiratory secretions, and indirectly transmitted by fomites. Latency is a trait common to most herpesviruses, although the site and duration vary greatly. For example, EBV will persist in an asymptomatic, latent form in the host immune system, primarily in EBV-specific cytotoxic T cells⁴² while latent HSV has been detected only in sensory neurons.^48,49 HHV-8 has been transmitted through organ transplantation⁵⁰ and blood transfusion;⁵¹ some evidence suggests non-sexual horizontal transmission.⁵²

Laboratory Safety

Clinical materials and isolates of herpesviruses may pose a risk of infection following ingestion, accidental parenteral inoculation, and droplet exposure of the mucous membranes of the eyes, nose, or mouth, or inhalation of concentrated aerosolized materials. HHV-8 may be present in human blood or blood products and tissues or saliva. Aerosol transmission cannot be excluded as a potential route of transmission. Clinical specimens containing the more virulent Herpesvirus simiae (B-virus) may be inadvertently submitted for diagnosis of suspected herpes simplex infection. HCMV may pose a special risk during pregnancy because of potential infection of the fetus. All human herpesviruses pose an increased risk to persons who are immunocompromised.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for activities utilizing known or potentially infectious clinical materials or cultures of indigenous viral agents that are associated or identified as a primary pathogen of human disease. Although there is little evidence that infectious aerosols are a significant source of LAI, it is prudent to avoid the generation of aerosols during the handling of clinical materials or isolates, or during the necropsy of animals. Primary containment devices (e.g., BSC) should be utilized to prevent exposure of workers to infectious aerosols. Additional containment and procedures, such as those described for BSL-3, should be considered when producing, purifying, and concentrating human herpesviruses, based on risk assessment.

Containment recommendations for herpesvirus simiae (B-virus, Monkey B virus) are described separately in another agent summary statement in this chapter.

Special Issues

Vaccine

A live, attenuated vaccine for varicella zoster is licensed and available in the United States. In the event of a laboratory exposure to a non-immune individual, varicella vaccine is likely to prevent or at least modify disease.⁴⁷

Treatment

Antiviral medications are available for treatment of several of the herpesviruses.

Transfer of Agent

Agent: Influenza

Influenza is an acute viral disease of the respiratory tract. The most common clinical manifestations are fever, headache, malaise, sore throat and cough. GI tract manifestations (nausea, vomiting and diarrhea) are rare but may accompany the respiratory phase in children. The two most important features of influenza are the epidemic nature of illness and the mortality that arises from pulmonary complications of the disease.⁵⁴

The influenza viruses are enveloped RNA viruses belonging to the Orthomyxoviridae. There are three serotypes of influenza viruses, A, B and C. Influenza A is further classified into subtypes by the surface glycoproteins that possess either hemagglutinin (H) or neuraminidase (N) activity. Emergence of completely new subtypes (antigenic shift) occurs at irregular intervals with Type A viruses. New subtypes are responsible for pandemics and can result from reassortment of human and avian influenza virus genes. Antigenic changes within a type or subtype (antigenic drift) of A and B viruses are ongoing processes that are responsible for frequent epidemics and regional outbreaks and make the annual reformulation of influenza vaccine necessary.

Influenza viral infections, with different antigenic subtypes, occur naturally in swine, horses, mink, seals and in many domestic and wild avian species. Interspecies transmission and reassortment of influenza A viruses have been reported to occur among, humans and wild and domestic fowl. The human influenza viruses responsible for the 1918, 1957 and 1968 pandemics contained gene segments closely related to those of avian influenza viruses.⁵⁵ Swine influenza has also been isolated in human outbreaks.⁵⁶ Control of influenza is a continuing human and veterinary public health concern.

Occupational Infections

LAI have not been routinely documented in the literature, but informal accounts and published reports indicate that such infections are known to have occurred, particularly when new strains showing antigenic shift or drift are introduced into a laboratory for diagnostic/research purposes.⁵⁶ Occupationally-acquired, nosocomial infections are documented.^57,58Laboratory animal-associated infections have not been reported; however, there is possibility of human infection acquired from infected ferrets and vice versa.

Natural Modes of Infection

Airborne spread is the predominant mode of transmission especially in crowded, enclosed spaces. Transmission may also occur through direct contact since influenza viruses may persist for hours on surfaces particularly in the cold and under conditions of low humidity.⁵⁵ The incubation period is from one to three days. Recommendations for treatment and prophylaxis of influenza are available.⁵⁹

Laboratory Safety

The agent may be present in respiratory tissues or secretions of humans and most infected animals and birds. In addition, the agent may be present in the intestines and cloacae of many infected avian species. Influenza viruses may be disseminated in multiple organs in some infected animal species. The primary laboratory hazard is inhalation of virus from aerosols generated by infecting animals or by aspirating, dispensing, mixing, centrifuging or otherwise manipulating virus-infected samples. In addition, laboratory infection can result from direct inoculation of mucus membranes through virus contaminated gloves following handling of tissues, feces or secretions from infected animals. Genetic manipulation has the potential for altering the host range, pathogenicity, and antigenic composition of influenza viruses. The potential for introducing influenza viruses with novel genetic composition into humans is unknown.

Containment Recommendations

BSL-2 facilities, practices and procedures are recommended for diagnostic, research and production activities utilizing contemporary, circulating human influenza strains (e.g., H1/H3/B) and low pathogenicity avian influenza (LPAI) strains (e.g., H1-4, h4, H8-16), and equine and swine influenza viruses. ABSL-2 is appropriate for work with these viruses in animal models. All avian and swine influenza viruses require an APHIS permit. Based on economic ramifications and source of the virus, LPAI H5 and H7 and swine influenza viruses may have additional APHIS permit-driven containment requirements and personnel practices and/or restrictions.

Non-Contemporary Human Influence (H2N2) Strains

Non-contemporary, wild-type human influenza (H2N2) strains should be handled with increased caution. Important considerations in working with these strains are the number of years since an antigenically related virus last circulated and the potential for presence of a susceptible population. BSL-3 and ABSL-3 practices, procedures and facilities are recommended with rigorous adherence to additional respiratory protection and clothing change protocols. Negative pressure, HEPA-filtered respirators or positive air-purifying respirators (PAPRs) are recommended for use. Cold-adapted, live attenuated H2N2 vaccine strains may continue to be worked with at BSL-2.

1918 Influenza strain

Any research involving reverse genetics of the 1918 influenza strain should proceed with extreme caution. The risk to laboratory workers is unknown at the present time, but the pandemic potential is thought to be significant. Until further risk assessment data are available, the following practices and conditions are recommended for manipulation of reconstructed 1918 influenza viruses and laboratory animals infected with the viruses. These practices and procedures are considered minimum standards for work with the fully reconstructed virus.

BSL-3 and ABSL-3 practices, procedures and facilities.
Large laboratory animals such as NHP should be housed in primary barrier systems in ABSL- 3 facilities.
Rigorous adherence to additional respiratory protection and clothing change protocols.
Use of negative pressure, HEPA-filtered respirators or PAPRs.
Use of HEPA filtration for treatment of exhaust air.
Amendment of personnel practices to include personal showers prior to exiting the laboratory.

Highly Pathogenic Avian Influenza (HPAI)

Manipulating HPAI viruses in biomedical research laboratories requires similar caution because some strains may pose increased risk to laboratory workers and have significant agricultural and economic implications. BSL-3 and ABSL-3 practices, procedures and facilities are recommended along with clothing change and personal showering protocols. Loose-housed animals infected with HPAI strains must be contained within BSL-3-Ag facilities (See Chapter 13). Negative pressure, HEPA-filtered respirators or positive airpurifying respirators are recommended for HPAI viruses with potential to infect humans. The HPAI are agricultural Select Agents requiring registration of personnel and facilities with the lead agency for the institution (CDC or USDA-APHIS). An APHIS permit is also required. Additional containment requirements and personnel practices and/or restrictions may be added as conditions of the permit.

Other Influenza Recombinant or Reassortant Viruses

When considering the biocontainment level and attendant practices and procedures for work with other influenza recombinant or reassortant viruses, the local IBC should consider but not limit consideration to the following in the conduct of protocol-driven risk assessment.

The gene constellation used.
Clear evidence of reduced virus replication in the respiratory tract of appropriate animal models, compared with the level of replication of the wildtype parent virus from which it was derived.
Evidence of clonal purity and phenotypic stability.
The number of years since a virus that was antigenically related to the donor of the hemagglutinin and neuraminidase genes last circulated.

If adequate risk assessment data are not available, a more cautious approach utilizing elevated biocontainment levels and practices is warranted. There may be specific requirements regarding the setting of containment levels if your institution is subject to the NIH Guidelines.

Special Issues

Occupational Health Considerations

Institutions performing work with HPAI and avian viruses that have infected humans; non-contemporary wild-type human influenza strains, including recombinants and reassortants; and viruses created by reverse genetics of the 1918 pandemic strain should develop and implement a specific medical surveillance and response plan. At the minimum these plans should:

require storage of baseline serum samples from individuals working with these influenza strains
strongly recommend annual vaccination with the currently licensed influenza vaccine for such individuals
provide employee counseling regarding disease symptoms including fever, conjunctivitis and respiratory symptoms
establish a protocol for monitoring personnel for these symptoms
establish a clear medical protocol for responding to suspected laboratory-acquired infections

Antiviral drugs (e.g., oseltamivir, amantadine, rimantadine, zanamivir) should be available for treatment and prophylaxis, as necessary.⁵⁹ It is recommended that the sensitivities of the virus being studied to the antivirals be ascertained. All personnel should be enrolled in an appropriately constituted respiratory protection program.

Influenza viruses may require USDA and/or USPHS import permits depending on the host range and pathogenicity of the virus in question.

Select Agent

Strains of HPAI and 1918 influenza 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

Agent: Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) is a rodent-borne viral infectious disease that presents as aseptic meningitis, encephalitis, or meningoencephalitis. The causative agent is the LCM virus (LCMV) that was initially isolated in 1933. The virus is the protypical member of the family Arenaviridae.

Occupational Infections

LAI with LCM virus are well documented. Most infections occur when chronic viral infection exists in laboratory rodents, especially mice, hamsters and guinea pigs.^60-62 Nude and severe combined immune deficient (SCID) mice may pose a special risk of harboring silent chronic infections. Inadvertently infected cell cultures also represent a potential source of infection and dissemination of the agent

Natural Modes of Infection

LCM and milder LCMV infections have been reported in Europe, the Americas, Australia, and Japan, and may occur wherever infected rodent hosts of the virus are found. Several serologic studies conducted in urban areas have shown that the prevalence of LCMV infection among humans ranges from 2% to 10%. Seroprevalence of 37.5% has been reported in humans in the Slovak Republic.⁶³

The common house mouse, Mus musculus, naturally spreads LCMV. Once infected, these mice can become chronically infected as demonstrated by the presence of virus in blood and/or by persistently shedding virus in urine. Infections have also occurred in NHP in zoos, including macaques and marmosets (Callitrichid hepatitis virus is a LCMV).

Humans become infected by inhaling infectious aerosolized particles of rodent urine, feces, or saliva, by ingesting food contaminated with virus; by contamination of mucous membranes with infected body fluids; or by directly exposing cuts or other open wounds to virus-infected blood. Four recipients of organs from a donor who had unrecognized disseminated LCMV infection sustained severe disease and three succumbed. The source of donor infection was traced to a pet hamster that was not overtly ill.⁶⁴

Laboratory Safety

The agent may be present in blood, CSF, urine, secretions of the nasopharynx, feces and tissues of infected animal hosts and humans. Parenteral inoculation, inhalation, contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals are common hazards. Aerosol transmission is well documented.⁶⁰ Of special note, tumors may acquire LCMV as an adventitious virus without obvious effects on the tumor. Virus may survive freezing and storage in liquid nitrogen for long periods. When infected tumor cells are transplanted, subsequent infection of the host and virus excretion may ensue. Pregnant women infected with LCMV have transmitted the virus to their fetus with death or serious central nervous system malformation as a consequence.⁶⁵

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are suitable for activities utilizing known or potentially infectious body fluids, and for cell culture passage of laboratory adapted strains. BSL-3 is required for activities with high potential for aerosol production, work with production quantities or high concentrations of infectious materials, and for manipulation of infected transplantable tumors, field isolates and clinical materials from human cases. Strains of LCMV that are shown to be lethal in nonhuman primates should be handled at BSL-3. ABSL-2 practices, containment equipment, and facilities are suitable for studies in adult mice with strains requiring BSL-2 containment. Work with infected hamsters also should be done at ABSL-3.

Special Issues

Vaccines

Vaccines are not available for use in humans.

Transfer of Agent

Agent: Poliovirus

Poliovirus is the type species of the Enterovirus genus in the familyPicornaviridae. Enteroviruses are transient inhabitants of the gastrointestinal tract, and are stable at acid pH. Picornaviruses are small, ether-insensitive viruses with an RNA genome. There are three poliovirus serotypes (P1, P2, and P3). Immunity to one serotype does not produce significant immunity to the other serotypes.

Occupational Infections

Laboratory-associated poliomyelitis is uncommon. Twelve cases, including two deaths, were reported between 1941 and 1976.^62,66 No laboratory-associated poliomyelitis has been reported for nearly 30 years. Both inactivated poliovirus vaccine (IPV) and oral poliovirus vaccine (OPV) are highly effective in preventing disease, but neither vaccine provides complete protection against infection. Poliovirus infections among immunized laboratory workers are uncommon but remain undetermined in the absence of laboratory confirmation. An immunized laboratory worker may unknowingly be a source of poliovirus transmission to unvaccinated persons in the community.⁶⁷

Natural Modes of Infection

At one time poliovirus infection occurred throughout the world. Transmission of wild poliovirus ceased in the United States in 1979 or possibly earlier. A polio eradication program conducted by the Pan American Health Organization led to elimination of polio from the Western Hemisphere in 1991. The Global Polio Eradication Program has dramatically reduced poliovirus transmission throughout the world.

Humans are the only known reservoir of poliovirus, which is transmitted most frequently by persons with inapparent infections. Person-to-person spread of poliovirus via the fecal-oral route is the most important route of transmission, although the oral-oral route may account for some cases.

Laboratory Safety

The agent is present in the feces and in throat secretions of infected persons and in lymph nodes, brain tissue, and spinal cord tissue in fatal cases. For non-immunized persons in the laboratory, ingestion or parenteral inoculation are the primary routes of infection. For immunized persons, the primary risks are the same, except for parenteral inoculation, which likely presents a lower risk. The importance of aerosol exposure is unknown. Laboratory animal- associated infections have not been reported, but infected nonhuman primates should be considered to present a risk.

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for all activities utilizing wild poliovirus infectious culture fluids, environmental samples, and clinical materials, in addition, potentially infectious materials collected for any purpose should be handled at BSL- 2. Laboratory personnel working with such materials must have documented polio vaccination. Persons who have had a primary series of OPV or IPV and who are at an increased risk can receive another dose of IPV, but available data do not indicate the need for more than a single lifetime IPV booster dose for adults. ⁶⁸ ABSL-2 practices, containment equipment, and facilities are recommended for studies of virulent viruses in animals. Laboratories should use authentic Sabin OPV attenuated strains unless there are strong scientific reasons for working with wild polioviruses. In anticipation of polio eradication, the WHO recommends destruction of all poliovirus stocks and potential infectious materials if there is no longer a programmatic or research need for such materials.⁶⁹Institutions/laboratories in the United States that currently retain wild poliovirus infectious or potential infectious material should be on the United States National Inventory maintained by CDC. When one year has elapsed after detection of the last wild poliovirus worldwide, CDC will inform relevant institutions/laboratories about additional containment procedures. Safety recommendations are subject to change based on international polio eradication activities.

Special Issues

When OPV immunization stops, global control and biosafety requirements for wild as well as attenuated (Sabin) poliovirus materials are expected to become more stringent, consistent with the increased consequences of inadvertent transmission to a growing susceptible community.

Transfer of Agent

Agent: Poxviruses

Four genera of the subfamily Chordopoxvirinae, family Poxviridae, (Orthopoxvirus, Parapoxvirus, Yatapoxvirus, and Molluscipoxvirus) contain species that can cause lesions on human skin or mucous membranes with mild to severe systemic rash illness in laboratorians. Species within the first three genera mostly arise as zoonotic agents.^70,71 Laboratory-acquired poxvirus infections of most concern are from the orthopoxviruses that infect humans: variola virus (causes smallpox; human-specific), monkeypox virus (causes smallpox-like disease, cowpox virus (causes skin pustule, generalized rash), and vaccinia virus (causes skin pustule, systemic illness).^70-75

Occupational Infections

Vaccinia virus, the leading agent of laboratory-acquired poxvirus infections, is used to make the current smallpox vaccine and may occur as a rare zoonosis.^70,71 Laboratory acquired infections with standard, mutant, or bioengineered forms of vaccinia virus have occurred, even in previously vaccinated laboratorians. In addition, vaccination with live vaccinia virus sometimes has side effects, which range from mild events (e.g., fever, fatigue, swollen lymph nodes) to rare, severe, and at times fatal outcomes (e.g., generalized vaccinia, encephalitis, vaccinia necrosum, eczema vaccinatum, ocular keratitis, corneal infection, fetal infection of pregnancy, and possibly myocardial infarction, myopericarditis, or angina), thus vaccination contraindications should be carefully followed.^70,73-75

Natural Modes of Infection

Smallpox has been eradicated from the world since 1980, but monkey pox virus is endemic in rodents in parts of Africa. Importation of African rodents into North America in 2003 resulted in an outbreak of monkeypox in humans.⁷² Molluscum contagiosum, a disease due to Molluscipoxvirus infection, results in pearly white lesions that may persist for months in persons immunocompromised for various reasons, including chronic illness, AIDS, other infections, medications, cancer and cancer therapies, or pregnancy.⁷⁰

Laboratory Safety

Poxviruses are stable in a wide range of environmental temperatures and humidity and may be transmitted by fomites.⁷⁰ Virus may enter the body through mucous membranes, broken skin, or by ingestion, parenteral inoculation or droplet or fine-particle aerosol inhalation. Sources of laboratory-acquired infection include exposure to aerosols, environmental samples, naturally or experimentally infected animals, infectious cultures, or clinical samples, including vesiculopustular rash lesion fluid or crusted scabs, various tissue specimens, excretions and respiratory secretions.

Containment Recommendations

Worldwide, all live variola virus work is to be done only within WHO approved BSL-4/ABSL-4 facilities; one is at the CDC in Atlanta and the other is at the State Research Center of Virology and Biotechnology (VECTOR) in Koltsovo, Russia.⁷⁶

In general, all persons working in or entering laboratory or animal care areas where activities with vaccinia, monkey pox, or cow pox viruses are being conducted should have evidence of satisfactory vaccination. Vaccination is advised every three years for work with monkeypox virus and every 10 years for cowpox and vaccinia viruses (neither vaccination nor vaccinia immunoglobulin protect against poxviruses of other genera).^73-75 Vaccination is not required for individuals working only in laboratories where no other orthopoxviruses or recombinants are handled.⁷⁵

ABSL-3 practices, containment equipment, and facilities are recommended for monkeypox work in experimentally or naturally infected animals. BSL-2 facilities with BSL-3 practices are advised if other work with monkeypox virus is performed by vaccinated personnel. These practices include the use of Class-I or -II BSCs and barriers, such as safety cups or sealed rotors, for all centrifugations. The NIH Guidelines have assessed the risk of manipulating attenuated vaccinia strains (modified virus Ankara (MVA), NYVAC, TROVAC, and ALVAC) in areas where no other human orthopoxviruses are being used and have recommended BSL-1.⁷⁶ However, higher levels of containment are recommended if these strains are used in work areas where other orthopoxviruses are manipulated. BSL-2 and ABSL-2 plus vaccination, are recommended for work with most other poxviruses.

Special Issues

Other Considerations

The CDC provides information on poxviruses, especially variola and monkeypox viruses, and on smallpox vaccination, and for reporting vaccination adverse events. Clinical and other laboratories using poxviruses and clinicians can phone the CDC Clinician Information Line (877-554-4625) and/or the CDC public information hotline (888-246-2675) concerning variola and other human poxvirus infections, smallpox vaccine, vaccinia immunoglobulin, poxvirus antiviral drugs, or other treatments or quarantine issues. Contact CDC regarding applications to transfer monkeypox viruses.

Transfer of Agent

Agent: Rabies Virus (and Related Lyssaviruses)

Rabies is an acute, progressive, fatal encephalitis caused by negative-stranded RNA viruses in the genus Lyssavirus, family Rhabdoviridae.⁷⁷ Rabies virus is the representative member (type species) of the genus. Members of the group include Australian bat lyssavirus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Lagos bat virus, and Mokola virus.

Occupational Infections

LAI are extremely rare; two have been documented. Both resulted from presumed exposure to high concentrations of infectious aerosols, one generated in a vaccine production facility,⁷⁸ and the other in a research facility.⁷⁹ Naturally or experimentally infected animals, their tissues, and their excretions are a potential source of exposure for laboratory and animal care personnel.

Natural Modes of Infection

The natural hosts of rabies are many bat species and terrestrial carnivores, but most mammals can be infected. The saliva of infected animals is highly infectious, and bites are the usual means of transmission, although infection through superficial skin lesions or mucosa is possible.

Laboratory Safety

When working with infected animals, the highest viral concentrations are present in central nervous system (CNS) tissue, salivary glands, and saliva, but rabies viral antigens may be detected in all innervated tissues. Accidental parenteral inoculation, cuts, or needle sticks with contaminated laboratory equipment, bites by infected animals, and exposure of mucous membranes or broken skin to infectious tissue or fluids, are the most likely sources for exposure of laboratory and animal care personnel. Infectious aerosols have not been a demonstrated hazard to personnel working with routine clinical materials and conducting diagnostic examinations. Fixed and attenuated strains of virus are presumed to be less hazardous, but the two recorded cases of laboratory-associated rabies resulted from presumed exposure to the fixed Challenge Virus Standard and Street Alabama Dufferin strains, respectively.

Containment Recommendations

BSL-2 and/or ABSL-2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious materials or animals. Pre exposure rabies vaccination is recommended for all individuals prior to working with lyssaviruses or infected animals, or engaging in diagnostic, production, or research activities with these viruses.⁸⁰ Rabies vaccination also is recommended for all individuals entering or working in the same room where lyssaviruses or infected animals are used.

Prompt administration of post exposure booster vaccinations is recommended following recognized exposures in previously vaccinated individuals per current guidelines.⁸¹ For routine diagnostic activities, it is not always feasible to open the skull or remove the brain of an infected animal within a BSC, but it is pertinent to use appropriate methods and personal protection equipment, including dedicated laboratory clothing, heavy protective gloves to avoid cuts or sticks from cutting instruments or bone fragments, and a face shield or PAPR to protect the skin and mucous membranes of the eyes, nose, and mouth from exposure to tissue fragments or infectious droplets.

If a Stryker saw is used to open the skull, avoid contacting brain tissue with the blade of the saw. Additional primary containment and personnel precautions, such as those described for BSL-3, are indicated for activities with a high potential for droplet or aerosol production, and for activities involving large production quantities or high concentrations of infectious materials.

Special Issues

Transfer of Agent

Agent: Retroviruses, Including Human and Simian Immunodeficiency Viruses (HIV and SIV)

The family Retroviridae is divided into two subfamilies, the Orthoretrovirinaewith six genera including the Lentivirus genus which includes HIV-1 and HIV-2. Other important human pathogens are human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2), members of the Deltaretrovirus genus, and theSpumaretrovirinae with one genus Spumavirus containing a variety of NHP viruses (foamy viruses) which can occasionally infect humans in close contact with NHP.

Occupational Infections

Data on occupational HIV transmission in laboratory workers are collected through two CDC- supported national surveillance systems: surveillance for:

AIDS and
HIV-infected persons who may have acquired their infection through occupational exposures.

For surveillance purposes, laboratory workers are defined as those persons, including students and trainees, who have worked in a clinical or HIV laboratory setting anytime since 1978. Cases reported in these two systems are classified as either documented or possible occupational transmission. Those classified as documented occupational transmission had evidence of HIV seroconversion (a negative HIV-antibody test at the time of the exposure which converted to positive) following a discrete percutaneous or mucocutaneous occupational exposure to blood, body fluids, or other clinical or laboratory specimens. As of June 1998, CDC had reports of 16 laboratory workers (all clinical) in the United States with documented occupational transmission.⁸²

Workers have been reported to develop antibodies to simian immunodeficiency virus (SIV) following exposures. One case was associated with a needle-stick that occurred while the worker was manipulating a blood-contaminated needle after bleeding an SIVinfected macaque monkey.⁸³ Another case involved a laboratory worker who handled macaque SIV-infected blood specimens without gloves. Though no specific incident was recalled, this worker had dermatitis on the forearms and hands while working with the infected blood specimens.⁸⁴ A third worker⁸⁵ was exposed to SIV-infected primate blood through a needle-stick and subsequently developed antibodies to SIV. To date there is no evidence of illness or immunological incompetence in any of these workers.

Natural Modes of Infection

Retroviruses are widely distributed as infectious agents of vertebrates. Within the human population spread is by close sexual contact or parenteral exposure through blood or blood products.

Laboratory Safety

HIV has been isolated from blood, semen, saliva, tears, urine, CSF, amniotic fluid, breast milk, cervical secretion, and tissue of infected persons and experimentally infected nonhuman primates.⁸⁶

Although the risk of occupationally acquired HIV is primarily through exposure to infected blood, it is also prudent to wear gloves when manipulating other body fluids such as feces, saliva, urine, tears, sweat, vomitus, and human breast milk. This also reduces the potential for exposure to other microorganisms that may cause other types of infections.

In the laboratory, virus should be presumed to be present in all blood or clinical specimens contaminated with blood, in any unfixed tissue or organ (other than intact skin) from a human (living or dead), in HIV cultures, in all materials derived from HIV cultures, and in/on all equipment and devices coming into direct contact with any of these materials.

SIV has been isolated from blood, CSF, and a variety of tissues of infected nonhuman primates. Limited data exist on the concentration of virus in semen, saliva, cervical secretions, urine, breast milk, and amniotic fluid. Virus should be presumed to be present in all SIV cultures, in animals experimentally infected or inoculated with SIV, in all materials derived from SIV cultures, and in/on all equipment and devices coming into direct contact with any of these materials.⁸⁷

The skin (especially when scratches, cuts, abrasions, dermatitis, or other lesions are present) and mucous membranes of the eye, nose, and mouth should be considered as potential pathways for entry of these retroviruses during laboratory activities. Whether infection can occur via the respiratory tract is unknown. The need for using sharps in the laboratory should be evaluated. Needles, sharp instruments, broken glass, and other sharp objects must be carefully handled and properly discarded. Care must be taken to avoid spilling and splashing infected cell-culture liquid and other potentially infected materials.⁸⁵

Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for activities involving blood-contaminated clinical specimens, body fluids and tissues. HTLV-1 and HTLV-2 should also be handled at this level. Activities such as producing research laboratory-scale quantities of HIV or SIV, manipulating concentrated virus preparations, and conducting procedures that may produce droplets or aerosols, are performed in a BSL-2 facility, using BSL-3 practices. Activities involving large-scale volumes or preparation of concentrated HIV or SIV are conducted at BSL-3. ABSL-2 is appropriate for NHP and other animals infected with HIV or SIV. Human serum from any source that is used as a control or reagent in a test procedure should be handled at BSL-2. In addition to the aforementioned recommendations, persons working with HIV, SIV, or other bloodborne pathogens should consult the OSHA Bloodborne Pathogen Standard.⁸⁸ Questions related to interpretation of this standard should be directed to federal, regional or state OSHA offices

Special Issues

It is recommended that all institutions establish written policies regarding the management of laboratory exposure to HIV and SIV; including treatment and prophylaxis protocols (See Chapter 7).

The risk associated with retroviral vector systems can vary significantly; especially lentiviral vectors. Because each gene transfer system can vary significantly, no specific guideline can be offered other than to have all gene transfer protocols reviewed by an IBC.

Transfer of Agent

Agent: Severe Acute Respiratory Syndrome (SARS) Coronavirus

SARS is a viral respiratory illness caused by a previously undescribed coronavirus, SARS- associated coronavirus (SARS-CoV) within the family Coronaviridae. SARS was retrospectively recognized in China in November 2002. Over the next few months, the illness spread to other south-east Asian countries, North America, South America, and Europe following major airline routes. The majority of disease spread occurred in hospitals, among family members and contacts of hospital workers. From November 2002 through July 2003, when the global outbreak was contained, a total of 8,098 probable cases of SARS were reported to the WHO from 29 countries.⁸⁹ In general, SARS patients present with fever (temperature greater than 100.4°F [>38.0°C]), malaise and myalgias quickly followed by respiratory symptoms including shortness of breath and cough. Ten to 20 percent of patients may have diarrhea. Review of probable cases indicates that the shortness of breath sometimes rapidly progresses to respiratory failure requiring ventilation. The case fatality rate is about 11%.

Occupational Infections

Healthcare workers are at increased risk of acquiring SARS from an infected patient especially if involved in pulmonary/respiratory procedures such as endotracheal intubation, aerosolization or nebulization of medications, diagnostic sputum induction, airway suctioning, positive pressure ventilation and high-frequency oscillatory ventilation.

Two confirmed episodes of SARS-CoV transmission to laboratory workers occurred in research laboratories in Singapore and Taiwan.^89,90 Both occurrences were linked to breaches in laboratory practices. Laboratory-acquired infections in China, during 2004, demonstrated secondary and tertiary spread of the disease to close contacts and healthcare providers of one of the employees involved.⁹¹ Although, to date, no laboratory-acquired cases have been associated with the routine processing of diagnostic specimens, SARS coronavirus represents an emerging infectious disease for which risk to the medical and laboratory community is not fully understood.

Natural Modes of Infection

The mode of transmission in nature is not well understood. It appears that SARS is transmitted from person to person through close contact such as caring for, living with, or having direct contact with respiratory secretions or body fluids of a suspect or probable case.⁹² SARS is thought to be spread primarily through droplets, aerosols and possibly fomites. The natural reservoir for SARS CoV is unknown at this time.

Laboratory Safety

SARS-CoV may be detected in respiratory, blood, or stool specimens. The exact mode of transmission of SARS-CoV laboratory-acquired infection has not been established, but in clinical settings, the primary mode of transmission appears through direct or indirect contact of mucous membranes with infectious respiratory droplets.^93,94

Containment Recommendations

In clinical laboratories, whole blood, serum, plasma and urine specimens should be handled using Standard Precautions which includes use of gloves, gown, mask, and eye protection. Any procedure with the potential to generate aerosols (e.g., vortexing or sonication of specimens in an open tube) should be performed in a BSC. Use sealed centrifuge rotors or gasketed safety carriers for centrifugation. Rotors and safety carriers should be loaded and unloaded in a BSC. Procedures conducted outside a BSC must be performed in a manner that minimizes the risk of personnel exposure and environmental release.

The following procedures may be conducted in the BSL-2 setting: pathologic examination and processing of formalin-fixed or otherwise inactivated tissues, molecular analysis of extracted nucleic acid preparations, electron microscopic studies with glutaraldehyde-fixed grids, routine examination of bacterial and fungal cultures, routine staining and microscopic analysis of fixed smears, and final packaging of specimens for transport to diagnostic laboratories for additional testing (specimens should already be in a sealed, decontaminated primary container).

Activities involving manipulation of untreated specimens should be performed in BSL-2 facilities following BSL-3 practices. In the rare event that a procedure or process involving untreated specimens cannot be conducted in a BSC, gloves, gown, eye protection, and respiratory protection (acceptable methods of respiratory protection include: a properly fit-tested, National Institute for Occupational Safety and Health (NIOSH)-approved filter respirator (N-95 or higher level) or a PAPR equipped with HEPA filters) should be used. All personnel that may use respiratory protective devices should be enrolled in an appropriately constituted respiratory protection program. Work surfaces should be decontaminated upon completion of work with appropriate disinfectants. All waste must be decontaminated prior to disposal.

SARS-CoV propagation in cell culture and the initial characterization of viral agents recovered in cultures of SARS specimens must be performed in a BSL-3 facility using BSL-3 practices and procedures. Risk assessment may dictate the additional use of respiratory protection.

Inoculation of animals for potential recovery of SARS-CoV from SARS samples, research studies and protocols involving animal inoculation for characterization of putative SARS agents must be performed in ABSL-3 facilities using ABSL-3 work practices. Respiratory protection should be used as warranted by risk assessment. In the event of any break in laboratory procedure or accidents (e.g. accidental spillage of material suspected of containing SARS-CoV), procedures for emergency exposure management and/or environmental decontamination should be immediately implemented and the supervisor should be notified. The worker and the supervisor, in consultation with occupational health or infection control personnel, should evaluate the break in procedure to determine if an exposure occurred (See Special Issues, below).

Special Issues

Occupational Health Considerations

Institutions performing work with SARS coronavirus should require storage of a baseline serum sample from individuals who work with the virus or virus-containing specimens. Personnel working with the virus or samples containing or potentially containing the virus should be trained regarding the symptoms of SARS-CoV infection and counseled to report any fever or respiratory symptoms to their supervisor immediately. They should be evaluated for possible exposure and the clinical features and course of their illness should be closely monitored. Institutions performing work with the SARS- CoV or handling specimens likely to contain the agent should develop and implement a specific occupational medical plan with respect to this agent. The plan, at a minimum, should contain procedures for managing:

identifiable breaks in laboratory procedures
exposed workers without symptoms
exposed workers who develop symptoms within ten days of an exposure
symptomatic laboratory workers with no recognized exposure

Further information and guidance regarding the development of a personnel exposure response plan is available from the CDC.⁹⁵ Laboratory workers who are believed to have had a laboratory exposure to SARS-CoV should be evaluated, counseled about the risk of SARS-CoV transmission to others, and monitored for fever or lower respiratory symptoms as well as for any of the following: sore throat, rhinorrhea, chills, rigors, myalgia, headache, and diarrhea.

Local and/or state public health departments should be promptly notified of laboratory exposures and illness in exposed laboratory workers.

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.

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The B Virus Working Group. Guidelines for prevention of Herpesvirus simiae (B virus) infection in monkey handlers. J Med Primatol. 1988;17(2):77-83.
Huff JL, Eberle R, Capitanio J, et al. Differential detection of B virus and rhesus cytomegalovirus in rhesus macaques. Journal of General Virology. 2003;84:83-92.
Roizman B, Pellett, P. The family Herpesviridae: a brief introduction. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Volume 2. Philadelphia: Lippincott Williams and Wilkins. 2001. p. 2381-98.
Heymann, D, editor. Control of communicable diseases. 18th ed. Washington, DC: America Public Health Association. 2004.
Straus S. Human herpesvirus types 6 and 7. In: Mandell G, Bennett J, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 6th ed. Volume 2. Philadelphia: Elsevier Inc. 2005. p. 1821-25.
Wyatt LS, Frenkel N. Human herpesvirus 7 is a constitutive inhabitant of adult human saliva. J Virol. 1992;66:3206-9.
Chang Y, Cesarman E, Pessin MS, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science. 1994;266:1865-69.
Dukers NHTM, Reeza G. Human herpesvirus 8 epidemiology: what we know and do not know. AIDS. 2003;17:1177-30.
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Regamey N, Tamm M, Wernli M, et al. Transmission of human herpesvirus 8 infection from renal transplant donors to recipients. N Engl J Med. 1998;339:1358-63.
Luppi M, Barozzi P, Guaraldi G, et al. Human herpesvirus 8-associated diseases in solid- organ transplantation: importance of viral transmission from the donor. Clin Infect Dis. 2003;37:606-7.
Mbulaiteye SM, Biggar RJ, Bakaki PM, et al. Human herpes 8 infection and transfusion history in children with sickle-cell disease in Uganda. J Natl Cancer Inst. 2003;95:1330-5.
Whitby D, Luppi M, Sabin C, et al. Detection of antibodies to human herpesvirus 8 in Italian children: evidence for horizontal transmission. Brit J Cancer. 2000;82:702-4.
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Dowdle WR, Hattwick MA. Swine influenza virus infections in humans. J Infect Dis. 1977;136 Suppl:S386-5399.
Stott DJ, Kerr G, Carman WF. Nosocomial transmission of influenza. Occup Med. 2002;52:249-53.
Horcajada JP, Pumarola T, Martinez JA, et al. A nosocomial outbreak of influenza during a period without influenza epidemic activity. Eur Respir J. 2003;21:303-7.
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Section Va: Reassortant Influenza A Viruses

1. Scope

All subtypes of influenza A virus occur naturally in wild birds. While wild birds usually do not become ill when infected with influenza A virus, infected domesticated poultry such as chickens do become ill and may die. Influenza A viruses are categorized as low pathogenicity (“LPAI”; temporary illness in domesticated poultry such as decreased egg production) or high pathogenicity (“HPAI”; 90% or higher mortality in domesticated poultry within 48 hours of initial infection.) Humans can become ill from exposure to avian influenza viruses, usually by close contact with infected poultry or their secretions. Some HPAI such as H5N1 have resulted in documented human fatalities in the past 10 years.

Work with HPAI is currently regulated under the Select Agent Program (Title 9, Code of Federal Regulations, Part 121.) Recently, reassortant viruses built on a backbone of LPAI such as A/PR/8/34, and containing hemagglutinin (HA) and neuraminidase (NA) genes from avian influenza subtype H5N1, were made available to the research community by the Centers for Disease Control and Prevention (CDC.) These viruses are exempt from the Select Agent Program. This SOP was designed to establish a system of information and safeguards that should be followed at the University of North Carolina when using these reassortant viruses.

2. Procedure

2.1 Agent

Reassortant avian influenza viruses containing one or more gene sequences from highly pathogenic avian influenza.

2.2 Employees at risk

Individuals actively working with or in rooms containing the following: cultures of reassortant viruses, experimentally infected laboratory animals, or experimentally infected materials of animal origin, such as poultry eggs, are a potential source of infection to exposed laboratory personnel.

2.3 Laboratory hazards

2.3.1

Ingestion, parenteral inoculation, and droplet or aerosol exposure of mucous membranes or broken skin with infectious fluids or tissues, are the primary hazards to laboratory personnel.

2.3.2

Human infections resulting from exposure to strains of the subtypes of LPAI used in these reassortant viruses have not been documented.

2.4 Required Procedures

2.4.1

All Principal Investigators (PIs) using reassortant influenza A viruses must complete an application for work with recombinant DNA and be approved by the Institutional Biosafety Committee.

2.4.2

All PIs who wish to work with reassortant influenza A viruses must obtain a permit from the United States Department of Agriculture (USDA). Information may be found at the USDA Import Export page and Import/Export forms. USDA will contact the investigator to schedule an inspection of the lab.

2.4.3

After a risk assessment by EHS, Biosafety Level 2 facilities with BSL-3 practices, also known at the University as Biosafety Level 2+, will be required for all activities involving the use or manipulation of reassortant influenza A viruses. The specific additional practice requirements for BSL-2+, involving reassortant influenza A viruses, include the following:

2.4.3.1 A SOP must be prepared for the BSL-2+ lab. EHS will assist you with preparing a SOP.
2.4.3.2 Access is restricted when work is in progress.
2.4.3.3 Principal Investigator is responsible for ensuring that all personnel demonstrate proficiency in the practices and operations of the facility prior to beginning work with the organisms.
2.4.3.4 Any vacuum line is protected with liquid disinfectant traps and/or HEPA filters.
2.4.3.5 Class II or III Biological Safety Cabinets (BSCs) are used for all manipulations involving infectious materials.
2.4.3.6 Centrifuge Safety Cups must be used for centrifugation outside of a biosafety cabinet. Safety Cups must only be opened in a Biosafety Cabinet.
2.4.3.7 Generation of aerosols must be minimized.
2.4.3.8 The use of sharps is restricted or eliminated.
2.4.3.9 Personnel should wear additional protective gear while handling reassortant viruses, infected animals, and infected materials of animal origin such as poultry eggs. Protective gear includes an N-95 respirator or equivalent, hair cover, liquid barrier gloves, a gown (preferably liquid barrier and disposable), eye protection, and shoe covers. Workers must be fit-tested to the N-95 respirator and enrolled in the University’s respiratory protection program.ABSL 3 containment and practices must be followed.
2.4.3.10 Concurrent work involving other influenza viruses is prohibited in the designated lab for handling reassortant viruses.
2.4.3.11 All surfaces, equipment, and wastes, must be disinfected at the end of each work session.
Potentially infectious materials associated with reassortant viruses may not be left in the lab.
2.4.3.12 After the work session with reassortant viruses is completed, at least 30 minutes must pass before other workers may enter the lab and handle other biological agents.
2.4.3.13 Each lab must be evaluated by EHS on a case-by-case basis for suitability for work with reassortant viruses. The lab must be maintained under negative pressure with respect to adjacent labs or corridors at all times.

2.4.4

Laboratories are inspected by EHS to verify appropriate BSL-2+ or BSL-3 containment and practices.

2.4.5

If a BSL-2+ facility is used, the facility should include an anteroom in which garb can be donned. If an anteroom is not available, a garb station outside of the laboratory and out of the public thoroughfare should be used to don garb. N-95 respirators may be kept within the laboratory, only if they can be donned prior to work with reassortant viruses. Anyone intending to enter the laboratory when work with reassortant viruses is in progress must don a respirator prior to entering the lab. All personal protective equipment except for the N-95 respirator should be removed in the lab before exiting. Protective equipment should be disposed as biohazardous.

2.4.6

Personnel should undergo an immunization review at the University Employee Occupational Health Clinic (UEOHC) and thereby be offered the seasonal influenza vaccine. However, the vaccine shall not be the live, attenuated version (nasal spray) as this is recommended only for those between the ages of 2 and 49.

2.4.7

Personnel declining to receive the seasonal influenza vaccine must complete a declination form for UEOHC records.

2.4.8

Personnel should monitor their body temperature daily and report any symptoms to their supervisor which may indicate an influenza infection, such as temperature over 100.4°F, sore throat, cough, labored breathing. The supervisor must contact the Biological Safety Officer (919-962-5507).

2.4.9

Staff must be advised that stocks of Tamiflu (oseltamivir) and other antiviral treatments are kept on hand by UEOHC, and that occupational exposures must be immediately reported to the PI and EHS.

2.4.10

All transport of infectious materials within the BSL-2+ or BSL-3 laboratory must be performed in leak-proof sealed containers.

2.4.11

Non-lab personnel, such as janitors or trades workers, may not enter the laboratory when reassortant viruses are in use. All work areas must be surface decontaminated prior to their entry.

2.4.12

If it is intended to inactivate the virus and then handle it further in another laboratory outside of the designated laboratory, then EHS and the Institutional Biosafety Committee must review and approve the inactivation protocol. Inactivated virus may be handled in a BSL-2 laboratory without respiratory protection.

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Biological Safety Manual - Chapter 08: Agent Summary Statements (Section V: Viral Agents)

Title

Table of Contents

Agent: Hantaviruses

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Transfer of Agent

Agent: Hendra Virus (Formerly Known as Equine Morbillivirus) and Nipah Virus

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Select Agent

Transfer of Agent

Agent: Hepatitis A Virus, Hepatitis E Virus

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Vaccines

Transfer of Agent

Agent: Hepatitis B Virus, Hepatitis C Virus (Formerly Known as nonA nonB Virus), Hepatitis D Virus

Occupational Infections

Laboratory Safety

Containment Recommendations

Special Issues

Vaccines

Transfer of Agent

Agent: Herpesvirus simiae (Cerocopithecine herpesvirus I, Herpes B Virus)

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Select Agent

Transfer of Agent

Agent: Human Herpes Virus

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Vaccine

Treatment

Transfer of Agent

Agent: Influenza

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Non-Contemporary Human Influence (H2N2) Strains

1918 Influenza strain

Highly Pathogenic Avian Influenza (HPAI)

Other Influenza Recombinant or Reassortant Viruses

Special Issues

Occupational Health Considerations

Select Agent

Transfer of Agent

Agent: Lymphocytic Choriomeningitis Virus

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Vaccines

Transfer of Agent

Agent: Poliovirus

Occupational Infections

Natural Modes of Infection

Laboratory Safety

Containment Recommendations

Special Issues

Transfer of Agent

Agent: Poxviruses