A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.

The efficacy of passive immunization for treatment of Lassa Fever (LF) is believed to depend on the titre of the neutralizing antibody infused. For the purpose of identifying optimal donors of LV-immune plasma, a population of LF-convalescent patients in Liberia was tested for prevalence of neutralizing antibody. Minimally protective titres, expressed as a log10 neutralization index, (LNI), were established in animal models as LNI greater than 2. LNI titres for 26 donors, tested eight or more months after illness, were modest: 16 titred 1 less than LNI less than 2, 4 titred 2 greater than LNI less than 3, and only 4 titred LNI greater than 3. Sequentially obtained plasma from six donors indicated that the LNI response was delayed relative to the indirect fluorescent antibody (IFA) response, that high titres (LNI greater than 3) occurred only after seven months and in only two of six patients. Most of the unselected LV-immune plasma will require concentration to therapeutically useful LNI titres. In a passive immunization experiment, guinea-pigs were protected by a late convalescent plasma (LNI = 4.8, IFA = 320) but not by an early plasma, (LNI = 0.6, IFA = 640), thus supporting the selection of immune plasma on the basis of the LNI. Cross serological testing with LV strains and convalescent plasma from patients in Sierra Leone, Liberia and Nigeria suggested that these LV strains were indistinguishable by cross-IFA, but were readily distinguishable by cross neutralization tests. Geographical matching of LV and plasma origins may thus be a factor in selection of optimal plasma for passive immunization of Lassa fever.

A model for studying the pathogenesis of virulent arenavirus infection was developed by adapting Pichinde virus to produce lethal infections of inbred guinea pigs. This adapted Pichinde virus retained low virulence for primates, thus potentially reducing the biohazard to investigators. Whereas all inbred (strain 13) guinea pigs were infected and killed by 3 plaque-forming units or more of adapted Pichinde virus injected subcutaneously, outbred (Hartley strain) guinea pigs were relatively resistant. All infected, inbred guinea pigs died at 13 to 19 days after inoculation, with viremias in excess of 5 log 10 plaque-forming units/ml, severe lymphopenia (<1,000/mm 3 ), and elevated serum glutamic oxaloacetic acid transaminase levels. Immunofluorescent antibody examination of tissues and infectivity titrations of tissue homogenates obtained at 3- to 4-day intervals demonstrated significant viral replication in all visceral tissues examined, but not in brain. Livers of all moribund guinea pigs contained moderate to severe hepatocellular necrosis and diffuse fatty change. Splenic red pulp and adrenal cortical tissues were engorged with blood and contained necrotic foci. Pancreatic acinar tissues were atrophied and vacuolated; lung sections typically contained areas of moderate to severe interstitial pneumonia. Inflammatory cells were conspicuously absent from all lesions. The virological and pathological features of adapted Pichinde infection in guinea pigs are remarkably similar to those described for Lassa virus infections in rhesus monkeys and humans, suggesting that this model might provide insight into the pathogenesis and treatment of Lassa fever in humans.

SUMMARYThe U937 monocytic cell line was used to determine whether antibodies could facilitate infection and replication of the arenaviruses, Pichinde virus (PV) and Lassa fever virus (LFV). When high dilutions of PV-immune serum were added to cultures simultaneously with PV inoculum, virus replication was dramatically (1000-fold) increased. Low dilutions of this antiserum neutralized the virus. LFV also replicated in U937 cells. The presence of LFV-specific immune serum in the growth medium increased the viral titre as much as 10000-fold. Addition of heat-aggregated IgG partially inhibited antibody-mediated enhancement, probably by inhibiting the binding of immune complexes to the monocytic cells.

Recent filoviral outbreaks in animal primates have raised public awareness of the potential for filoviruses to become a public health concern; methods that efficiently identify these viruses are therefore of high priority. An indirect immunoelectron microscopy method, which uses homologous guinea pig polyclonal antiserum, successfully identified Ebola-related (Reston) virus particles in serum and tissue culture fluid specimens with infectivity titres of 300 plaque forming units (pfu) per ml or more. The sensitivity of this procedure is sufficient to show virus in most acute phase sera, and is equal to that of the antigen capture enzyme linked immunosorbent assay (ELISA). The immunoelectron microscopy fluid technique can differentiate among antigenically distinct filoviruses in less than three hours. It should be valuable in the rapid diagnosis of potential filoviral infections.We recently showed that "post-embedment" immunoelectron microscopy of MA-104 cells, inoculated with serum from sick animal primates, is useful in diagnosing filoviral (Marburg, Ebola, or Reston) infections.' Since the recent importation of Ebola-related Reston virus into the United States,2 development of rapid diagnostic procedures for these viruses is now of special concern. In a refinement of the immunoelectron microscopy Positive gold sphere staining of a Reston virion was observed with immunoelectron microscopy performed as described. Preparation was stained by negative contrast with 1% phosphotungstic acid (pH = 6-6).procedure, we adapted the method of Lin3 to show the presence of Ebola-related virus (Reston virus) in fluid specimens. This adaptation is more rapid and simpler to perform than thin section examination of inoculated cell cultures. MethodsOur immunoelectron microscopy procedure critically depends on the use of guinea pig antisera, collected and pooled 99 days after subcutaneous inoculation of guinea pigs with 500 plaque forming units (pfu) of Reston virus. The immunoflourescent antibody titre of this pool was 1/ 160 against Reston virus.About 1-5 cc of fluid, such as serum, plasma, and tissue culture supernatant, was centrifuged in a conical Eppendorf microcentrifuge tube at 12 000 x g for 15 minutes. The supernatant was removed, and the virus pellet resuspended in 5-10 ,ul of phosphate buffered saline (PBS), and transferred to a 300-mesh, nickel electron microscopy grid pre-coated with Formvar and carbon. After blotting excess fluid the grid was completely immersed for 45 minutes at 23'C in a dilution (1 in 150) of guinea pig antiserum against Reston virus in TRI S-buffer containing 0 1 % bovine serum albumin and 0-05% Tween-20 (pH = 8-1). The grid was washed with TRIS-bovine serum albumin-Tween buffer (pH = 7.2) for five minutes and transferred for 45 minutes at 23°C to anti-guinea pig IgG labelled with 10 nm gold spheres (Janssen Life Sciences), diluted 1 in 10 in the TRIS-buffer (pH = 8-1). The grid was washed by successive immersion in three drops of TRIS-bovine serum albumin-Tween buffer (pH = 7-2), two ...