Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Lassa virus (Website 2):
      1. Common Name: Lassa virus
      2. GenBank Taxonomy No.: 11620
      3. Description: Lassa fever is caused by Lassa virus, a member of the arenavirus group, which is transmitted to human beings from the rodent reservoir host, Mastomys natalensis, by direct contact with infected tissues or indirectly, possibly by food contaminated with excreta, and possibly by aerosols arising from these animals(Gear and Isaacson, 1988). Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone(Bowen et al., 2000).
      4. Variant(s):
         • Lassa virus (STRAIN GA391) (Website 3):
           • Common Name: Lassa virus (STRAIN GA391)
           • GenBank Taxonomy No.: 11621
           • Parents: Lassa virus
           • Description: GA391 is of Nigerian origin (Clegg et al., 1991).
         • Lassa virus (strain Josiah) (Website 4):
           • Common Name: Lassa virus (strain Josiah)
           • GenBank Taxonomy No.: 11622
           • Parents: Lassa virus
           • Description: Strain Josiah was isolated in Sierra Leone(Clegg et al., 1991).

1. Lassa virus Information
   1. Stage Information:
      1. Viron:
         • Size: Morphologically, arenavirus virions consist of enveloped particles that vary in diameter from approximately 60 to more than 300 nm, with a mean particle size of 92 nm as determined by electron microscopy.
         • Shape: The virions are approximately spherical, enveloped particles that range in diameter from 50 to 300 nm. The surface of the virion is smooth with T-shaped spikes, composed of viral glycoproteins, extending 7-10 nm from the envelope.
         • Picture(s):
           • Lassa virus image (Website 12)
             
             
             
             Description: Lassa virus, the causative agent of Lassa fever, an important hemorrhagic disease of West Africa. Thin section of virions in a space between cellsLassa virus buds from the surface membrane of cells where it is then free to invade other nearby cells and is free to enter the bloodstream. Magnification approximately x55,000. Micrograph from F. A. Murphy, School of Veterinary Medicine, University of California, Davis.

1. Genome of Lassa virus
   1. Description: The arenavirus genome consists of two single-stranded RNA molecules, designated L and S, that contain essentially nonoverlapping sequence information. There are minor differences in the lengths of the genomic RNA segments for the individual viruses (L approximately 7,200 bases and S approximately 3,400 bases), but the general organization of the viral genomes, based on current sequence information, is well preserved across the virus family(Southern, 1996). The genomic RNA consists of two segments, S (3.4 k) and L (7.2 kb), both of which are arranged in an ambisense orientation. The S segment encodes the nucleocapsid protein (NP) in negative, antimessage sense at the 3'-end and the viral glycoprotein precursor, GP-C in message sense at the 5'-end. The L RNA segment contains the L protein (polymerase) gene at the 3'-end in negative polarity and the zinc-binding (Z) protein at the 5'-end in message polarity. Posttranslational modification of the cell-associated GP-C precursor yields the structural glycoproteins GP-1 (44 kd) and GP-2 (35 kd), which are assembled into a tetrameric virion spike. GP-1 contains determinants that interact with viral receptors and is recognized by neutralizing antibody. GP-2 contains sites that promote acid-dependent membrane fusion necessary for viral entry. The nucleocapsid protein is an internal RNA-binding protein that complexes with genomic RNA(Peters et al., 1996).
   2. L Segment(Website 9)
      1. GenBank Accession Number: NC_004297
      2. Size: 7279 bp ss-RNA(Website 9).
      3. Gene Count: 2 genes(Peters et al., 1996).
      4. Description: The L RNA segment contains the L protein (polymerase) gene at the 3'-end in negative polarity and the zinc-binding (Z) protein at the 5'-end in message polarity(Peters et al., 1996).
   3. S Segment(Website 10)
      1. GenBank Accession Number: NC_004296
      2. Size: 3402 bp ss-RNA(Website 10).
      3. Gene Count: 2 genes(Peters et al., 1996).
      4. Description: The S segment encodes the nucleocapsid protein (NP) in negative, antimessage sense at the 3'-end and the viral glycoprotein precursor, GP-C in message sense at the 5'-end(Peters et al., 1996). Posttranslational modification of the cell-associated GP-C precursor yields the structural glycoproteins GP-1 (44 kd) and GP-2 (35 kd), which are assembled into a tetrameric virion spike. GP-1 contains determinants that interact with viral receptors and is recognized by neutralizing antibody. GP-2 contains sites that promote acid-dependent membrane fusion necessary for viral entry(Peters et al., 1996).

1. General biosafety information
   1. Level: Like Lassa virus, Junin, Machupo, Guanarito, and Sabia viruses are infectious by aerosol and the human and rodent specimens should be processed with appropriate precautions in BSL 4 laboratories(Buchmeier et al., 2001).
   2. Precautions: The patient should be isolated in a single room with an adjoining anteroom serving as its only entrance. The anteroom should contain supplies for routine patient care, as well as gloves, gowns, and masks for the staff. The Appendix lists suggested supplies for the anteroom. Hand-washing facilities should be available in the anteroom, as well as containers of decontaminating solutions. If possible, the patient's room should be at negative air pressure compared with the anteroom and the outside hall, and the air should not be recirculated. However, this is not absolutely required, and does not constitute a reason to transfer the patient. If a room such as described is not available, use adjacent rooms to provide safe and adequate space(MMWR, 1988). Strict barrier-nursing techniques should be enforced: all persons entering the patient's room should wear disposable gloves, gowns, masks, and shoe covers. Protective eye wear should be worn by persons dealing with disoriented or uncooperative patients or performing procedures that might involve the patient's vomiting or bleeding (for example, inserting a nasogastric tube or an intravenous or arterial line). Protective clothing should be donned and removed in the anteroom. Only essential medical and nursing personnel should enter the patient's room and anteroom. Isolation signs listing necessary precautions should be posted outside the anteroom(MMWR, 1988). Lipid-containing viruses, including the enveloped viruses, are among the most readily inactivated of all viral agents. Suitable disinfectant solutions include 0.5% sodium hypochlorite (10% aqueous solution of household bleach), as well as fresh, correctly prepared solutions of glutaraldehyde (2% or as recommended by the manufacturer) and phenolic disinfectants (0.5%-3%). Soaps and detergents can also inactivate these viruses and should be used liberally(MMWR, 1988). Laboratory personnel accidentally exposed to potentially-infected material (for example, through injections or cuts or abrasions on the hands) should immediately wash the infected part, apply a disinfectant solution such as hypochlorite solution, and notify the patient's physician. The person should then be considered as a high-risk contact and placed under surveillance. Accidental spills of potentially contaminated material should be liberally covered with disinfectant solution, left to soak for 30 minutes, and wiped up with absorbent material soaked in disinfectant(MMWR, 1988).
   3. Disposal: The patient should use a chemical toilet. All secretions, excretions, and other body fluids (other than laboratory specimens) should be treated with disinfectant solution. All material used for patients, such as disposable linen and pajamas, should be double-bagged in airtight bags. The outside bags should be sponged with disinfectant solution and later incinerated or autoclaved. Disposable items worn by staff, such as gowns, gloves, etc., should be similarly treated. Disposable items used in patient care (suction catheters, dressings, etc.) should be placed in a rigid plastic container of disinfectant solution. The outside of the container should be sponged with disinfectant, and the container should be autoclaved, incinerated, or otherwise safely discarded(MMWR, 1988). All unnecessary handling of the body, including embalming, should be avoided. Persons who dispose of the corpse must take the same precautions outlined for medical and laboratory staff. The corpse should be placed in an airtight bag and cremated or buried immediately(MMWR, 1988). Disposable items, such as pipette tips, specimen containers, swabs, etc., should be placed in a container filled with disinfectant solution and incinerated. Clothes and blankets that were used by the patient should be washed in a disinfectant, such as hypochlorite solution. Nondisposable items such as endoscopes used in patient care must be cleaned with decontaminating fluids (for example, gluteraldehyde or hypochlorite). Laboratory equipment must be treated similarly. All non- disposable materials that withstand autoclaving should be autoclaved, after they have been soaked in disinfectant solution. The patient's bed and other exposed surfaces in the hospital room, or in vehicles used to transport the patient, should be decontaminated with disinfectant solution(MMWR, 1988).

1. Vero Cell Culture :
   1. Description: The specific diagnosis is readily made by the isolation and identification of the virus. This is usually done by the inoculation of blood from the patient into Vero cell cultures. The virus is also readily isolated from urine, throat washings, and from pleural, peritoneal and pericardial effusions. The viremia may persist for 2 to 3 weeks, and virus may be detected in the urine of some patients for as long as 32 days after the onset of illness(Gear and Isaacson, 1988). Arenaviruses are most often quantitated in vitro by plaque or quantal assay on Vero or L cells. Plaque formation may require long incubation periods of up to 7 days for members of the Tacaribe group. More commonly however, 4-5 days are sufficient for plaque development followed by neutral red vital staining or formalin fixation and crystal violet staining to visualize plaques. Plaques may vary widely in size and appearance(Peters et al., 1996). Plaques obtained from a high multiplicity of inoculum or persistent infections in cell culture, both of which lead to high concentrations of defective interfering virus, may be turbid or may even exhibit a 'bull's-eye' appearance, presumably resulting from alternating cycles of interfering virus production as the plaque expands(Peters et al., 1996). Cytopathic effect (CPE) in cell monolayers is not typical of arenavirus/cell interactions. Assay on Vero monolayers in screw cap tissue culture tubes has been utilized occasionally where the plaque assay is impracticable or when assaying slow-growing or highly virulent arenaviruses. This assay offers a higher degree of containment and greater resistance to dehydration than the plaque assay. Cytopathic effect is evident as cell rounding and detachment from the substrate, in exceptional cases proceeding to total exfoliation of the culture(Peters et al., 1996).
   2. Medium: Vero cells (ATCC, CCL 81) were grown in Minimum Essential Medium with Earle's salts supplemented with non-essential amino acids, 1% penicillin, 1% streptomycin, 1% -glutamine and 10% heat-inactivated fetal bovine serum and were maintained in the same medium containing 2% fetal bovine serum(Lozano et al., 1997).

1. Outbreak Locations:
   1. Lassa fever is an endemic disease in portions of West Africa. It is recognized in Guinea, Liberia, Sierra Leone, as well as Nigeria. However, because the rodent species which carry the virus are found throughout West Africa, the actual geographic range of the disease may extend to other countries in the region(Website 8).
2. Transmission Information:
   1. From: Human(Website 8). , To: Human(Website 8). , With Destination:Human(Website 8). (Website 8)
      Mechanism: Lassa fever may also spread through person-to-person contact. This type of transmission occurs when a person comes into contact with virus in the blood, tissue, secretions, or excretions of an individual infected with the Lassa virus. The virus cannot be spread through casual contact (including skin-to-skin contact without exchange of body fluids). Person-to-person transmission is common in both village and health care settings, where, along with the above-mentioned modes of transmission, the virus also may be spread in contaminated medical equipment, such as reused needles. This is called nosocomial transmission(Website 8).
   2. From: Mastomys(Gear and Isaacson, 1988). , To: Human(Gear and Isaacson, 1988). , With Destination:Human(Gear and Isaacson, 1988). (Gear and Isaacson, 1988)
      Mechanism: Lassa fever is caused by Lassa virus, a member of the arenavirus group, which is transmitted to human beings from the rodent reservoir host, Mastomys natalensis, by direct contact with infected tissues or indirectly, possibly by food contaminated with excreta, and possibly by aerosols arising from these animals(Gear and Isaacson, 1988).
3. Environmental Reservoir:
   1. Multimammate Rat(Website 8, Peters et al., 1996, Fisher-Hoch, 1993):
      1. Description: The reservoir, or host, of Lassa virus is a rodent known as the "multimammate rat" of the genus Mastomys. It is not certain which species of Mastomys are associated with Lassa; however, at least two species carry the virus in Sierra Leone. Mastomys rodents breed very frequently, produce large numbers of offspring, and are numerous in the savannas and forests of West, Central, and East Africa. In addition, Mastomys generally readily colonize human homes. All these factors together contribute to the relatively efficient spread of Lassa virus from infected rodents to humans(Website 8).
      2. Survival: Lassa virus, like LCMV, induces chronic viremic infection in neonatal Mastomys and transient, immunizing infection in adults. This type of infection, which is also efficiently transmitted to the fetus, would favor a maintenance cycle in which congenital, vertical transmission is the major feature(Peters et al., 1996). Studies of wild-caught Mastomys show that over half the captured animals in some foci may be chronically infected, and antibody and virus may be present at the same time in about one-third of these(Fisher-Hoch, 1993).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Emergency Contact: If clinicians feel that VHF is a likely diagnosis, they should take two immediate steps: 1) isolate the patient, and 2) notify local and state health departments and CDC(MMWR, 1988). Report incidents to state health departments and the CDC (telephone {404} 639-1511; from 4:30 p.m. to 8 a.m., telephone {404} 639-2888). Information on investigating and managing patients with suspected viral hemorrhagic fever, collecting and shipping diagnostic specimens, and instituting control measures is available on request from the following persons at Centers for Disease Control (CDC) in Atlanta, Georgia; for all telephone numbers, dial 404-639 + extension: Epidemic Intelligence Service (EIS) Officer, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases (ext. 1344); Chief, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases: Joseph B. McCormick, M.D. (ext. 3308); Senior Medical Officer, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases: Susan P. Fisher-Hoch, M.D. (ext. 3308); Director, Division of Viral Diseases, Center for Infectious Diseases (ext. 3574). After regular office hours and on weekends, the persons named above may be contacted through the CDC duty officer (ext. 2888)(MMWR, 1988).
      2. Delivery Mechanism: The VHF agents are all highly infectious via the aerosol route, and most are quite stable as respirable aerosols. This means that they satisfy at least one criterion for being weaponized, and some clearly have the potential to be biological warfare threats. Most of these agents replicate in cell culture to concentrations sufficiently high to produce a small terrorist weapon, one suitable for introducing lethal doses of virus into the air intake of an airplane or office building. Some replicate to even higher concentrations, with obvious potential ramifications. Since the VHF agents cause serious diseases with high morbidity and mortality, their existence as endemic disease threats and as potential biological warfare weapons suggests a formidable potential impact on unit readiness. Further, returning troops may well be carrying exotic viral diseases to which the civilian population is not immune, a major public health concern(Website 7).
      3. Containment: Patients with VHF syndrome generally have significant quantities of virus in their blood, and perhaps in other secretions as well (with the exceptions of dengue and classic hantaviral disease). Well-documented secondary infections among contacts and medical personnel not parenterally exposed have occurred. Thus, caution should be exercised in evaluating and treating patients with suspected VHF syndrome. Over-reaction on the part of medical personnel is inappropriate and detrimental to both patient and staff, but it is prudent to provide isolation measures as rigorous as feasible. At a minimum, these should include the following: stringent barrier nursing; mask, gown, glove, and needle precautions; hazard-labeling of specimens submitted to the clinical laboratory; restricted access to the patient; and autoclaving or liberal disinfection of contaminated materials, using hypochlorite or phenolic disinfectants. For more intensive care, however, increased precautions are advisable. Members of the patient care team should be limited to a small number of selected, trained individuals, and special care should be directed toward eliminating all parenteral exposures. Use of endoscopy, respirators, arterial catheters, routine blood sampling, and extensive laboratory analysis increase opportunities for aerosol dissemination of infectious blood and body fluids. For medical personnel, the wearing of flexible plastic hoods equipped with battery-powered blowers provides excellent protection of the mucous membranes and airways(Website 7).

1. Organism Detection Test:
   1. Electron Microscopy :
      1. Description: When the identity of a VHF agent is totally unknown, isolation in cell culture and direct visualization by electron microscopy, followed by immunological identification by immunohistochemical techniques is often successful(Website 7, Mekki and van der Groen, 1981). Yellow fever, dengue (types 1, 2 and 4), Chikungunya, Rift Valley fever, Ebola, Marburg, and Lassa viruses were inoculated into susceptible cell cultures and daily investigated by indirect immunofluorescence (IFA) and electron microscopy (EM) with a view to achieve an early detection-identification of these agents. Compared to the other cell lines tested (Vero, BHK-21 and Aedes albopictus), CV-1 cells were found to be more sensitive. Viral antigens were detected by IFA from a few hours post inoculation (CHIK and RVF) to a maximum of 3 days (YF and EBO). For most of the viruses studied, the cytopathic effect (CPE) commenced 2-3 days after the detection of viral antigens. Virus particles were detected by EM only in the case of EBO, MBG and LAS, before any CPE was observed in cell cultures(Mekki and van der Groen, 1981).
   2. IFA :
      1. Description: A method is described for preparation of polyvalent antigens for use in rapid screening for immunofluorescent antibodies to Lassa, Marburg, and Ebola viruses. The technique uses mixtures of specifically infected Vero cells placed on Teflon-templated microscopy slides. It was found to be as sensitive as the use of monovalent antigens for detection and quantitation of antibodies to these highly hazardous human pathogen(Johnson et al., 1981). The most reliable and safe routine method for the laboratory at present is detection of virus-specific antibody by IFA(McCormick and Fisher-Hoch, 2002). The advantages of IFA are its simplicity, low cost, ease of manipulation, its ability to control the background reaction, its overall reliability, and its flexibility, since it allows screening for several antigens in one test. Its disadvantages are slightly, but not significantly, lower sensitivity than ELISA, its modest subjectivity for the complete novice, and the cross-reactivity of the reagents with IgM, IgG, and IgA(McCormick and Fisher-Hoch, 2002). Where conditions are optimal and reagents are available, ELISA should be used. Where the simplicity and reliability of IFA are advantageous, it remains a functional and reasonable alternative(McCormick and Fisher-Hoch, 2002).
      2. False Negative: About two-thirds of clinical infections with Lassa fever could be accurately diagnosed by measuring IgM antibodies on the day of admission(Johnson et al., 1981).
   3. Viral Isolation (Peters et al., 1996):
      1. Description: Lassa virus is easily isolated from blood or serum during the febrile phase of the disease up to 14 days post onset, even after the appearance of antibody. Viremia is usually higher in fatal cases. Virus can also be detected in necropsy tissues(Peters et al., 1996). Routine virus isolation may be accomplished easily from serum or tissues in cell cultures, but should be performed in BSL4 laboratory facilities(McCormick and Fisher-Hoch, 2002).
2. Immunoassay Test:
   1. Reversed Passive Hemagglutination :
      1. Description: Conditions were defined for functional covalent coupling of anti-Lassa virus globulins to glutaraldehyde-fixed chicken erythrocytes. Tolylene-2,4-diisocyanate in a reaction mixture containing not more than 0.01 M NaCl produced uniformly good conjugates which were used in reversed passive hemagglutination (RPH) and reversed passive hemagglutination inhibition (RPHI) tests to detect Lassa virus antigens in infected cell cultures and specific antigens in Vero cell cultures. Identical results were obtained with this method and with immunofluorescent-antibody (IFA) staining in the detection and identification of Lassa virus isolated from human and rodent specimens from West Africa. The RPHI method was equal to IFA for serological diagnosis of acute human Lassa virus infection and superior to IFA, complement fixation, and a radioimmunoassay procedure for detection of Lassa virus antibodies in a human population where this infection is endemic(Goldwasser et al., 1980).
   2. ELISA :
      1. Description: We compared ELISA and IFA testing on sera from 305 suspected cases of Lassa fever by using virus isolation with a positive reverse transcription-PCR (RT-PCR) test as the "gold standard." Virus isolation and RT-PCR were positive on 50 (16%) of the 305 suspected cases. Taken together, Lassa virus antigen and IgM ELISAs were 88% (95% confidence interval [CI], 77 to 95%) sensitive and 90% (95% CI, 88 to 91%) specific for acute infection. Due to the stringent gold standard used, these likely represent underestimates. Diagnosis could often be made on a single serum specimen. Antigen detection was particularly useful in providing early diagnosis as well as prognostic information. Level of antigenemia varied inversely with survival. Detection by ELISA of IgG antibody early in the course of illness helped rule out acute Lassa virus infection. The presence of IFA during both acute and convalescent stages of infection, as well as significant interobserver variation in reading the slides, made interpretation difficult. However, the assay provided useful prognostic information, the presence of IFA early in the course of illness correlating with death. The high sensitivity and specificity, capability for early diagnosis, and prognostic value of the ELISAs make them the diagnostic tests of choice for the detection of Lassa fever(Bausch et al., 2000). The use of ELISA in Africa is replete with problems resulting in a significant misrepresentation of reality(McCormick and Fisher-Hoch, 2002). Where conditions are optimal and reagents are available, ELISA should be used. Where the simplicity and reliability of IFA are advantageous, it remains a functional and reasonable alternative(McCormick and Fisher-Hoch, 2002).
      2. False Negative: ELISA of IgM proved to be the single most sensitive assay in detecting acute infection overall, identifying 36 (72%) of the 50 cases. However, antigen detection was more sensitive early in the course of the disease, identifying 15 (30%) of the 50 on the first blood draw. In practice, ELISAs for antigen and IgM are used in tandem, with a case considered positive if ELISA antigen and/or IgM are present. Preformed on all 305 suspected cases, ELISA for antigen and IgM (ELISA Ag/IgM) detected 44 (88%) of the 50 culture-confirmed cases for a sensitivity and specificity of 88% (95% confidence interval, 77 to 95%) and 90% (95% confidence interval, 88 to 91%), respectively(Bausch et al., 2000).
   3. Immunoblot assay (ter Meulen et al., 1998):
      1. Description: The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 g of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 g. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens(ter Meulen et al., 1998).
      2. False Positive: One serum sample from the 244 German controls (area of nonendemicity) reacted weakly (faint 1+ reaction) in the LVA blot. This 0.41% rate of false positivity closely parallels the rate of 1% recently reported for an immunoblot assay for hantavirus(ter Meulen et al., 1998).
      3. False Negative: Of the IIF-positive sera from Guinea and Liberia, 9.3 and 25%, respectively, did not react in the LVA blot. Lack of reactivity did not depend on the LVA titer measured in the IIF assay, as sera with a low titer of 1/20 and with a high titer of 1/160 tested negative(ter Meulen et al., 1998).
3. Nucleic Acid Detection Test: