Pirital virus (Arenaviridae) infection in the syrian golden hamster, Mesocricetus auratus: a new animal model for arenaviral hemorrhagic fever.

Adult Syrian golden hamsters inoculated intraperitoneally with Pirital virus, a recently discovered member of the Tacaribe complex of New World arenaviruses, developed a progressively severe, fatal illness with many of the pathologic features observed in fatal human cases of Lassa fever and other arenaviral hemorrhagic fevers. Most of the animals became moribund by Day 5 and were dead by Day 7 after inoculation. The most consistent histopathologic changes included interstitial pneumonitis, splenic lymphoid depletion and necrosis, and multifocal hepatic necrosis without significant inflammatory cell infiltration. The liver changes ranged from single cell death by apoptosis to coagulative necrosis of clusters of hepatocytes. Immunohistochemical studies of the liver demonstrated the presence and accumulation ot Pirital virus antigen within hepatocytes as well as Kupffer cells. An in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay showed progressively increasing apoptotic activity in the liver of infected hamsters. A human hepatoblastoma cell line (Hep G2/C3A) inoculated with Pirital virus also developed progressive cell destruction and accumulation of viral antigen, as demonstrated by immunofluorescence. Results of this pilot study suggest that the Pirital virus-hamster model is a very promising new small animal model for studying the pathogenesis of arenavirus infections, particularly, the mechanism of direct virus-induced hepatic injury. It may also be useful for testingantiviral agents for treatment of arenaviral hemorrhagic fevers.

Diagnosis of Oropouche virus infection using a recombinant nucleocapsid protein-based enzyme immunoassay.

Oropouche (ORO) virus is an emerging infectious agent that has caused numerous outbreaks of an acute febrile (dengue-like) illness among humans in Brazil, Peru, and Panama. Diagnosis of ORO virus infection is based mainly on serology. Two different antigens, hamster serum antigen (HSA) and Vero cell lysate antigen (VCLA), are currently used in enzyme immunoassays (EIAs) in Brazil and Peru, respectively, to investigate the epidemiology of ORO virus infection. Both antigens involve use of infectious virus, and for this reason their use is restricted. Consequently, the frequency and distribution of ORO virus infection are largely unexplored in other countries of South America. This report describes the use of a bacterially expressed recombinant nucleocapsid (rN) protein of ORO virus in EIAs for the diagnosis of ORO virus infection. The data revealed that the purified rN protein is comparable to the authentic viral N protein in its antigenic characteristics and is highly sensitive and specific in EIAs. Among 183 serum samples tested, a high degree of concordance was found between rN protein-based EIA and HSA- and VCLA-based EIAs for the detection of both ORO virus-specific immunoglobulin M (IgM) and IgG antibodies. The high sensitivity, specificity, and safety of the rN protein-based EIA make it a useful diagnostic technique that can be widely used to detect ORO virus infection in South America.

Allpahuayo virus: a newly recognized arenavirus (arenaviridae) from arboreal rice rats (oecomys bicolor and oecomys paricola) in northeastern peru.

Allpahuayo virus was initially isolated from arboreal rice rats (Oecomys bicolor and Oecomys paricola) collected during 1997 at the Allpahuayo Biological Station in northeastern Peru. Serological and genetic studies identified the virus as a new member of the Tacaribe complex of the genus Arenavirus. The small (S) segment of the Allpahuayo virus prototype strain CLHP-2098 (Accession No. AY012686) was sequenced, as well as that of sympatric isolate CLHP-2472 (Accession No. AY012687), from the same rodent species. The S segment was 3382 bases in length and phylogenetic analysis indicated that Allpahuayo is a sister virus to Pichinde in clade A. Two ambisense, nonoverlapping reading frames were identified, which result in two predicted gene products, a glycoprotein precursor (GPC) and a nucleocapsid protein (NP). A predicted stable single hairpin secondary structure was identified in the intergenic region between GPC and NP. Details of the genetic organization of Allpahuayo virus are discussed.

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

Evidence of sexual reproduction in the protozoan parasite Leishmania (Kinetoplastida: Trypanosomatidae).

Parasitic protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) are generally thought to multiply by binary fission; however, data from quantitative microspectrophotometry indicate that nuclear fusion or sexual reproduction takes place in the intracellular amastigote form. Among several different Leishmania species, the mean +/- SD nuclear DNA content of all promastigotes (extracellular form) and of some amastigotes (intracellular form) in macrophages was 0.098 +/- 0.032 relative units; in contrast, other amastigotes within the same macrophage had a mean +/- SD nuclear DNA content of 0.219 +/- 0.050. The latter population of amastigotes are apparently produced when the nuclei of a pair of 0.098 amastigotes fuse. These 0.219 amastigotes later go through what is probably the typical meiotic cycle to reform the 0.098 condition we observed among promastigotes. The demonstration of sexual reproduction in Leishmania has important implications for the future direction of research on this medically important parasite.

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Vesicular stomatitis virus, New Jersey serotype: replication in and transmission by Lutzomyia shannoni (Diptera: Psychodidae).

Laboratory-reared female sand flies (Lutzomyia shannoni) were experimentally infected, orally and by intrathoracic inoculation, with the New Jersey serotype of vesicular stomatitis (VSNJ) virus. Virus replication occurred in the insects following infection by both routes. Virus titers greater than 10(4) plaque forming units of VSNJ virus were present in heads of orally infected sand flies 12 days after virus ingestion, confirming that a persistent disseminated infection had occurred. Both orally and parenterally infected Lu. shannoni transmitted VSNJ virus by bite to susceptible rodents and by transovarial transmission to a small percentage of their F1 progeny. The significance of these findings in the epizootiology of VSNJ virus on Ossabaw Island, Georgia, an enzootic focus of this virus, is discussed.

Natural infection of humans, animals, and phlebotomine sand flies with the Alagoas serotype of vesicular stomatitis virus in Colombia.

Five isolations of the Alagoas serotype of vesicular stomatitis virus (Rhabdoviridae: Vesiculovirus) were made from naturally infected phlebotomine sand flies (Lutzomyia spp.) collected in Colombia. These are the first isolations of Alagoas virus from an arthropod. Replication of the virus occurred in laboratory-reared sand flies (Lutzomyia longipalpis) after inoculation. Bite and transovarial transmission of the virus was also demonstrated in experimentally infected sand flies. Alagoas virus neutralizing antibodies were found in sera of humans and animals living near the insect collection site; antibody rates among human residents of two nearby towns were 63% and 83%, respectively. Results of comparative serologic studies demonstrated that Alagoas virus is closely related antigenically to Indiana, Cocal, and Maraba viruses and that these four agents form a complex within the vesicular stomatitis virus serogroup. The antigenic similarity among these four viruses makes their differentiation difficult; it also raises doubts about the accuracy of current laboratory methods used for identifying isolates in this serogroup. A discussion follows on the significance of human antibodies to these agents and on the role of sand flies in their ecology.

A simple method for experimental infection of phlebotomine sand flies with Leishmania.

Mouse macrophages, grown in continuous cell culture at 37 degrees C, were inoculated with the promastigote stage of various human pathogenic Leishmania species. Under these culture conditions, the parasites rapidly entered the cells and transformed into amastigotes. Two or 3 days after inoculation, the infected macrophages were mixed with washed human erythrocytes and were fed to female sand flies (Phlebotomus papatasi and Lutzomyia longipalpis) through a chick skin membrane. Within 7-10 days after feeding, large numbers of promastigotes were observed in the anterior portion of the insects' guts, indicating that the infected sand flies were capable of transmitting the parasites by bite. This relatively simple and rapid technique should facilitate studies on the biology of Leishmania in their insect vectors. It also eliminates the need for animals as a source of amastigotes.