Punta Toro Virus Research Articles

Prophylaxis with cationic liposome‐DNA complexes protects hamsters from phleboviral disease: importance of CpG motifs?

Cationic liposome‐DNA complexes formed with plasmid DNA (CLDC), CpG‐free plasmid DNA, or CpG‐containing oligodeoxynucleotides (ODN) were evaluated for their ability to elicit protective immunity against Punta Toro virus (PTV) challenge in the hamster infection model. Liposome‐DNA formulations were administered by the intraperitoneal route 4 h prior to inoculation of a highly lethal PTV inoculum. Survival outcomes, as well as reduction of viral burden and liver disease, were assessed. A dose of CLDC containing 30 μg of plasmid significantly improved survival outcome, decreased systemic and liver viral loads, and reduced liver pathology compared to animals receiving placebo. Known mouse‐ and human‐reactive ODNs delivered in liposome complexes failed to protect challenged hamsters. Treatment with liposomes containing non‐CpG motif‐containing plasmid reduced liver virus titers compared with untreated or CpG treated, but did not protect hamsters from death. This suggests that while elements other than CpG motifs may stimulate a response sufficient to control viral replication in the target organ, the empty vector plasmid DNA backbone with CpG islands appears to be critical to stimulating protection against mortality in the PTV hamster model. Supported by contract grant NO1‐AI‐15435 from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Prophylactic and therapeutic intervention of Punta Toro virus ( Phlebovirus, Bunyaviridae) infection in hamsters with interferon alfacon-1

Punta Toro virus (PTV) is a member of the Bunyaviridae family, genus Phlebovirus, related to the highly pathogenic Rift Valley fever virus (RVFV). It produces a disease in hamsters that models severe Rift Valley fever (RVF) in humans. The recent outbreak of RVF in Kenya stresses the need to identify prophylactic and therapeutic measures for preventing and treating severe forms of disease. To this end, interferon (IFN) alfacon-1 (consensus IFN-α) was evaluated in cell culture against RVFV and PTV, and in the hamster PTV infection model. Survival outcome following treatment initiated pre- and post-virus challenge and the suppression of viral burden and liver disease in infected hamsters was determined. Pre-treatment of cell cultures with IFN alfacon-1 induced marked antiviral activity against both viruses. Intraperitoneal treatment of hamsters initiated 4 h prior to infection with PTV was highly protective and greatly limited liver disease and systemic and liver viral burden. Complete protection from a highly lethal challenge dose was afforded by treatment initiated 36 h following viral inoculation. Although efficacy was much reduced, IFN alfacon-1 therapy was still beneficial when started as late as 3–5 days post-virus exposure. These studies suggest that IFN alfacon-1 may be an effective treatment for early intervention following infection with RVFV.

In Vitro and In Vivo Activities of T-705 against Arenavirus and Bunyavirus Infections

There is a need for the development of effective antivirals for the treatment of severe viral diseases caused by members of the virus families Bunyaviridae and Arenaviridae. The pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has demonstrated remarkable antiviral activity against influenza virus and, to a lesser degree, against some other RNA viruses (Y. Furuta, K. Takahashi, Y. Fukuda, M. Kuno, T. Kamiyama, K. Kozaki, N. Nomura, H. Egawa, S. Minami, Y. Watanabe, H. Narita, and K. Shiraki, Antimicrob. Agents Chemother., 46:977-981, 2002). Here, we report that T-705 is highly active against a panel of bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly fever viruses) and arenaviruses (Junin, Pichinde, and Tacaribe viruses) by cytopathic effect and virus yield reduction cell-based assays. The 50% effective concentrations for T-705 ranged from 5 to 30 microg/ml and 0.7 to 1.2 microg/ml against the bunyaviruses and arenaviruses examined, respectively. We also demonstrate that orally administered T-705 is efficacious in treating Punta Toro virus in the mouse and hamster infection models, as well as Pichinde virus infection in hamsters. When administered twice daily for 5 to 6 days, beginning 4 h pre- or 24 h post-Punta Toro virus challenge, a 30-mg/kg of body weight/day dose provided complete protection from death and limited viral burden and liver disease. A dose of 50 mg/kg/day was found to be optimal for treating Pichinde infection and limiting viral replication and disease severity. In general, T-705 was found to be more active than ribavirin in cell-based assays and in vivo, as reflected by substantially greater therapeutic indexes. Our results suggest that T-705 may be a viable alternative for the treatment of life-threatening bunyaviral and arenaviral infections.

TLR3 Is Essential for the Induction of Protective Immunity against Punta Toro Virus Infection by the Double-Stranded RNA (dsRNA), Poly(I:C12U), but not Poly(I:C): Differential Recognition of Synthetic dsRNA Molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Enantioselective synthesis and antiviral activity of purine and pyrimidine cyclopentenyl C-nucleosides

The enantiomerically pure carbocyclic purine and pyrimidine C-nucleosides 1– 4 were synthesized via the key intermediate, 2,3-(isopropylidenedioxy)-4-(trityloxymethyl)-4-cyclopenten-1-ol ( 5), which was prepared from d-ribose in eight steps. Synthesized compounds were evaluated as potential antiviral agents against HIV, SARSCoV, Punta Toro, West Nile, and Cowpox viruses. However, only 9-deazaneplanocin A ( 1) exhibited moderate anti-HIV activity.

The S segment of Punta Toro virus (Bunyaviridae, Phlebovirus) is a major determinant of lethality in the Syrian hamster and codes for a type I interferon antagonist.

Two strains of Punta Toro virus (PTV), isolated from febrile humans in Panama, cause a differential pathogenesis in Syrian hamsters, which could be a useful model for understanding the virulence characteristics and differential outcomes in other phleboviral infections such as Rift Valley fever virus. Genetic reassortants produced between the lethal Adames (A/A/A) and nonlethal Balliet (B/B/B) strains were used in this study to investigate viral genetic determinants for pathogenesis and lethality in the hamster model. The S segment was revealed to be a critical genome segment, determining lethality with log(10) 50% lethal doses for each PTV genotype as follows (L/M/S convention): A/A/A, <0.7; B/A/A, <0.7; A/B/A, 1.5; B/B/A, 2.2; B/A/B, 4.7; A/B/B, >4.7; A/A/B, >4.7; B/B/B, >4.7. In addition, the Adames strain inhibits the induction of alpha/beta interferon (IFN-alpha/beta) in vivo and in vitro and inhibits the activation of the IFN-beta promoter. Expression of the PTV Adames NSs protein, encoded by the S RNA segment, inhibited the virus-mediated induction of an IFN-beta promoter-driven reporter gene, suggesting that PTV NSs functions as a type I IFN antagonist. Taken together, these data indicate a mechanism of pathogenesis in which the suppression of the type I IFN response early during PTV infection leads to early and uncontrolled viral replication and, ultimately, hamster death. This study contributes to our understanding of Phlebovirus pathogenesis and identifies potential targets for immune modulation to increase host survival.

TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection

TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3(-/-) mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3(-/-) mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-gamma, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3(-/-) mice.

Recombinant Eimeria Protozoan Protein Elicits Resistance to Acute Phlebovirus Infection in Mice but Not Hamsters

A protein antigen from an Eimeria protozoan has recently been reported to induce antitumor activity in mice. This activity most likely results from the strong induction of interkeukin-12 (IL-12) and gamma interferon (IFN-gamma), which are also essential factors in the establishment of protective immunity against viral infection. We evaluated recombinant Eimeria antigen (rEA) as a potential immunotherapeutic agent in mouse and hamster models of acute phleboviral disease. Punta Toro virus (PTV) was highly sensitive to a single dose of nanogram quantities of rEA in the mouse infection model. Intraperitoneal treatment with rEA also reduced virus load and liver damage associated with PTV infection. IL-12 was elicited following exposure of uninfected mice to quantities of rEA of 10 ng or greater, and the levels peaked at between 3 and 8 h postexposure. IFN-gamma release was induced more slowly and required less rEA (1 ng) to produce a significant rise in systemic levels. The induction of IL-12 and IFN-gamma involved in the coordination of innate and adaptive immune responses to microbial pathogens required myeloid differentiation factor 88, a signaling adaptor shared by most members of the Toll-like receptor (TLR) family. Despite encouraging results in the murine system, rEA failed to protect hamsters challenged with PTV. Our findings suggest that hamsters may lack functional TLR11, which has recently been shown to recognize a profilin-like protein homologous to rEA from the protozoan Toxoplasma gondii. Further investigation into the immunostimulatory capacity of rEA in other mammalian systems is necessary.

Protective immunity against acute phleboviral infection elicited through immunostimulatory cationic liposome-DNA complexes

Cationic liposome-DNA complexes (CLDC) have been demonstrated to induce potent antitumor activities. The ability of these complexes to elicit protective immunity against viral infections has not been fully explored. Here we report findings on the use of CLDC as an antiviral agent in a mouse model of acute phleboviral (Punta Toro virus) disease. CLDC treatment of mice challenged with Punta Toro virus (PTV) resulted in dramatic increases in survival and reduced viral burden and other parameters indicative of protection against disease. CLDC were effective when administered by intraperitoneal and intravenous routes and elicited protective immunity when given within 1 day of virus challenge. Treatments administered 36 h or longer after challenge, however, were not effective in preventing mortality or disease. CLDC treatment induced release of a number of potential antiviral cytokines including IFN-γ, IL-12, and IFN-α. Taken together, our findings indicate that non-specific immunotherapy with CLDC appears to be an effective treatment for blocking PTV-induced disease and suggests that further exploration in other viral disease models may be warranted.

Synthesis and Biological Activity of Isoxazolidinyl Polycyclic Aromatic Hydrocarbons: Potential DNA Intercalators

Isoxazolidinyl polycyclic aromatic hydrocarbons (isoxazolidinyl-PAHs) have been synthesized in good yields by 1,3-dipolar cycloaddition methodology promoted by microwave irradiation. The structures of the obtained cycloadducts have been determined by NOE experiments and supported by computational studies at the AM1 level. The cytotoxicity and antiviral activity of the synthesized compounds have been investigated. In particular, compounds 7c and 7e show high levels of cytotoxicity on MOLT-3 leukemia cells; 7e exerts a remarkable enhancing activity on apoptosis caused by anti-fas antibody addition. Furthermore, compounds 7b and 8b exhibit specific antiviral effects against the Punta Toro virus.

Apoptosis of Hepatocytes Caused by Punta Toro Virus ( Bunyaviridae: Phlebovirus) and Its Implication for Phlebovirus Pathogenesis

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.

Analogues of Acyclic Nucleosides Derived from Tris-(Hydroxymethyl)Phosphine Oxide or Bis-(Hydroxymethyl)Phosphinic Acid Coupled to DNA Nucleobases

A series of novel acyclic nucleoside analogues containing bis-(hydroxymethyl)phosphinic acid (BHPA) or tris(hydroxymethyl)phosphine oxide (THPO) coupled with DNA nucleobases or with 5-fluorouracil were prepared and their antiviral activity was studied against cytomegalovirus (CMV), varicella-zoster virus (VZV), parainfluenza-virus type 3, reovirus-type 1, sindbis, coxsackie B4, punta toro, vesicular stomatitis and respiratory syncytial virus, herpes simplex virus-type 1 (KOS) and type 2 (G), vaccinia virus and herpes simplex virus-1 (TK- KOS ACVr). No specific antiviral effects were noted for any of test compounds against viruses evaluated, except thymine, cytosine and adenine derivatives of BHPA exerting borderline activity against respiratory syncytial virus at the 80 mg/ml concentration.

Synthesis, antiviral and cytotoxic activity of 6-bromo-2,3-disubstituted-4(3H)-quinazolinones.

In the present study, a series of 6-bromo-2,3-disubstitued-4(3H)-quinazolinones was synthesized by condensation of 6-bromo-2-substituted-benzoxazin-4-one with trimethoprim, pyrimethamine and lamotrigine. The chemical structures of the synthesized compounds were confirmed by means of IR, (1)H-NMR and mass spectral and elemental analysis. The antiviral activity and cytotoxicity of the compounds were tested in E(6)SM (Herpes simplex-1 KOS, Herpes simplex-1 TK-KOS ACV, Herpes simplex-2 G, Vaccinia virus, Vesicular stomatitis virus, Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus) and HeLa cell culture (Vesicular stomatitis virus, Coxsackie virus B4 and Respiratory syncyticla virus). Investigation of anti-HIV activity was done against replication of HIV-1 (HTLV-III B LAI) in MT-4 cells. 6-Bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the most potent antiviral activity with a MIC of 1.92 microg/ml against vaccinia virus in E(6)SM cell culture. The other compounds did not exhibit antiviral activity nor afford significant cytoprotection to the E(6)SM and HeLa cell culture when challenged with the viruses. The study implies that 4 may possess activity against Pox viruses including variola. In the anti-HIV study, 6-bromo-2-methyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (3) and 6-bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the least cytotoxic concentration (0.424, 0.461 microg/ml) which is an index of the infective viability of mock infected MT-4 cells with HIV-1. None of the compounds exhibited significant anti-HIV activity.

5'-Nor carbocyclic 5'-deoxy-5'-(isobutylthio)adenosine and a 2',3'-dideoxy-2',3'-didehydro derivative.

The inhibition of biochemical processes requiring S-adenosylmethionine as a co-factor have led to many nucleoside-based medicinal agents. Included in this group are 5'-deoxy-5'-(isobutylthio)adenosine (SIBA), a nucleoside with antiparasitic, antiviral and antiproliferative effects, and 5'-noraristeromycin, a carbocyclic-derived nucleoside with potent antiviral properties. This report brings together the structural components of these two compounds by describing both enantiomers of carbocyclic 5-nor SIBA (3 and 4). Owing to the recent interest in 2',3'-dideoxy-2',3'-didehydro nucleosides as antiviral agents, this derivative of 3 (5) is also described. All three compounds were screened against a variety of viruses and were found to be inactive at high concentrations or at limiting concentrations for the screening methods. The viruses subjected to 3-5 were herpes simplex virus types 1 and 2, human cytomegalovirus, vaccinia virus, vesicular stomatitis virus, respiratory syncytial virus, varicelIa zoster virus, coxsackie virus, parainfluenza-3 virus, sindbis virus, punta toro virus, reovirus-1, human immunodeficiency virus, influenza virus types A and B, adenovirus type 1 and measles virus. These results suggest that the C-5' methylene of the C-5' thio-based carbocyclic nucleosides is important for their antiviral properties.

Broad-spectrum antiviral activity and mechanism of antiviral action of the fluoroquinolone derivative K-12.

The fluoroquinolone derivatives have been shown to inhibit human immunodeficiency virus (HIV) replication at the transcriptional level. We confirmed the anti-HIV activity of the most potent congener, 8-difluoromethoxy-1-ethyl-6-fluoro-1,4-dihydro-7-[4-(2- methoxyphenyl)-1-piperazinyl]-4-quinolone-3-carboxylic acid (K-12), in both acutely and chronically infected cells. K-12 was active against different strains of HIV-1 (including AZT- and ritonavir-resistant HIV-1 strains), HIV-2 and simian immunodeficiency virus, in MT-4, CEM, C8166 and peripheral blood mononuclear cells. In all of these antiviral assay systems, K-12 showed a similar activity (EC50 0.2-0.6 microM). K-12 inhibited Moloney murine sarcoma virus-induced transformation of C3H/3T3 cells with an EC50 of 6.9 microM. Also, K-12 proved inhibitory to herpesvirus saimiri, human cytomegalovirus, varicella-zoster virus and herpes simplex virus types 1 and 2 (in order of decreasing sensitivity), but was not inhibitory (at subtoxic concentrations) to human herpesvirus type 8 (as evaluated in BCBL-1 cells), vaccinia virus, Sindbis virus, vesicular stomatitis virus, respiratory syncytial virus, Coxsackie virus, Punta Toro virus, parainfluenza virus or reovirus. Time-of-addition experiments and quantitative transactivation bioassays indicated that K-12 inhibits the Tat-mediated transactivation process in HIV-infected cells.

Completion of molecular characterization of Toscana phlebovirus genome: nucleotide sequence, coding strategy of M genomic segment and its amino acid sequence comparison to other phleboviruses

The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10–14 non-templated nucleotides at the 5′ end and it is truncated at the 3′ end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Uukuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS G N and G C glycoproteins were placed at residues 297 and 936 of the precursor.

Detection of Rift Valley fever virus in mosquitoes by RT-PCR

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.

Use of the yellow fever virus vaccine strain 17D for the study of strategies for the treatment of yellow fever virus infections

We have employed the attenuated vaccine strain 17D of yellow fever virus (YFV) to evaluate the inhibitory effect of a selected series of compounds on YFV in Vero cells. Use of the vaccine strain does not require high-level microbiological containment facilities and should allow extensive screening. In addition, YFV may serve as a model for other flaviviruses including hepatitis C virus (HCV), and thus strategies for the treatment of YFV infections may apply to flavivirus infections in general. In the present study, several compounds belonging to different classes of nucleoside analogues and polyanions were evaluated for their inhibitory effect on the replication of YFV. Compounds that are targeted at: (i) IMP dehydrogenase (ribavirin, EICAR, tiazofurin, selenazofurin and mycophenolic acid), (ii) OMP decarboxylase (pyrazofurin and 6-azauridine), (iii) CTP synthetase (carbodine and cyclopentenyl cytosine), (iv) dihydrofolate reductase (methotrexate) and the (v) sulfated polymers (dextran sulfate and PAVAS) proved inhibitory to the replication of YFV. Mycophenolic acid (EC 50: 0.08 μg/ml), EICAR (EC 50: 0.8 μ/ml) and methotrexate (EC 50: 0.07 μg/ml) were the most effective. The finding that EICAR and mycophenolic acid, despite their potent anti-YFV activity, had little or no effect on the replication of the bunyavirus Punta Toro or herpes simplex virus in Vero cells, indicates that their anti-YFV activity is rather specific and does not merely result from cytotoxicity. Inhibitors of S-adenosylhomocysteine hydrolase (SAH hydrolase) and thymidylate synthase were found to be devoid of anti-YFV activity.

Antiviral activities of tragacanthin polysaccharides on Punta Toro virus infections in mice.

Tragacanthin polysaccharides from Astragalus brachycentrus (AV208) and Astragalus echidnaeformis (AV212) plants, which are devoid of in vitro antiviral activity, were evaluated in a mouse model of Punta Toro virus (PTV) infection. The PTV (a phlebovirus member of the Bunyaviridae family of viruses) is a model for studying the treatment of Rift Valley fever and hantavirus infections. Single intraperitoneal treatments with 12.5-200 mg/kg/day doses of AV212 given 24 h before or 4 and 24 h after virus inoculation protected the majority of mice from mortality. Single treatments with AV208 were ineffective when given 24 h before the virus challenge; however, protection was afforded when treatments were administered at 4 and 24 h following virus inoculation. In a follow-up study, AV208 treatments of 1.6-50 mg/kg/day given 24 h subsequent to virus exposure caused reductions in mortality, liver infection scores, liver and spleen virus titers, and serum transaminases. The polysaccharides did not activate lymphocytes or natural killer cells, nor was interferon induced in treated mice. However, mice pretreated with fumed silica (a macrophage poison) and infected with the PTV were not protected by subsequent administration of AV208 or AV212 at 50 mg/kg, providing evidence that activation of peritoneal macrophages by the polysaccharides affords protection to infected animals. These compounds should be considered for the potential treatment of significant human infections induced by bunyaviruses and hantaviruses.

Tropism of Bunyaviruses: Evidence for a G1 Glycoprotein-Mediated Entry Pathway Common to the California Serogroup

The California serogroup is composed of antigenically and biologically related viruses within theBunyavirusgenus of the Bunyaviridae. We used a large panel of murine cells to study their tissue tropisms and found virtually identical patterns of viral replication among all of the members of this serogroup, in contrast to other members of the family (Bunyamwera, Cache Valley, and Punta Toro viruses). By analyzing the nonpermissive infections with both an RNA dot-blot and a virus binding assay, we determined that tropism for cultured cells was determined at the level of entry. A truncated soluble form of the La Crosse G1 glycoprotein (sG1) was expressed in a baculovirus system and, despite slight differences in glycosylation, was shown to resemble native G1 by immunoprecipitation with six monoclonal antibodies. sG1 bound to permissive but not to nonpermissive cell lines, as demonstrated by flow cytometry. The sG1 effectively blocked infection of permissive cell lines with all of the California serogroup viruses, but did not block infection of two other bunyaviruses. These results indicate that the California serogroup bunyaviruses share a common receptor on vertebrate cells which may differ from the receptor used by other Bunyaviridae and demonstrate that the G1 glycoprotein is the virus attachment protein. sG1 will be a useful reagent in the search for a putative receptor molecule.