Experimental infection of nonhuman primates with sandfly fever virus.

Due to the lack of an animal model, previous studies of sandfly fever have relied upon human challenge trials. We examined the infectivity and potential pathogenicity of sandfly fever virus in cynomolgus monkeys (Macaca fascicularis). Three different preparations of sandfly fever virus. Sicilian strain, and a placebo were compared by different routes of administration. The most notable postchallenge clinical event was a decrease in lymphocytes in the intramuscularly challenged monkeys. Plaque-reduction neutralization responses peaked earlier in animals challenged intravenously as compared with those in animals challenged intramuscularly. There was no evidence for neurotropism or meningeal inflammation. Sandfly fever virus was infectious for cynomolgus monkeys, but produced no detectable clinical disease that might serve as a marker for animal modeling studies. On the other hand, the preclinical data provide supportive evidence for safe parenteral administration of a Sicilian strain of sandfly fever virus inoculum to humans as a challenge model for sandfly fever disease.

A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus.

To investigate the ability of a vaccinia virus-vectored vaccine expressing the M and the S segments of Hantaan (HTN) virus (C. S. Schmaljohn, S. E. Hasty, and J. M. Dalrymple, Vaccine 10:10-13, 1992) to elicit a protective immune response against other hantaviruses, we vaccinated hamsters with the recombinant vaccine and challenged them with HTN, Seoul (SEO), or Puumala (PUU) virus. Neutralizing antibodies to HTN virus were found in all vaccinated hamsters both before and after challenge. Neutralizing antibody titers to SEO virus were present at low levels or were undetectable after two immunizations with the vaccine but were positive in all vaccinated hamsters after challenge with SEO virus and were also positive in control animals that were not challenged. Neutralizing antibodies to PUU virus were observed only in hamsters previously challenged with PUU virus. To assay for virus in the blood and tissues of the hamsters, we developed a nested reverse transcriptase (RT)-PCR with cross-reactive outer primers and serotype-specific inner primers. The RT-PCR specifically detected as little as 1 PFU of virus in serum containing high-titer neutralizing antibodies and was more sensitive than immunofluorescent antibody staining for detecting virus in lung and kidney specimens of infected hamsters. By using the RT-PCR, we found that vaccinated hamsters, challenged with HTN or SEO virus, neither were viremic nor had evidence of virus in their lungs or kidneys. In contrast, vaccinated hamsters challenged with PUU virus were viremic and had PUU virus-specific nucleic acid in their organs.

Prevalence of hepatitis B and C viruses in healthy Indonesian blood donors.

Blood samples were collected from 7572 healthy volunteer blood donors from 21 of the 27 Indonesian provinces, and tested for antibodies to hepatitis C virus (anti-HCV) using the new second-generation enzyme immunosorbent assay, and also tested for hepatitis B surface antigen (HBsAg). We detected anti-HCV in 2.1% of the blood donors. No statistically significant difference was found between males and females or between locations, but there was a statistically significant increasing likelihood of anti-HCV prevalence with increasing age. HBsAg was found in 8.8% of the 3839 tested donors. There was no statistically significant difference between sexes or age groups, but there was a statistically significant higher prevalence in the islands of Sulawesi and eastern Indonesia. Only 7 individuals, from 5 locations, were both anti-HCV and HBsAg positive. Based on responses to a questionnaire, a history of surgery, blood transfusion, intravenous medication, and acupuncture were identified as risk factors for the presence of anti-HCV. No such risk factor was identified for HBsAg prevalence. The combined data suggest separate modes of transmission for the 2 viruses, and indicate the need for continued surveillance for these agents in Indonesian blood banks.

Prevalence of hemagglutination-inhibition and neutralizing antibodies to arboviruses in horses of java.

A study was conducted to measure the prevalence of hemagglutination-inhibition (HI) and neutralizing antibodies against two arboviruses (Chikungunya and Japanese encephalitis virus) in horses of Java, Indonesia. Blood specimens were collected from a sample of 112 horses at two stables: Pulo Mas, a racing track-horse complex, located in a residential area in North Jakarta, and Pamulang, a riding school, located in a rural environment of West Jaya. Sera were tested by the HI assay and plaque reduction neutralization test. JEV antibodies were detected by HI in 58 (52%) of the horses, while only 11 (10%) had Chikungunya antibodies by HI. The proportion of Pamulang horses infected with JEV (66%) was significantly higher than found among Pulo Mas horses (40%) screened (p < 0.01). Of the 58 horses with JEV antibodies by HI, 52 (90%) were found to have specific neutralization antibodies to JEV. HI and neutralization tests on horse sera indicated that the risk to alpha virus infections was minimal in horses surveyed from Java. However, there was a high risk of JEV infection among the same population.

Comparative sensitivity of laboratory methods to diagnose dengue virus infections at Husada Hospital, Jakarta.

Several methods are available for diagnosis of dengue virus infections including a new commercially available dengue blot IgG assay. We conducted a study to compare the sensitivity of the dengue blot with the conventional diagnostic methods. Serum samples from suspected dengue patients were collected for virus isolation and the following serological assays: the hemagglutination-inhibition assay, an IgM/IgG enzyme-linked immunosorbent assay, and the dengue blot. When suspected dengue samples were tested by all methods, viral isolation detected the fewest dengue infections (10.5%), while the IgM/IgG ELISA was the most successful (46.3%) in diagnosing dengue infections. In a specific comparison between the IgM/IgG ELISA and the dengue blot, the dengue blot had an overall sensitivity of 48.8%, with a specificity of 88.7%. When patients were classified by their serological response, the dengue blot had a sensitivity of only 1.7% in those patients with a primary or recent dengue infection, however in secondary infections, the sensitivity of the dengue blot improved to 93.5%. Testing convalescent samples from patients with primary infections, only slightly changed the sensitivity of the dengue blot. The diagnosis of dengue is needed rapidly by clinicians to insure prompt treatment of patients. The dengue blot provides a rapid and easily performed assay, especially sensitive in secondary dengue infections which are most common in hospitalized cases in Asia.

Prevalence of hepatitis C viral antibody in transfused and nontransfused Egyptian children.

Hepatitis C (HCV) virus is recognized as the major cause of what was previously referred to as parenterally acquired (blood-mediated) non-A, non-B hepatitis. A study involving 252 transfused and nontransfused Egyptian children was conducted from November 1990 through February 1991 to determine the prevalence of HCV and the role of blood and blood and blood product transfusions in the spread of the virus. Serum specimens were assayed by a second generation enzyme immunoassay and were considered reactive only after supplemental testing using the second generation recombinant immunoblot assay. Prevalence among 84 young study subjects with hematologic disorders was 55% (46 of 84), while no HCV antibodies were detected among the two nonhematologic pediatric populations studied: 84 hospital admissions and 84 acutely ill but otherwise healthy outpatients (seeking treatment for symptoms associated with a new condition less than three weeks old in the absence of any chronic health problem). Ninety-two percent (77 of 84) of the hematology-related cases had medical histories of multiple transfusions. Positive antibody responses (46) were significantly associated with increased duration of illness (P < 0.001) and the volume and number of transfusions (P < 0.01) when compared with negative ones (38). However, prior hospitalization and/or surgery were not related to HCV antibody status. The high prevalence of HCV antibody among multiply transfused infants and children suggests that blood and blood product supplies should be regularly screened for HCV antibody.

Prevalence of serum antibodies to human T lymphotropic virus-1 in an isolated tribe in the highlands of Irian Jaya, Indonesia.

There have been several recent reports on the high prevalence of serum antibodies to human T lymphotropic virus-1 (HTLV-1) in isolated populations residing in the coastal areas and highlands of Papua New Guinea. In the absence of significant cases of clinical disease, it has been surmised that this reactivity might be the consequence of serologic recognition of yet undefined human retroviruses or parasite antigens. These observations prompted an investigation of the prevalence of anti-HTLV-1 antibodies among members of the Ngalum tribe that dwells in a secluded highland valley in the eastern Jayawijaya Mountains of Irian Jaya, Indonesian New Guinea. Of 165 tribespeople, 85 (52%) were positive for IgG antibodies to HTLV-1 in an indirect enzyme-linked immunosorbent assay. Eighty-two were more than 10 years of age. On the Western blot, all positive sera reacted strongly with the p19 core antigen, but recognition of the envelope antigens, gp46 and gp21, was conspicuously absent. Thirty-four of the 85 villagers with these indeterminant blots had active Plasmodium falciparum infections, but antibody absorption studies with HTLV-1 and P. falciparum erythrocytic stage antigens failed to confirm suspected serologic cross-reactivities. Thirty-three others had acute malaria and/or high titers of anti-malaria antibodies but were seronegative for HTLV-1. We suspect that indeterminant Western blots for HTLV-1 reflect antibody responses to related latent retroviruses that are activated as a consequence of immunosuppression following malaria infection and chloroquine therapy.

Infection of inbred rat strains with Rift Valley fever virus: development of a congenic resistant strain and observations on age-dependence of resistance.

A congenic rat strain (WF.LEW) was derived from the susceptible Wistar-Furth (WF) (background strain) and the resistant LEW (donor strain) inbred strains and was used to evaluate the phenotypic expression of a dominant Mendelian gene that confers resistance to fatal hepatic disease caused by the ZH501 strain of Rift Valley fever virus (RVFV). Resistance to hepatic disease developed gradually with age, with full expression at approximately 10 weeks in the WF.LEW and LEW rat strains. The ZH501 strain caused fatal hepatitis in WF rats regardless of age. However, resistance to the SA75 RVFV strain (relatively non-pathogenic for adult rats), was age- and dose-dependent in both WF and LEW rats. The resistance gene transferred to the newly derived WF.LEW congenic rat strain appears to amplify age-dependent resistance of adult rats, resulting in protection against fatal hepatic disease caused by the virulent ZH501 strain. The congenic rat strain will be a valuable asset in elucidating the mechanism of resistance to Rift Valley fever virus governed by the dominant Mendelian gene.

Pathogenesis of Rift Valley fever in rhesus monkeys: role of interferon response.

Rhesus monkeys inoculated intravenously with Rift Valley fever (RVF) virus presented clinical disease syndromes similar to human cases of RVF. All 17 infected monkeys had high-titered viremias but disease ranged from clinically inapparent to death. Three (18%) RVF virus-infected monkeys developed signs of hemorrhagic fever characterized by epistaxis, petechial to purpuric cutaneous lesions, anorexia, and vomiting prior to death. The 14 remaining monkeys survived RVF viral infection but, 7 showed clinical signs of illness characterized by diminished food intake, cutaneous petechiae, and occasional vomiting. The other 7 monkeys showed no evidence of clinical disease. All monkeys had detectable serum interferon 24-30 h after infection, but 4 of 7 monkeys that did not develop clinical illness had serum interferon titers within 12 h after infection. In lethally infected macaques, indices of hepatic function and blood coagulation were abnormal within 2 days, implicating early pathogenetic events as critical determinants of survival. Serum transferase values were elevated in proportion to severity of clinical disease and outcome of infection. Both myocardial damage and laboratory evidence consistent with disseminated intravascular coagulation were present in fatal infections. All surviving monkeys developed neutralizing antibodies to RVF virus 4-7 days after infection, and this coincided with termination of viremia. Two fatally infected monkeys were viremic until death on days 6 and 8, and the third cleared viremia on day 5 and developed antibody on day 6 but died on day 15. There was a significant correlation between a delayed interferon response and mortality, suggesting that the early appearance of interferon was influential in limiting the severity of disease.

Hemostatic derangement produced by Rift Valley fever virus in rhesus monkeys.

Rift Valley fever (RVF) is an important cause of disease in animals and humans in sub-Saharan Africa. In a small percentage of human cases, the disease is complicated by hemorrhage, which often is associated with a fatal outcome. Inoculation of rhesus monkeys with the Zagazig Hospital strain of RVF virus produced a clinical picture similar to illness in humans. Ten of 17 monkeys developed clinical evidence of hemostatic impairment. When coagulation tests were performed, this group of monkeys had significant abnormalities, including evidence for disseminated intravascular coagulation. These abnormalities were much less pronounced in the remaining seven monkeys-whose only sign of illness was transient fever-and, in general, they paralleled the level of viremia and the degree of elevation in levels of serum hepatic enzymes. Autopsy of the three monkeys with severe disease revealed hepatic necrosis.

Prevention of Rift Valley fever in rhesus monkeys with interferon-alpha.

Prophylactic and therapeutic efficacy of recombinant leukocyte A interferon (rIFN-alpha A) and Sendai virus-induced human leukocyte interferon (HuIFN-alpha) administered intramuscularly to Rift Valley fever virus (RVFV)-infected rhesus monkeys was studied. Clinical, virologic, immunologic, and hemostatic parameters were monitored. Five daily inoculations of 5 X 10(5) units of either interferon product per kilogram of body weight, initiated 24 hours before or 6 hours after RVFV infection, prevented or greatly suppressed viremia. No clinical signs of disease or laboratory evidence of impaired hemostasis was observed. Serum neutralizing antibody to RVFV was detected within 6 days of virus inoculation. Prophylactic administration of 5 X 10(4) or 5 X 10(3) units of rIFN-alpha A per kilogram also limited viremia, hepatocellular damage, and hemostatic derangement. Untreated, RVFV-infected, control monkeys developed high-titered viremia, clinical disease, and impaired hemostasis. These data suggest that rIFN-alpha A and HuIFN-alpha are effective in protecting RVFV-infected rhesus monkeys from viremia and hepatocellular damage and may be beneficial in human RVF infection.

Expression of the Hantaan virus M genome segment by using a vaccinia virus recombinant.

A cDNA containing the complete open reading frame of the Hantaan virus (HTN) M genome segment has been cloned into vaccinia virus. This recombinant virus expresses two glycoproteins which are similar to the HTN structural glycoproteins, G1 and G2, in molecular weight, cleavage pattern, and cellular distribution. Both HTN and recombinant vaccinia virus glycoproteins are exclusively associated with the Golgi apparatus of the cell. Despite this intracellular restriction, mice inoculated with the recombinant vaccinia virus raised neutralizing antibodies against HTN. The specificity of virus neutralization appears to reside in the HTN glycoproteins, since a vaccinia virus recombinant expressing the HTN nucleocapsid protein was unable to elicit a neutralizing antibody response.

Pathogenicity and immunogenicity of a mutagen-attenuated Rift Valley fever virus immunogen in pregnant ewes.

A mutagenized clone of Rift Valley fever virus (RVFV; MV P12) used in inoculation of 3 pregnant ewes was immunogenic, nonpathogenic, and nonabortogenic. In contrast, inoculation of a matched group of 3 pregnant ewes with parent RVFV induced clinical disease and abortions. Ewes given MV P12 delivered healthy lambs that had RVFV antibody titers of less than 1:10 at birth, increasing to greater than or equal to 1:80 after ingestion of colostrum. Ewes inoculated with parent RVFV developed marked viremia, followed by RVFV antibody titers greater than or equal to 1:1,280; ewes inoculated with MV P12 developed low viremia titers and RVFV antibody titers of 1:80 to 1:320. Postpartum challenge exposure of the previously MV P12-inoculated ewes with virulent Zagazig human 501 strain RVFV indicated that the ewes were protected from clinical disease. The RVFV-susceptible female Culex pipiens that fed on the MV P12-inoculated ewes failed to transmit RVFV to hamsters; mosquitoes that fed on the parent RVFV-inoculated ewes became infected and transmitted RVFV to hamsters.

Coding strategy of the S genome segment of Hantaan virus.

Hantaan virus is the type species of the recently recognized Hantavirus genus of Bunyaviridae. The small (S) RNA segment of the negative-sense, tripartite genome was molecularly cloned and the nucleotide sequence was determined. The RNA sequence derived from the cDNA copy was found to contain 1696 nucleotides. A single open reading frame of sufficient size to encode the virus nucleocapsid protein was detected in the cDNA corresponding to viral complementary-sense RNA. RNA transcripts of the cDNA were synthesized with SP6 polymerase and were used to program cell-free reticulocyte lysate translation systems. Viral complementary-sense transcripts served as efficient messages in translation systems and generated Hantaan nucleocapsid protein. No translation products were detected when lysates were programmed with viral-sense transcripts. This coding assignment of the nucleocapsid protein to the viral complementary-sense RNA of the S genome segment is consistent with those of other members of this family. Unlike other Bunyaviridae, which encode both a nucleocapsid protein and a nonstructural (NSs) protein of similar sizes, a NSs protein has not been identified for Hantaan virus. Furthermore, other than the nucleocapsid protein gene sequence, the only potential open reading frame in Hantaan S RNA encoded a short, 48-amino acid polypeptide which initiated two codons beyond the termination of the nucleocapsid protein in the same reading frame. These data demonstrate that the coding strategy of the Hantaan virus S RNA is different than those reported for other viruses in this family.