Punta Toro Virus Research Articles

EIF5A is activated by virus infection or dsRNA and facilitates virus replication through modulation of interferon production.

Active hypusine-modified initiation elongation factor 5A is critical for cell proliferation and differentiation, embryonic development, and innate immune response of macrophages to bacterial infection. Here, we demonstrate that both virus infection and double-stranded RNA viral mimic stimulation induce the hypusination of eIF5A. Furthermore, we show that activation of eIF5A is essential for the replication of several RNA viruses including influenza A virus, vesicular stomatitis virus, chikungunya virus, mayaro virus, una virus, zika virus, and punta toro virus. Finally, our data reveal that inhibition of eIF5A hypusination using the spermidine analog GC7 or siRNA-mediated downmodulation of eIF5A1 induce upregulation of endoplasmic reticulum stress marker proteins and trigger the transcriptional induction of interferon and interferon-stimulated genes, mechanisms that may explain the broad-spectrum antiviral activity of eIF5A inhibition.

A facile one pot multi component synthesis of alkyl 4-oxo-coumarinyl ethylidene hydrazono-thiazolidin-5-ylidene acetates and their antiviral activity

An efficient one-pot synthesis of alkyl 4-oxo-coumarinyl ethylidene hydrazono-thiazolidin-5-ylidene acetate derivatives has successfully been achieved via a three component cyclization reaction of various substituted 3-acetyl coumarins, thiosemicarbazide and dialkyl acetylenedicarboxylates, in presence of acetic acid. The isolated products were obtained in pure form with high yields through simple workup. The newly synthesised compounds structure was established on the basis of spectral (IR, 1H NMR, 13C NMR, ESI- mass) elemental analysis and single crystal X-ray data. All synthesised compounds were screened for their antiviral activity against a broad spectrum of human viruses in different cell cultures. Of the novel synthesised compounds, thirteen compounds exerted activity against Punta Toro virus, including compound IV-19, for which an antiviral potency was noted against a broad panel of DNA and RNA viruses as well.

Further Characterization of Rio Grande Virus and Potential for Cross Reactivity with Rift Valley Fever Virus Assays.

Phleboviruses (genus Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses (Equus caballus) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Labyrinthopeptin A1 inhibits dengue and Zika virus infection by interfering with the viral phospholipid membrane

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC50 around 1 μM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses.

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole derivatives.

A series of indole-based spirothiazolidinones have been designed, synthesized and evaluated, in vitro, for their antitubercular, antiviral, antibacterial, and antifungal activities. The structures of the new compounds were established by IR, 1H NMR, 13C NMR (proton decoupled, APT, and DEPT), electrospray ionization mass spectrometry, and microanalysis. Compounds bearing a phenyl substituent at position 8 of the spiro ring, exhibited significant antitubercular activity against Mycobacterium tuberculosis H37Rv ATCC 27294 at concentrations of 3.9 and 7.8 µM. Still, some of the tested compounds displayed activity on mycobacteria with MIC values of 16 and 31 µM. Four of the indole-spirothiazolidinone derivatives were found to be moderately active against Punta Toro virus, yellow fever virus or Sindbis virus in Vero cells. The antiviral EC50 values were in the range of 1.9–12 µM and the selectivity index (ratio of cytotoxic to antivirally effective concentration) was above 10 in some cases. The most potent effect was seen with the compound that is methylated at positions 2 and 8 of the spirothiazolidinone system.Graphic abstract Electronic supplementary materialThe online version of this article (10.1007/s00706-019-02457-9) contains supplementary material, which is available to authorized users.

Asymmetric Primaquine and Halogenaniline Fumardiamides as Novel Biologically Active Michael Acceptors.

Novel primaquine (PQ) and halogenaniline asymmetric fumardiamides 4a–f, potential Michael acceptors, and their reduced analogues succindiamides 5a–f were prepared by simple three-step reactions: coupling reaction between PQ and mono-ethyl fumarate (1a) or mono-methyl succinate (1b), hydrolysis of PQ-dicarboxylic acid mono-ester conjugates 2a,b to corresponding acids 3a,b, and a coupling reaction with halogenanilines. 1-[bis(Dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was used as a coupling reagent along with Hünig′s base. Compounds 4 and 5 were evaluated against a panel of bacteria, several Mycobacterium strains, fungi, a set of viruses, and nine different human tumor cell lines. p-Chlorofumardiamide 4d showed significant activity against Staphylococcus aureus, Streptococcus pneumoniae and Acinetobacter baumannii, but also against Candida albicans (minimum inhibitory concentration (MIC) 6.1–12.5 µg/mL). Together with p-fluoro and p-CF3 fumardiamides 4b,f, compound 4d showed activity against Mycobacterium marinum and 4b,f against M. tuberculosis. In biofilm eradication assay, most of the bacteria, particularly S. aureus, showed susceptibility to fumardiamides. m-CF3 and m-chloroaniline fumardiamides 4e and 4c showed significant antiviral activity against reovirus-1, sindbis virus and Punta Toro virus (EC50 = 3.1–5.5 µM), while 4e was active against coxsackie virus B4 (EC50 = 3.1 µM). m-Fluoro derivative 4a exerted significant cytostatic activity (IC50 = 5.7–31.2 μM). Acute lymphoblastic leukemia cells were highly susceptible towards m-substituted derivatives 4a,c,e (IC50 = 6.7–8.9 μM). Biological evaluations revealed that fumardiamides 4 were more active than succindiamides 5 indicating importance of Michael conjugated system.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50~1.5µM, minimal cytotoxic concentration=60µM, selectivity index=40).

Clinical Manifestations of Punta Toro Virus Species Complex Infections, Panama, 2009.

An investigation in Panama found that Punta Toro virus species complex (PTVs) may contribute to febrile illnesses with symptoms mirroring those of dengue fever. However, further studies are needed to determine if PTV infection causes only a mild disease or if it can have more serious manifestations in some patients.

Synthesis and in vitro antiviral evaluation of 4-substituted 3,4-dihydropyrimidinones

A series of 4-substituted 3,4-dihydropyrimidine-2-ones (DHPM) was synthesized, characterized by IR, 1H NMR, 13C NMR and HRMS spectra. The compounds were evaluated in vitro for their antiviral activity against a broad range of DNA and RNA viruses, along with assessment for potential cytotoxicity in diverse mammalian cell lines. Compound 4m, which possesses a long lipophilic side chain, was found to be a potent and selective inhibitor of Punta Toro virus, a member of the Bunyaviridae. For Rift Valley fever virus, which is another Bunyavirus, the activity of 4m was negligible. DHPMs with a C-4 aryl moiety bearing halogen substitution (4b, 4c and 4d) were found to be cytotoxic in MT4 cells.

SEROSURVEY OF SELECTED ARBOVIRAL PATHOGENS IN FREE-RANGING, TWO-TOED SLOTHS (CHOLOEPUS HOFFMANNI) AND THREE-TOED SLOTHS (BRADYPUS VARIEGATUS) IN COSTA RICA, 2005-07.

We screened for antibodies to 16 arboviruses in four populations of free-ranging sloths in Costa Rica. Blood samples were taken from 16 Hoffman's two-toed sloths (HTSs; Choloepus hoffmanni ) and 26 brown-throated sloths (BTSs; Bradypus variegatus ) over a 3-yr period. We used serologic assays to detect antibodies against 10 arboviruses previously described in sloths (St. Louis encephalitis [SLEV], Changuinola, Venezuelan equine encephalitis, Ilheus [ILHV], Oropouche, Mayaro, Utinga, Murutucu, Punta Toro, and vesicular stomatitis [VSV] viruses) and six arboviruses not described in sloths (Rio Grande, West Nile [WNV], eastern equine encephalitis, Piry, Munguba, and La Crosse viruses). Overall, 80% of sloths had detectable antibodies to SLEV, 67% had antibodies to ILHV, 32% to Punta Toro virus, 30% to Changuinola virus, 15% to WNV, 14% to VSV, 11% to Venezuelan equine encephalitis virus, and 10% to Rio Grande virus. No samples had detectable antibodies to the remaining eight viruses. We found a significant increase in prevalence of antibody to VSV in HTSs between 2005 and 2007, and for WNV antibody between 2005 and 2006. We found no significant differences in the prevalences of antibodies to the sampled viruses between the two locations. Antibody prevalences were significantly higher in HTSs than in BTSs for SLEV in 2005. Antibody-positive results for ILHV were likely due to cross-reaction with SLEV. The novel finding of antibodies to Rio Grande virus in sloths could be due to cross-reaction with another phlebovirus. These findings might have implications for land management and domestic animal health. Due to the nature of the study, we could not determine whether sloths could represent amplification hosts for these viruses, or whether they were only exposed and could be used as sentinel species. Further studies are needed to fully characterize arboviral exposure in sloths.

Phleboviruses and the Type I Interferon Response.

The genus Phlebovirus of the family Bunyaviridae contains a number of emerging virus species which pose a threat to both human and animal health. Most prominent members include Rift Valley fever virus (RVFV), sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV), Toscana virus (TOSV), Punta Toro virus (PTV), and the two new members severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV). The nonstructural protein NSs is well established as the main phleboviral virulence factor in the mammalian host. NSs acts as antagonist of the antiviral type I interferon (IFN) system. Recent progress in the elucidation of the molecular functions of a growing list of NSs proteins highlights the astonishing variety of strategies employed by phleboviruses to evade the IFN system.

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR

ABSTRACTRift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro virus and Pichinde virus.

BackgroundResearch with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices.MethodsTotal nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV.ResultsOptimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors.ConclusionsPTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users.

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

The Intrathecal Antibody Response in Multiple Sclerosis Brain Does Not React Against Measles Virus

liminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora. RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12. The ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays.

Further Characterization of Rio Grande Virus and Potential for Serological Cross Reactivity with other Phleboviruses

Members of the genus Phlebovirus (family Bunyaviridae ) are new and emerging disease pathogens of humans and animals. Newly identified viruses include Heartland virus (HRTV), Lone Star virus in the USA, and Severe Fever with Thrombocytopenia Syndrome virus in Asia. Assays to support surveillance, epidemiologic studies, and diagnosis of these viruses may also detect related viruses within the genus, confounding interpretation. Rio Grande virus (RGV) was isolated in 1973 from southern plains woodrats ( Neotoma micropus ) in the United States and has been preliminarily identified as a phlebovirus transmitted by the sand fly Lutzomyia anthophora . RGV is not known to cause disease in humans, but it could be detected by assays designed for HRTV or other phleboviruses. The goal of this study was to determine antigenic cross-reaction between RGV and other phleboviruses. A commercially available ELISA based sand fly fever antigen detection kit was tested for the ability to detect RGV and other New and Old-World phleboviruses, including attenuated Rift valley fever virus (RVFV) strain MP12, Punta Toro virus (PTV), Toscana virus, Aguacate virus, Anhanga virus, Arumowot virus, and Chagres virus. Immunocytochemistry and Western blotting were used to detect cross reactions between RGV, MP12, and PTV using rabbit anti-RVFV nucleocapsid protein and glycoproteins GC and GN, mouse monoclonal anti-PTV, and sheep polyclonal anti-MP12. The ELISA test detected cross reactivity for all phleboviruses excluding RGV, but Western blotting detected the presumed RGV nucleocapsid protein (N) using rabbit anti-RVFV-N serum, RGV-infected cells were also identified when labeled with this antibody. Our findings demonstrate assay specific antigenic cross reactivity between these phleboviruses, thus further characterization of the molecular targets of the cross-reaction is required for proper interpretation of serological assays.

Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae).

Punta Toro virus (PTV), a member of the PTV complex, is a relatively common causative agent of febrile illness in Panama that is often misdiagnosed as 'dengue' or 'influenza'. Currently, only two named members make up this species complex, PTV and Buenaventura virus (BUEV). Genomic and antigenic characterization of 17 members of the PTV complex, nine of which were isolated from human acute febrile illness cases, reveals that this species complex is composed of six distant viruses. We propose to add four additional new viruses, designated Leticia virus, Cocle virus, Campana virus and Capira virus.

Design, antiviral and cytostatic properties of isoxazolidine-containing amonafide analogues

A novel series of 5-arylcarbamoyl- and 5-arylmethyl-2-methylisoxazolidin-3-yl-3-phosphonates have been synthesized via cycloaddition of N-methyl-C-(diethoxyphosphoryl)nitrone with N-substituted naphthalimide acrylamides and N-allylnaphthalimides. All cis- and trans-isoxazolidine phosphonates obtained herein were assessed for antiviral activity against a broad range of DNA and RNA viruses. Isoxazolidines trans-9d and trans-9f exhibited the highest activity (EC50=8.9μM) toward cytomegalovirus. Compounds cis- and trans-9d as well as cis- and trans-9f were found potent against HSV and Vaccinia viruses (EC50 in the 45–58μM range), whereas isoxazolidines 10a and 10d suppressed replication of Coxsackie B4 and Punta Toro viruses (EC50 in the 45–73μM range). Antiproliferative evaluation of all obtained isoxazolidines revealed the promising activity of cis-9b, cis-9d, trans-9d, cis-9e, trans-9e, cis-9f and trans-9f toward tested cancer cell lines with IC50 in the 1.1–19μM range.

Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment

Rift Valley fever is a zoonotic, arthropod-borne disease that affects livestock and humans. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus) is primarily transmitted through mosquito bites, but can also be transmitted by exposure to infectious aerosols. There are presently no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. In addition, efficacy has also been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, hamsters challenged with the highly pathogenic ZH501 strain of RVFV were used to evaluate the activity of favipiravir against lethal infection. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2–3days of infection. Oral favipiravir (200mg/kg/day) prevented mortality in 60% or greater of hamsters challenged with RVFV when administered within 1 or 6h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden observed in the brain in moribund animals. When combined, T-705 and ribavirin treatment started 24h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.