Preparation and use of erythrocyte-globulin conjugates to Lassa virus in reversed passive hemagglutination and inhibition.

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.     

Inactivated Lassa virus elicits a non protective immune response in rhesus monkeys.

We attempted to protect three rhesus monkeys from Lassa fever by vaccination with a preparation of purified whole Lassa virus which had been inactivated by gamma irradiation. The vaccinated monkeys developed antibodies against the three major viral proteins of Lassa virus demonstrated by radioimmunoprecipitation. When the three vaccinated monkeys and two unvaccinated control monkeys were challenged all five became severely ill and died. Prior to death a secondary, high-titer antibody response to Lassa virus was observed in the three vaccinated monkeys, whereas the two unvaccinated monkeys developed a primary, low-titer antibody response. Though titers of Lassa virus in serum reached peak levels earlier following challenge in the non vaccinated, at the time of death serum and organ virus titers did not differ significantly. Changes in platelet aggregation, leukocyte counts, and liver enzymes, abnormalities of which have been associated with severity of Lassa fever, were found to be comparable in the two groups. The humoral antibody response measured in these animals following vaccination, although of the same magnitude as found in humans recovered from Lassa fever, was insufficient to protect the animals from this fatal disease. Evidence is now accumulating that the cell-mediated immune response must be activated in order to protect against challenge with arenaviruses.     

Standardization of a plaque assay for Lassa virus

The plaque reduction neutralization test (PRNT) has been used routinely in serological studies with such arenaviruses as Junin, Machupo, and Parana. However, difficulties have been encountered in using the PRNT for LCM virus, while conflicting views have been expressed about the reliability and efficacy of the test with Lassa virus. We have therefore investigated and evaluated the plaque assay for Lassa virus. In addition, the suitability of the PRNT for determining the potency of a serum and its efficacy in passive immunization for the treatment of Lassa fever was also investigated. The Lassa virus plaque assay satisfied the criteria proposed by Cooper [1961] for determining satisfactory plaque technique. Lassa virus plaques appear within 3 days of inoculating Vero cell cultures. By day 5, the plaques are clearly defined, discrete, and measure 1.5 to 2.0 mm. In the plaque reduction neutralization test, the use of native non-inactivated serum was required for a reliable and reproducible determination of serum antibody titer. The potency and suitability of a serum for Lassa fever serotherapy was determined by the use of a constant serum-varying virus (CS-VV) and/or a constant virus-varying serum (CV-VS) PRN technique.     

Indirect immunoenzyme method for the laboratory diagnosis of Lassa and Ebola hemorrhagic fevers

Conditions for performing solid-phase indirect enzyme-immunoassay (SPEIA) for the detection of Lassa and Ebola virus antigens and antibodies to them using horseradish peroxidase-labeled antispecific globulins were developed. The method is highly sensitive, specific, and reproducible. By this method, antigens of Lassa and Ebola viruses could be detected in tissue culture fluid of the infected cell cultures and in animal organ suspensions. Detection of antibodies to Lassa and Ebola viruses in human convalescent sera and in normal donors by means of SPEIA opens possibilities for its use in large-scale diagnostic and seroepidemiological surveys.     

A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa.     

Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP)

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.     

Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus

A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.     

Pigeon herpes virus. II. Susceptibility of avian and mammalian cell cultures to infection with pigeon herpes virus

Duck embryo fibroblasts, chicken embryo liver cell cultures, chick kidney cell cultures and 7 mammalian cell lines were tested for susceptibility to pigeon herpes virus (HVP). All avian cell cultures were susceptible to HVP but there were variations in the cytophatic response. The baby hamster kidney cell line was susceptible to infection with HVP: morphological modifications were supported by the demonstration of HVP-specific antigen and the increase in virus titre. All other mammalian cell lines were refractory to infection with HVP.     

Detection of Lassa virus antigens and Lassa virus-specific immunoglobulins G and M by enzyme-linked immunosorbent assay.

Rapid diagnosis of Lassa fever is desirable for the timely therapeutic intervention and implementation of strict quarantine procedures both in West Africa field hospitals where the disease is endemic and at international crossroads. An enzyme-linked immunosorbent assay (ELISA) to measure Lassa virus antigens in viremic sera was developed in which experimentally infected monkeys were used as a model for the human disease. In this test, Lassa virus antigens in test sera were captured in wells of microtiter plates by monkey anti-Lassa virus immunoglobulin. Guinea pig anti-Lassa virus immunoglobulin was then added, and binding of specific immunoglobulin was quantitated by the addition of rabbit anti-guinea pig immunoglobulin followed by alkaline phosphatase-labeled anti-rabbit immunoglobulin. This test detected viremia titers as low as 2.1 log10 PFU/ml in experimentally infected monkey sera, a titer often exceeded in patients with Lassa fever. Inactivation of infectious virus by beta-propiolactone or gamma-irradiation did not diminish reactivity. Antigen-ELISA concentrations increased with infectivity for the first 10 days after infection but then declined while infectivity titers remained high, suggesting that the presence of humoral antibody in viremic sera diminishes the sensitivity of the antigen ELISA. Lassa virus-specific immunoglobulin M (IgM) titers measured in an IgM capture ELISA were detectable within 10 days of infection and peaked after 36 days but remained detectable for 1.5 years. The Lassa virus-specific IgG ELISA response was slightly delayed, peaking on day 73 but declining only slightly thereafter. These studies in a realistic primate model suggest that the antigen detection ELISA or the IgM capture ELISA described, in which beta-propiolactone-inactivated sera are used, should be useful for the rapid diagnosis of human Lassa fever.     

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.     

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.     

Experimental Lassa virus infection in the squirrel monkey.

Experimental Lassa virus infection was investigated in a nonhuman primate in order to elucidate the target organs of the viral infection and the course of pathologic events. Four squirrel monkeys (Saimiri scirreus) were inoculated intramuscularly with Lassa virus and sacrificed for organ titrations and histopathology, one each day, on Days 7, 12, 14, and 28 after inoculation. The animals showed a variable clinical course, with an incubation period of 8 to 18 days. The virus was demonstrated to be virtually pantropic; however, lymph node, liver, and kidney were key early targets. After the onset of overt disease, patterns of lymphoreticulotropism, hepatotropism, nephrotropism, adrenotropism, and persistent viremia were evident. Complement-fixing antibody failed to develop after 28 days of infection. Histopathologic findings included germinal center necrosis in spleen and lymph node; myocarditis; acute arteritis; renal tubular necrosis and regeneration; hepatocytic regeneration; chronic inflammation of choroid plexus, ependyma, and meninges; and cerebral perivascular cuffing. There is a relationship between many of these lesions and certain features of other arenavirus infections. The model offers the opportunity to pursue investigations of experimental pathogenesis, transmissibility, and efficacy of immunotherapy.     

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies

Summary Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd, specific for the viral nucleoprotein (p 60). Three domains (A, B, C) were identified on the surface of the Lassa virus nucleoprotein using an ELISA-inhibition test. All domains carried different Lassa virus specific epitopes. In addition, the A-domain carried a group specific epitope present within the arenavirus family as a whole as shown by cross-reaction in immunofluorescence tests. The B-domain only carries Lassa virus specific epitopes, whereas the C-domain has a type specific and a subgroup specific (Lassa, LCM) epitope.     

The isolation and characteristics of reassortants between the Lassa and Mopeia arenaviruses

Reassortants with a mixed phenotype were produced by combined inoculation of Vero cells with Lassa and Mopeya viruses. These reassortants produced small plaques (Mopeya virus phenotype) and were not pathogenic for newborn mice (Lassa virus phenotype). The genotype of the reassortants was studied by dot hybridization experiments on filters using cDNA-probes differentiating genome segments of these viruses. The reassortants were shown to have Mopeya virus L-RNA and Lassa virus S-RNA.     

Pathogenesis of Lassa virus infection in guinea pigs.

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.     

Mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever

Arenaviruses are transmitted from rodents to human beings by blood or mucosal exposure. The most devastating arenavirus in terms of human disease is Lassa fever virus, causing up to 300,000 annual infections in West Africa. We used a model for Lassa fever in which Rhesus macaques were infected with a related virus, lymphocytic choriomeningitis virus (LCMV). Our goals were to determine the outcome of infection after mucosal inoculation and later lethal challenge, to characterize protective immune responses, and to test cross-protection between a virulent (LCMV-WE) and an avirulent (LCMV-ARM) strain of virus. Although intravenous infections in the monkey model were uniformly lethal, intragastric infections recapitulated the spectrum of clinical outcomes seen in human exposure to Lassa fever virus: death, recovery from disease, and most often, subclinical infection. Plaque neutralization, ELISA, lymphocyte proliferation, and chromium-release assays were used to monitor humoral and cellular immune responses. Cross protection between the two strains was observed. The three out of seven monkeys that experienced protection were also the three with the strongest cell-mediated immunity.     

Egtved virus: Demonstration of virus antigen by the fluorescent antibody technique in tissues of rainbow trout affected by viral haemorrhagic septicaemia and in cell cultures infected with Egtved virus

Summary The use of a direct fluorescent antibody technique for the demonstration of Egtved virus in tissue sections from diseased trout and in infected cell cultures is described.Specific fluorescence indicating the presence of Egtved virus could be demonstrated in kidney, spleen, liver, and heart from fish attacked by VHS.In cell cultures Egtved virus could be demonstrated eight hours after infection as a granular, mainly cytoplasmic fluorescence resembling the one described for vesicular stomatitis virus.Another trout pathogen, IPN virus, could be demonstrated in cell cultures by means of a similar technique. No cross-reaction between VHS and IPN conjugates was observed.     

Lassa and Mozambique viruses: cross protection in experiments on mice and action of immunosuppressants on experimental infections

The pathogenicity of Mozambique virus for random-bred mice and CBA mice was studied. In contrast to Lassa virus, intracerebral inoculation of newborn zandom-bred mice with Mozambique virus (1000 PFU/mouse) results in death of the animals. The pathogenic properties of both viruses for adult CBA mice were found to be similar: intracerebral inoculation of the viruses caused death of the animals within 6-8 days, but not intraperitoneal inoculation. With the latter, they produced a population of immunocompetent cells protecting syngeneic recipient mice against the lethal intracerebral inoculation of the homologous virus. Cross-protection experiments demonstrated that intraperitoneal inoculation of Lassa virus protected 70% of mice against the lethal infection with Mozambique virus, and intraperitoneal inoculation of Mozambique virus protected 45% of mice against Lassa virus. Cyclophosphamide exerted no protective effect in Mozambique virus-infected mice. Cyclosporin A exerted no therapeutic effect in mice lethally infected with Lassa or Mozambique virus.     

Protection of Lassa virus-infected guinea pigs with Lassa-immune plasma of guinea pig, primate, and human origin

Lassa virus-immune plasma has been used to treat human Lassa fever patients; however, criteria for plasma selection were based arbitrarily on available serologic tools and protective efficacy was never directly assessed. To test the validity of plasma therapy for Lassa virus infections in an animal model, and to develop biologically relevant criteria for selection of protective immune plasma, inbred, strain 13 guinea pigs were infected with a lethal dose of Lassa virus and treated with various Lassa-immune plasmas obtained from guinea pigs, primates, and convalescent human patients. Neutralizing antibody titers were determined in a virus dilution, plaque reduction test, and were expressed as a log10 plaque-forming units (PFU) neutralization index (LNI). All guinea pigs treated with immune plasma 6 ml/kg/treatment on days 0, 3, and 6 after virus inoculation were protected, provided the LNI exceeded 2.0. Plasmas obtained from donors in early convalescence (32-45 days) had low titers of N-antibody (LNI less than 2) and failed to confer protection, despite high titers of Lassa antibody measured in the indirect fluorescent antibody (IFA) test. Higher doses of marginally titered plasma conferred increased protection. The degree of protection and suppression of viremia was closely associated with LNI an not IFA titers. Administration of low-titered plasma did not result in immune enhancement. A high dose of human plasma from Liberia (12 ml/kg/treatment) was required to confer complete protection to guinea pigs infected with a Lassa virus strain from Sierra Leone (LNI = 1.6), while a lower dose (3 ml/kg/treatment) was sufficient for protection against a Liberian strain (LNI = 2.8), suggesting that a geographic matching of immune plasma and Lassa virus strain origin may increase treatment success. These studies support the concept of plasma therapy for Lassa infection and suggest that the plaque reduction neutralization test is more appropriate than the IFA test for predicting protective efficacy of passively administered plasma.