Spotted fever rickettsiae in ticks from the northern Sinai Governate, Egypt.

A field study was initiated in 1988 to investigate whether spotted fever group rickettsiae occur in geographic areas in Egypt that are adjacent to an area in the southern Israeli Negev that has a defined focus of spotted fever disease. Ticks were collected from dogs, sheep, and camels at four study sites in the northern Sinai. Tick hemolymph was processed for rickettsial detection by staining with fluorescein isothiocyanate-conjugated antibody to Rickettsia rickettsii. Of the 442 hemolymphs examined, 15 contained immunofluorescent rickettsiae. Eight hemolymph test-positive (HT+) ticks were Rhipicephalus sanguineus removed from dogs; the other HT+ ticks comprised three Hyalomma species, H. anatolicum, H. impeltatum, and H. dromedarii. Both HT+ and HT- ticks were tested for rickettsial DNA using the polymerase chain reaction (PCR). Eight of 10 HT+ field-collected ticks were PCR positive (PCR+). All laboratory colony R. rickettsii-infected ticks were PCR+. No HT- ticks from field or laboratory isolates were PCR+.

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.

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Physiological and immunologic disturbances associated with shock in a primate model of Lassa fever.

The degree of cell and organ damage in clinical and histological studies of patients dying of Lassa fever has been insufficient to explain the catastrophic shock characteristic of the fatal illness. To explore this issue further, we conducted a study of the evolution of shock in three Lassa virus-infected rhesus monkeys. By the sixth day after infection, a marked, progressive reduction of in vitro platelet aggregation occurred despite normal numbers of circulating platelets and a normal platelet survival time and was accompanied by loss of prostacyclin production by postmortem endothelium. Both of these functions recovered rapidly in a surviving animal. There was no evidence of disseminated intravascular coagulation, nor were clotting factors significantly abnormal. We observed association of viral antigen with neutrophils and progressive neutrophilia. Viremia was not reduced by a brisk antibody response in our animals, and there was a general depression of response to mitogens in mixed lymphocyte stimulation assays. Our findings suggest that shock in Lassa fever is due to biochemical dysfunctions of platelets and endothelial cells and results from loss of intravascular plasma volume, effusions, and hemorrhage.

Kinetic study of platelets and fibrinogen in Lassa virus-infected monkeys and early pathologic events in Mopeia virus-infected monkeys.

The rhesus monkey, an established model of Lassa fever, was used to study hematologic and hemostatic aspects of Lassa fever and whether Mopeia (also known as Mozambique) virus induces any cellular damage in this model. Six days after subcutaneous injection of 10(3.48) plaque forming units (PFU) of Lassa virus (Josiah strain) one group of monkeys received an intravenous injection of 111In-labeled allogeneic platelets and another group received 125I-labeled alogeneic fibrinogen. Lassa virus-infected monkeys developed a severe clinical illness with high viremia and typical pathology. Lassa antigen was found in most tissues using a Lassa nucleocapsid-specific monoclonal antibody. Platelet counts remained within normal limits. Platelet and fibrinogen kinetics were similar in infected and control animals. Hematologic and hemostatic changes indicate that disseminated intravascular coagulation plays no role in this model of Lassa fever. Levels of plasma fibronectin were reduced in Lassa-infected monkeys. Mopeia virus-infected monkeys were normothemic, aviremic, and there was no detection of Mopeia antigen in any tissues using polyclonal or monoclonal antibodies. Mopeia virus was recovered from the spleen of one monkey. Mopeia virus was associated with hepatocellular and renal tubular damage.

Production and characterization of monoclonal antibodies to Rickettsia rickettsii.

Five mouse ascitic fluids (MAFs) containing monoclonal antibody to Rickettsia rickettsii were produced from three original fusions by murine hybridoma technology. The five MAFs were fractionated and purified; each contained monoclonal antibody of the immunoglobulin G2a subclass. Each monoclonal antibody-containing MAF was titrated by indirect immunofluorescence against three R. rickettsii isolates from humans and four other spotted fever group rickettsiae. Each MAF was also titrated in the complement fixation, latex agglutination, microagglutination, and indirect hemagglutination tests. Two of the MAFs were examined for their ability to prevent fever and rickettsemia in susceptible guinea pigs after a 1:100 dilution of each was mixed with viable R. rickettsii, and all five MAFs were titrated in the mouse toxicity phenomenon assay. All MAFs had high indirect immunofluorescence titers to the three strains of R. rickettsii (1:200,000 to 1:800,000), reduced indirect immunofluorescence titers to R. montana, and were nonreactive with R. akari, R. sibirica, and R. conorii. Each MAF was able to fix complement in the presence of spotted fever group antigen reagent and agglutinate a suspension of purified R. rickettsii, and each was negative in both the latex agglutination and the indirect hemagglutination tests. The two MAFs which were tested proved to be capable of preventing rickettsemia and death in guinea pigs, and each MAF was able to prevent death in mice at dilutions ranging from 1:40 to 1:80.

Rocky Mountain spotted fever vaccine in an animal model.

Formalin-killed, purified Rickettsia rickettsii vaccine was evaluated in a guinea pig model of R. rickettsii infection. Vaccinated guinea pigs were partially protected by the vaccine when challenged with virulent, viable rickettsiae. Greater protection was observed when higher doses of vaccine were given and when frequent booster injections were administered. Stimulation of cell-mediated immunity to the vaccine antigens was variable and also appeared to be achieved more reproducibly with booster vaccinations. Serum antibody was elicited by high doses of vaccine and by booster vaccinations. The presence of serum antibody was useful in predicting immunity to challenge with R. rickettsii.

Correlation of rickettsial titers, circulating endotoxin, and clinical features in Rocky Mountain spotted fever.

Blood rickettsial titers, skin biopsy results, and circulating endotoxin measurements were correlated with the clinical course of disease in patients with Rocky Mountain spotted fever (RMSF). Nine of 11 patients with documented RMSF had Rickettsia rickettsii isolated from plasma samples. Of the eight patients in whom rickettsial titers were measured, seven had 10(0.7) to 10(1.2) median tissue culture infective doses (TCID50) per milliliter; all seven had mild to moderately severe disease. One patient with fulminant, fatal untreated RMSF had 10(3) TCID50/mL of postmortem plasma. Two patients from whom rickettsiae were not isolated had positive direct immunofluorescent stains of skin biopsy material for R rickettsii. Circulating endotoxin was present in two patients, one with documented rickettsemia and one with a positive skin biopsy alone. Only low levels of circulating rickettsiae are present in patients with moderately severe disease. Measurement of plasma endotoxin is not useful in the early diagnosis of RMSF.

Experimental infection of rhesus monkeys with Lassa virus and a closely related arenavirus, Mozambique virus.

As a model for the pathogenesis of Lassa fever in humans, nine rhesus monkeys were inoculated with Lassa virus. Three monkeys had had a previous asymptomatic experimental infection with Mozambique virus, a closely related arenavirus; these monkeys were protected from illness and viremia and manifested only mild pathologic lesions. The other animals developed severe disease and viremia. At necropsy, hepatocellular necrosis, interstitial pneumonia, a unique pulmonary arteritis, adrenal gland necrosis, encephalitis, and uveitis were prominent pathologic lesions which correlated with the organ titers of virus. One animal infected with Lassa virus developed prolonged viremia, a typical immune response, and sudden onset of lower limb paralysis after recovery; at necropsy chronic proliferative arteritis of the spinal cord, brain, and heart was evident. Similarities and differences in the pathologic lesions in this model and Lassa fever in humans indicate that care must be taken in interpreting the results of experiments concerning immune prophylaxis, pathogenesis, and treatment in rhesus monkeys.

Interrelationships among arenaviruses measured by indirect immunofluorescence.

An evaluation of reciprocal indirect immunofluorescent antibody titers for at least one representative of each known arenavirus serotype leads to the conclusion that the Old World aranaviruses are more closely related to each other than to other members of the group. These relationships are particularly evident in evaluations of human convalescent sera. New World arenaviruses cross-react strongly with each other in most instances but only weakly with with Old World arenaviruses.

Indirect immunofluorescence for the diagnosis of Lassa fever infection.

The indirect immunofluorescent technique is a rapid method for identification of Lassa virus and Lassa virus antibody. In the study reported here, Lassa virus antigen was detected by this method in Vero cell cultures within 24 hours of their inoculation with an infected human blood specimen. A diagnosis could be made from field-collected specimens within 3 days of their receipt.Fluorescent antibodies against Lassa virus were detected in human serum as early as 7 to 10 days after onset of illness, and were detected as long as 61 months after infection. Complement fixing antibodies were not as long lasting.No antigenic differences were noted by the indirect immunofluorescence technique between several Lassa virus strains isolated from Nigeria, Liberia, and Sierra Leone over a 6-year period.

Comparative pathology of Lassa virus infection in monkeys, guinea-pigs, and Mastomys natalensis.

Experimental Lassa virus infections of squirrel monkeys, guinea-pigs, and the African multimammate rat, Mastomys natalensis, were studied virologically and pathologically. In the monkeys, early viral lymphoreticulotropism, hepatotropism, nephrotropism, and viraemia were noted. At the time of death, viral titres in nearly all target organs were associated with necrotic changes: splenic lymphoid necrosis, renal tubular necrosis, myocarditis, arteritis, and hepatocytic regeneration. In convalescent monkeys, organ titres diminished slowly, and viraemia persisted at 28 days. At this time, renal and splenic regeneration was occurring and a new lesion, choriomeningitis, was present.Guinea-pigs infected with Lassa virus developed respiratory insufficiency with pulmonary oedema, alveolar hyaline membranes, myocarditis, and focal calcification of myocardial fibres and hepatocytes. Dying animals contained Lassa virus in virtually every organ tested, whereas survivors at 56 days were free of virus and had high complement-fixing antibody titres.Infection of neonatal Mastomys did not cause any clinical disease or pathological lesions despite the presence of virus in the blood, lymph nodes, liver, spleen, lung, brain, urine, and throat secretions throughout the 74-day study. Infected adult Mastomys also remained normal but had virus in many organs. In one animal, virus persisted until the termination of the study at 103 days. Several animals developed a mild meningoencephalitis. The pattern of infection and virus shedding in M. natalensis is ideal for maintenance of the virus in nature; together with the epidemiological field data this emphasizes the incidental nature of the exposure and infection of man.