Punta Toro Virus Research Articles

A Comparison of Pyrimidinone Analogue Immunomodulators for Treatment ofPhlebovirusInfections in Mice

The substituted pyrimidinone immunomodulators 5-bromo-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (ABPP), 5-iodo-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (AIPP), 5-bromo-2,3-dihydro-2-imino-6-methyl-4(1H)-pyrimidinone (ABMP), 5-chloro-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (ACPP), 5-bromo-2,3-dihydro-2-iminp-6-(3-fluorophenyl)-4(1H)-pyrimidinone (ABmFPP) and 5-chloro-2,3-dihydro-2-imino-6-(2,3-difluorophenyl)-4(1H)-pyrimidinone (AComF2PP) were evaluated against the hepatotropic and immunosuppressive infection of mice induced by Punta Toro virus, a Phlebovirus related to Rift Valley fever virus. Using intraperitoneal (i.p.) treatments once daily for 3 days beginning 1 day prior to virus exposure, the order of efficacy was ABPP > ABmFPP > AComF2PP > AIPP > ABMP > ACPP, with prevention of death as end-point. Using single i.p. treatment 1 day prior to virus exposure, antiviral activity was seen in the order ABPP > ABmFPP > ABMP > AIPP > ACPP > AComF2PP. Three active pyrimidonones, ABPP, ABMP and AIPP, were given by gavage (p.o.) using the above 3-day treatment schedule. In this experiment, expanded parameters were used, with prevention of death seen as well as decreased hepatic icterus, as evidenced by lowered liver scores and serum glutamic oxalacetic and pyruvic transaminases. In addition, recoverable virus titres were reduced in the livers and sera of the mice. Greatest activity was seen using ABPP, followed by ABMP and then by AIPP. ABPP in doses of 100–800 mg kg−1was markedly effective when administered p.o. in single treatments given as late as 24 hr after virus inoculation; a single dose, 400 mg kg−1, prevented death when given as late as 48 hr after virus exposure. In comparison of three once-daily, every-other-day, or every 2-day p.o. treatments, ABPP was most effective using the every-other-day regimen.

Inhibition of Phlebovirus Infections in vivo by Tiazofurin and Selenazofurin

The c-nucleosides tiazofurin and selenazofurin were evaluated in mice against the Adames strain of Punta Toro virus, a Phlebovirus related to Rift Valley fever and sandfly fever viruses. When administered subcutaneously (s.c.) twice a day for 5 days starting 4h before virus inoculation, tiazofurin reduced mortality, hepatic icterus scores, serum aspartate aminotransferase (AST) levels, and serum virus titres at 250–500 mg kg−1 day−1. Liver virus titres were not reduced in these treated animals, however. Oral tiazofurin treatment was effective by the same criteria at 375 and 750 mg kg−1, except that liver virus titres were reduced at 750 mg kg−1. Similar effects were observed with selenazofurin given s.c. (80–160 mg kg−1) or orally (80–320 mg kg−1) for 5 days. Selenazofurin suppressed liver virus titres by oral but not s.c. treatment. In a time-course study tiazofurin was effective s.c. at 250–1000 mg kg−1 when administered once only at 48 h after virus inoculation, whereas treatments at 4, 24, 72 and 96 h were less or not effective. By comparison with published results, tiazofurin and selenazofurin do not appear to be as active or selective as ribavirin and ribamidine against Punta Toro virus infections in mice.

Effects of 7-thia-8-oxoguanosine Alone of combination with ribavirin on punta toro virus infections in mice : D.F. Smee, J.H. Huffman, J. Coombs, J.W. Huggins ∗, and R.W. Sidwell. Antiviral Program, Utah State University, Logan, Utah, and ∗U.S. Army Medical Research Institute of Infectious Disease, Ft. Detrick, Maryland, U.S.A.

Effects of 7-thia-8-oxoguanosine Alone of combination with ribavirin on punta toro virus infections in mice : D.F. Smee, J.H. Huffman, J. Coombs, J.W. Huggins ∗, and R.W. Sidwell. Antiviral Program, Utah State University, Logan, Utah, and ∗U.S. Army Medical Research Institute of Infectious Disease, Ft. Detrick, Maryland, U.S.A.

The S RNA segment of Sandfly fever Sicilian virus: Evidence for an ambisense genome

The complete nucleotide sequence of the S RNA segment of Sandfly Fever Sicilian (SFS) virus ( Phlebovirus, Bunyaviridae) was determined from overlapping cDNA clones and by primer extension. The RNA is 1746 nucleotides in length and has two large open reading frames (ORF), one of which (24.8 kDa) is viral-complementary in sense, and the other (30.4 kDa) is in the viral sense. This ambisense genome arrangement has been seen in another member of the Phlebovirus genus, Punta Toro (PT) virus ( T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293–306 ), but not in representatives of either the Bunyavirus or H antavirus genera of the Bunyaviridae. Comparison of the predicted amino acid sequences for SFS virus with the recognized products of PT S RNA ( T. Ihara, Y. Matsuura, and D. H. L. Bishop, 1985, Virology 147, 317–325 ; H. A. Overton, T. Ihara, and D. H. L. Bishop, 1987, Virology 157, 338–350 ) indicated that the 24.8-kDa ORF encodes the nucleoprotein (N) of SFS virus, and the 30.4-kDa ORF codes for a nonstructural protein (NSs). Subgenomic messenger RNAs, from which these two proteins are presumably translated, were detected in virus-infected cellS.

In Vitro and in Vivo Phlebovirus Inhibition by Nucleosides Related to Ribavirin

Abstract Eleven compounds were compared to ribavirin for their in vitro and in vivo inhibition of Punta Toro virus (PTV), a phlebovirus in the Bunyaviridae virus family.

Effects of ribamidine, a 3-carboxamidine derivative of ribavirin, on experimentally induced Phlebovirus infections

Ribamidine (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine) was inhibitory in rhesus monkey kidney (LLC-MK 2 derivative) cells to Adames and Balliet strains of Punta Toro virus (PTV), a Phlebovirus related to Rift Valley fever and sandfly fever viruses. The 50% effective dose was 8 and 12 μg/ml against each respective virus strain; the 50% cytotoxic dose was 320 μg/ml, giving selectivity indices of 40 and 27 against each virus strain. The virus ratings were 1.2 and 1.0, respectively. In radiolabel uptake studies, ribamidine had a moderate effect on [ 3H]leucine uptake at dosages down to 1 μg/ml, but [ 3H]thymidine, [ 32P], and [ 3H]uridine were inhibited at high (100–1000 μg/ml) doses only. Subcutaneous (s.c.) and oral treatments of Adames PTV-infected mice were equally highly effective, as evidenced particularly by 100% survivors. Reduced hepatic icterus, serum oxalic acid transaminase, serum glutamic pyruvic acid transaminase, and recoverable virus titers from livers and sera of infected mice were also seen as a result of ribamidine treatment. Twice daily treatment for 5 days could be started as late as 72 h post-virus inoculation (p.v.i.) with significant inhibition of PTV infection seen. Single s.c. treatments administered as late as 48 h p.v.i. were similarly effective. Using the chronic therapy schedule, the maximum tolerated dose was 1000 mg/kg/day and the minimum effective dose was 31.3 to 62.5 mg/kg/day. Using single treatment, a maximum tolerated dose was >1000 mg/kg, and the minimum effective dose was 125 mg/kg. Ribamidine s.c. treatment of mice infected intracerebrally with the Balliet strain of PTV resulted in a moderate infection-inhibitory effect, seen especially by reduced virus titers in the brains of the infected, treated mice.

Antigenic analysis of Punta Toro Virus and identification of protective determinants with monoclonal antibodies

Hybridomas producing monoclonal antibodies to the three major structural proteins of Punta Toro virus (PTV) were established by fusion of spleen cells with Sp2/0-Ag-14 mouse plasmacytoma cells. Thirty-six independently derived monoclonal antibodies were evaluated in neutralization, hemagglutination inhibition, and ELISA assays and the isotype, antigen specificities, and cross-reactivities were determined. These antibodies were also assessed for their ability to provide protection in a murine model. Both G1- and G2-specific antibodies were obtained which neutralized virus infectivity in vitro and inhibited hemagglutination, whereas nucleocapsid-specific antibodies exhibited neither activity. All of the anti-G1 antibodies were PTV-specific, whereas anti-G2 and anti-nucleocapsid antibodies exhibited varying patterns of cross-reactivity with heterologous phleboviruses. All of the G1-reactive monoclonal antibodies, which bound to epitopes in two distinct topological sites as determined by competitive binding assays, provided efficient protection to both immunocompetent and immunosuppressed mice. In contrast, of the 23 G2-reactive antibodies, only 8 were able to protect immunocompetent mice and only one was able to protect immunosuppressed animals. The degree of protection achieved in vivo did not correlate directly with the neutralization titers determined in vitro.

Antigenic analysis of Punta Toro Virus and identification of protectivedeterminants with monoclonal antibodies1, 2

Antigenic analysis of Punta Toro Virus and identification of protectivedeterminants with monoclonal antibodies1, 2

Intracellular accumulation of punta toro virus glycoproteins expressed from cloned cDNA

The Punta Toro virus (PTV) middle size (M) RNA encodes two glycoproteins, G1 and G2, and possibly a nonstructural protein, NS M. A partial cDNA clone of the M segment which contains G1 and G2 glycoprotein coding sequences but lacks most of the NS M sequences was inserted into the genome of vaccinia virus under the control of an early vaccinia promoter. Cells infected with the recombinant virus were found to synthesize two polypeptides with molecular weights of 65,000 (G1) and 55,000 (G2) that reacted specifically with antibody against PTV. Studies using indirect immunofluorescence microscopy revealed that these proteins accumulated intracellularly in the perinuclear region. The results of endoglycosidase H digestion of these glycoproteins suggested that both G1 and G2 glycoproteins were transported from the RER to the Golgi complex. These proteins were not chased out from the Golgi region during a 6-hr incubation in the presence of cycloheximide. Surface immune precipitation and 125I-protein A binding assays also demonstrated that the majority of the G1 and G2 glycoproteins are retained intracellularly. These results indicate that the PTV glycoproteins contain the necessary information for retention in the Golgi apparatus.

In vitro and in vivo Phlebovirus inhibition by ribavirin

Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was markedly inhibitory in vitro to Adames and Balliet strains of Punta Toro virus (PTV), a Phlebovirus related to Rift Valley fever and sandfly fever viruses. By using inhibition of viral cytopathic effect in LLC-MK2 cells with both virus strains, the 50% effective dose was 4 to 10 micrograms/ml and the virus rating was 1.3. The Adames strain of PTV infection in mice was established for evaluation of the in vivo antiviral efficacy of ribavirin. The drug was administered subcutaneously (s.c.) twice daily for 5 to 7 days beginning 4 h pre-virus inoculation, 24 h post-virus inoculation, or 36 h post-virus inoculation, with increased survivors, reduced hepatic icterus, reduction of serum glutamic oxalic acid transaminase and serum glutamic pyruvic acid transaminase, and inhibition of infectious virus from sera and livers of infected mice. The minimum effective dose was 4.7 mg/kg per day, with a maximum tolerated dose of 75 mg/kg per day. When the same treatment schedule beginning 4 h pre-virus inoculation, 4 h post-virus inoculation, or 24 h post-virus inoculation was used, orally administered ribavirin was effective at doses as low as 6.3 mg/kg per day. Single s.c. ribavirin treatments at doses of 175 to 700 mg/kg administered from 4 to 48 h post-virus inoculation were also effective. No effect was seen when ribavirin was administered s.c. to mice infected intracerebrally with the PTV strain Balliet, even though treatment was begun 36 h before virus exposure.

Punta Toro virus infection of C57BL/6J mice: a model for phlebovirus-induced disease

Punta Toro virus infections of inbred strains of mice have been characterized and evaluated as a model in which to study various aspects of the host response to phlebovirus infections and the requirements for protective immunity. The Adames strain of Punta Toro virus was found to be strongly hepatotropic and lymphotropic and the outcome of infection was largely a function of age. C57BL/6J mice of less than 5 weeks of age uniformly developed fulminant hepatocellular necrosis with mean survival times of 4.2 days. Resistance to lethal infection increased with age such that >95% of 8-week-old mice survived challenge. The kinetics of viremia, antibody production, and hematological changes in 4- and 8-week animals indicated that the survival of the older animals is related to their ability to delay virus replication and the development of hepatic lesions during the initial 48 h of infection and their ability to terminate virus replication and clear virus from the circulation 4 to 5 days after infection. The mechanisms responsible for this resistance were studied using anti-interferon serum, immunosuppression, and passive immunization.

Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2

We have determined the complete nucleotide sequence of the virion M RNA segment of Uukuniemi virus ( Uukuvirus genus, Bunyaviridae) from cloned cDNA. The RNA that encodes the two membrane glycoproteins G1 and G2 is 3231 residues long (mol wt 1.1 × 10 6). The 5′ and 3′ ends of the RNA are partially complementary to each other for some 30 bp, enabling the formation of a stable panhandle structure (ΔG = −40 kcal/mol) and the circularization of the molecule. The extreme 5′ and 3′ terminal nucleotides are identical for 10 to 13 residues to those of the M RNA of Punta Toro and Rift Valley fever viruses, two members of the Phlebovirus genus. A single open reading frame comprising 1008 amino acid residues (mol wt 113,588) was found in the mRNA-sense strand between nucleotides 18 and 3042. This probably corresponds to the previously identified 110,000-Da precursor (pl 10) of G1 and G2. By comparing the partial aminoterminal sequences of purified G1 and G2 with the deduced protein sequence we confirmed that the gene order is NH 2-G1-G2-COOH. Both mature G1 and G2 are preceded by a stretch of 17 predominantly hydrophobic amino acids likely to represent the signal sequences. At their COON-terminal ends, G1 and G2 have a hydrophobic stretch of amino acids, 19 and 27 residues, respectively, that probably anchors the proteins to the lipid bilayer. The sequence indicates that mature G2 is 495 amino acids long (mol wt 54,869), whereas the exact size of G1 is unclear, since the location of the COON-terminus of G1 is not known. An upper value of 479 amino acids (mol wt 55,181) can, however, be suggested. Both G1 and G2 contain four potential glycosylation sites for Asn-linked glycans and both are unusually rich in cysteines, 6.1% in G1 and 5.4% in G2. Comparison of the amino acid sequence of the M RNA product of Uukuniemi virus with that of Punta Toro and Rift Valley fever viruses showed in both cases a weak homology that was more pronounced for the proteins located at the COON-terminal end of the precursor. This suggests a distant evolutionary relationship between the Phlebo- and Uukuvirus genera.

Identification of the N and NS s proteins coded by the ambisense S RNA of punta toro phlebovirus using monospecific antisera raised to baculovirus expressed N and NS s proteins

An essentially complete DNA copy of the ambisense S RNA species of Punta Toro (PT) phlebovirus (T. Ihara, Akashi, and D. H. L. Bishop, 1984, Virology 136, 293–306) has been inserted in either orientation into Autographa callfornica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5′ coding region of the AcNPV polyhedrin gene (G. E. Smith, M. D. Summers, and M. J. Fraser, 1983, Mol. Cell. Biol. 3, 2156–2165). The two types of recombinant viruses were used to infect Spodoptera frugiperda cells and the expressed PT viral proteins characterized. Recombinant AcNPV having the S DNA in one orientation expressed PT virus N protein in amounts estimated to represent some 50% of the infected cell extracts, whereas recombinants with the S DNA in the other orientation expressed the putative PT virus NS s protein in lower quantities. Antisera that were monospecific with respect to each of the two PT proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NS s proteins in PT virus-infected Vero cells.

Characterization of Punta Toro S mRNA species and identification of an inverted complementary sequence in the intergenic region of Punta Toro phlebovirus ambisense S RNA that is involved in mRNA transcription termination

The transcription termination sites for the subgenomic N and NS S mRNA species coded by the 1904 nucleotide, ambisense S RNA species of Punta Toro (PT) phlebovirus (Bunyaviridae, Ihara et al., 1984) have been identified by Northern analyses using a series of synthetic oligodeoxynucleotides. For the viral-complementary N mRNA species the oligonucleotides that were used represented viral RNA sequences; for the viral-sense NS S mRNA species they represented viral-complementary sequences. The results have been confirmed by employing the same oligodeoxynucleotides to backcopy purified mRNA preparations in the presence of appropriate chain-terminating dideoxynucleotides. The results obtained have allowed the 3′ termini of both mRNA species to be mapped to a common region of the viral S RNA between RNA residues 977 and 1017. Based on these results and the presence of short non-viral sequences at the 5′ ends of the PT mRNA species (Ihara et al., 1985), the estimated sizes of the PT N and NS S mRNA species are of the order of 1000 and 900 nucleotides, respectively. Computer analysis of DNA sequences representing the centrally located intergenic region (i.e., residues 768–1127) revealed a long inverted complementary sequence corresponding to nucleotides 886–1092. The peak of this potential hairpin structure, residue 996, correlates to the region of the genome identified by Northern analyses to be involved in the termination of mRNA transcription. Neither the N nor NS S mRNA species appeared to be polyadenylated to the extent that is characteristic of most eukaryotic mRNA species, although from the indicated sequences the viral mRNA species contain 3′ proximal regions that are rich in short stretches of adenylic acid residues. This is particularly true for the N mRNA species, permitting its purification by selective oligo(dT) chromatography. In vitro translation of purified N and NSS mRNA species by rabbit reticulocyte lysates resulted in the synthesis of proteins that were identified as the viral N and NS S species, respectively, by comparison with proteins obtained from PT virus infected Vero cells.

Analyses of the mRNA transcription processes of Punta Toro phlebovirus (Bunyaviridae)

The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293–306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NS s, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NS s mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NS s mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5′ ends of the N and NS s mRNA species have shown them to be heterogeneous in sequence and some 11–18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409–6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.

Regulation of the mRNA transcription and translation processes of the ambisense S RNA of punta toro phlebovirus (bunyaviridae)

Regulation of the mRNA transcription and translation processes of the ambisense S RNA of punta toro phlebovirus (bunyaviridae)

Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of rift valley fever virus

The complete sequence of Punta Toro virus ( Phlebovirus, Bunyaviridae) middle size (M), RNA has been determined. The RNA is 4330 nucleotides long (mol wt 1.46 × 10 6, base composition: 26.7% A, 33.6% U, 18.5% G, 21.2% C) and has 3′- and 5′-terminal sequences that, depending on the arrangement, are complementary for some 15 residues. The viral RNA codes in its viral-complementary sequence for a single primary gene product (the viral glycoprotein precursor) that is comprised of 1313 amino acids (146,376 Da) and is abundant in cysteine residues but has few potential asparagine-linked glycosylation sites. The 5′-noncoding region of the Punta Toro M viral-complementary RNA is short (16 nucleotides); the 3′-noncoding sequence is much longer (372 nucleotides). The latter is rich in short stretches of adenylate residues, like the 3′-noncoding regions of the Punta Toro S mRNA species (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293–306). No other large open reading frame has been identified in either the viral, or viral-complementary, M RNA sequences. Limited amino-terminal sequence analyses of the two viral glycoproteins have indicated the gene order and potential cleavage sites in the glycoprotein precursor. The data suggest the existence of a 30 × 10 3-Da polypeptide (designated NS M) in the glycoprotein precursor that precedes the G1 protein (i.e., gene product order: NS M-G1-G2). Examination of the sequence of the Punta Toro M gene product reveals the presence of multiple hydrophobic sequences including a 19-amino acid, carboxy-proximal, hydrophobic region (G2). This hydrophobic sequence is followed by a 13-amino acid-terminal sequence rich in charged amino acids. The size and constitution of the carboxy-terminal region is consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. Other regions of the glycoprotein precursor contain sequences of amino acids with a predominantly hydrophobic character (23, 50, and 20 amino acids in length). Their functions are unknown. The amino terminus of the G1 protein is located near the end of the 23-amino acid-long hydrophobic sequence of the presumptive precursor, the hydrophobic 50-amino acid sequence lies within G1, and the amino terminus of G2 is located in the middle of the 20-amino acid-long hydrophobic sequence. From the recently determined sequence of the M RNA gene product of Rift Valley fever virus ( Phlebovirus, Bunyaviridae; M. S. Collett, A. F. Purchio, K. Keegan, S. Frazier, W. Hayes, D. K. Anderson, M. D. Parker, C. Schmaljohn, and J. M. Dalrymple, 1985, Virology 144, 228–245) considerable homology with the Punta Toro M RNA gene product has been detected, except for the indicated NS M protein. No homology has been identified between snowshoe hare bunyavirus ( Bunyavirus, Bunyaviridae) glycoprotein precursor and that of Punta Toro virus.

Novel coding strategy (ambisense genomic RNA) revealed by sequence analyses of punta toro phlebovirus S RNA

Sequence analyses of Punta Toro viral S RNA species indicate the existence of a novel coding strategy for RNA viruses that involves both viral complementary and viral sense mRNA species. The Punta Toro nucleocapsid protein (N, 26.9 × 10 3 Da) is coded by a discrete viral complementary mRNA species corresponding to the 3′ half of the viral S RNA. A second, presumably nonstructural, gene product (NS, 29.1 × 10 3 Da) is coded by a viral sense mRNA species that corresponds to the 5' half of the viral RNA. The ambisense nature of the S RNA is unique by comparison with any other viral RNA and raises questions concerning the family assignment of phleboviruses.

Viruses isolated from Panamanian sloths.

Seven virus strains were isolated in Vero cells from whole blood samples from 80 wild-caught sloths, Bradypus variegatus and Choloepus hoffmanni, from Central Panamá. Four strains of at least two different serotypes are related to Changuinola virus; two of these were associated with prolonged or recrudescent viremias. One strain is an antigenic subtype of Punta Toro virus, and another, described here as Bradypus-4 virus, is a new, antigenically ungrouped virus. A second new virus from sloths, Utive virus, forms an antigenic complex within the Simbu serogroup with Utinga and Pintupo viruses. Tests on sequential plasma samples from radio-marked free-ranging sloths and from recently captured animals maintained in captivity showed that both species develop neutralizing antibodies following naturally acquired virus infections. Antibodies against the Changuinola and Simbu serogroup viruses are widespread in both sloth species and are especially prevalent in Choloepus, but are virtually absent in all other wild vertebrate species tested.

Morphogenesis of sandfly fever viruses (Bunyaviridae family)

The events occurring in the morphogenesis of sandfly fever viruses have been examined by thin-section electron microscopy and by an analysis of the association of virus-specific polypeptides with membranes of infected cells. Two representative sandfly fever viruses have been studied, Karimabad virus (KV) and Punta Toro virus (PTV), which appeared indistinguishable both in terms of virion structure, as monitored by negative staining, and morphogenesis, as observed in thin sections of infected Vero cells. Ammonium molybdate negative staining of purified, glutaraldehyde-fixed virions revealed essentially spherical particles, 87 nm in diameter, in which the surface proteins were constructed into closely packed, hollow, cylindrical subunits measuring 10–11 nm in diameter and 9–10 nm in length. These surface units are located peripherally to a 7-nm membrane bilayer which surrounds a nucleoid of variable electron density. As seen in thin sections of infected cells, the assembly of these particles was first detected at 12 hr after infection, occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Morphologically mature particles were formed by continuous involution (budding) of modified membrane segments into the lumen of these vesicles. Viral ribonucleoprotein (RNP), which was not observed free in the cytoplasm, condensed at the cytoplasmic face of these vesicles at areas at which viral spike structures could be observed at the contralateral (luminal) face. Neither RNP nor spike structures could be observed on adjacent sections of the vesicular membrane, or other membranes, which were not directly involved in the assembly of a budding virion. Analysis of the viral polypeptides in unfractionated membrane vesicles prepared from infected cells demonstrated that KV-specific envelope and nucleocapsid proteins rapidly became membrane bound, whereas a nonstructural polypeptide was found only in cytoplasmic fractions. Chymotrypsin treatment of these vesicles has indicated that at least one viral glycoprotein is inserted into cellular membranes such that approximately 12% of its sequence remains at the cytoplasmic membrane face. Proteolytic removal of this sequence generated an immunoprecipitable, glycosylated fragment which was protected from further proteolysis by its orientation within the vesicle. These morphologic and biochemical data have been used to construct a model for the assembly of these viruses. Virus particles are released from infected cells by exocytosis, a process which does not appear to result in significant modification of cell surface membranes with viral antigens.