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 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

Activation of platelet-derived growth factor receptor ß in the severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging viral infectious disease with a high case fatality rate, is caused by the SFTS virus (SFTSV). Although several cellular molecules involved in viral entry have been identified, the entry mechanisms of SFTSV remain unclear. In this study, we screened the protein kinase inhibitors in inhibitory effects on the infection of Vero cells with SFTSV using InhibitorSelect™ Protein Kinase Library Series (Merck & Co., Inc., Kenilworth, NJ, USA). Several types of inhibitors targeted to platelet-derived growth factor receptor ß (PDGFRß) inhibited the infection of Vero, Huh7, and NIH3T3 cells with SFTSV in a dose-dependent manner within the noncytotoxic range. In addition, these protein kinase inhibitors also inhibited the infection of the target cells with SFTSV glycoprotein (SFTSV-GP) pseudotyped virus (SFTSVpv). Activation of PDGFRß phosphorylation was detected in SFTSV-treated cells. The infectivities of SFTSVpv were specifically decreased not only in NIH3T3 cells treated with siRNA for PDGFRß but also in NIH3T3 cells treated with anti-PDGFRß neutralizing antibody in a dose-dependent manner. SFTSV growth and entry of SFTSVpv were also inhibited by Akt inhibitors. Activation of Akt phosphorylation was also detected in SFTSV-treated cells. These data indicate that PDGFRß is one of the important host factors in the entry steps of SFTSV.

Rescue of infectious Arumowot virus from cloned cDNA: Posttranslational degradation of Arumowot virus NSs protein in human cells.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East, affecting both humans and ruminants. There are no licensed vaccines or antivirals available for humans, whereas research using RVF virus (RVFV) is strictly regulated in many countries with safety concerns. Nonpathogenic Arumowot virus (AMTV), a mosquito-borne phlebovirus in Africa, is likely useful for the screening of broad-acting antiviral candidates for phleboviruses including RVFV, as well as a potential vaccine vector for RVF. In this study, we aimed to generate T7 RNA polymerase-driven reverse genetics system for AMTV. We hypothesized that recombinant AMTV (rAMTV) is viable, and AMTV NSs protein is dispensable for efficient replication of rAMTV in type-I interferon (IFN)-incompetent cells, whereas AMTV NSs proteins support robust viral replication in type-I IFN-competent cells. The study demonstrated the rescue of rAMTV and that lacking the NSs gene (rAMTVΔNSs), that expressing green fluorescent protein (GFP) (rAMTV-GFP) or that expressing Renilla luciferase (rAMTV-rLuc) from cloned cDNA. The rAMTV-rLuc and the RVFV rMP12-rLuc showed a similar susceptibility to favipiravir or ribavirin. Interestingly, neither of rAMTV nor rAMTVΔNSs replicated efficiently in human MRC-5 or A549 cells, regardless of the presence of NSs gene. Little accumulation of AMTV NSs protein occurred in those cells, which was restored via treatment with proteasomal inhibitor MG132. In murine MEF or Hepa1-6 cells, rAMTV, but not rAMTVΔNSs, replicated efficiently, with an inhibition of IFN-ß gene upregulation. This study showed an establishment of the first reverse genetics for AMTV, a lack of stability of AMTV NSs proteins in human cells, and an IFN-ß gene antagonist function of AMTV NSs proteins in murine cells. The AMTV can be a nonpathogenic surrogate model for studying phleboviruses including RVFV.

Experimental Infection of Sand Flies by Massilia Virus and Viral Transmission by Co-Feeding on Sugar Meal.

Background: Massilia virus (MASV) is a phlebovirus isolated from Phlebotomus perniciosus in various regions of southwestern Europe. It is closely related to human pathogens such as Toscana virus and sandfly fever Naples virus. The natural cycle of phleboviruses is poorly understood. Indeed, experimental studies demonstrate that transovarial and sexual transmission are not efficient enough for the maintenance of the virus in nature and to date there is no convincing evidence that a species of vertebrates is the reservoir of the virus. Here, we studied various transmission routes of MASV taking advantage of experimental colonies representing different species of sand flies. Methodology/Principal findings: In P. perniciosus, four sources of infection were compared: (i) Virus-seeded larval food to the first instar larvae (L1), or (ii) to the fourth instar larvae (L4), (iii) virus-seeded blood meal to adult females, and (iv) virus-seeded sugar meal to adults of both sexes. From 875 adults emerged from infected L1 and L4, only three were positive. In females infected by bloodmeal the infection rate was high before defecation, then it decreased drastically; MASV RNA was detected in only 5 out of 27 post-defecation. Surprisingly, the most efficient route of infection was observed after intake of virus-seeded sugar meal: 72% of females (79/110) and 52% of males (51/99) were found to be MASV RNA-positive. In addition, MASV-infected sandflies regurgitated virus particules into the sugar drop and MASV RNA was detectable in this drop for at least 24 h after regurgitation. MASV RNA was detected in about one third of the P. perniciosus exposed to this sugar drop contaminated by regurgitation. Sugar meal infection was also tested with six other species of sand flies. In males, there were no significant differences in infection rates when compared to P. perniciosus. In females, most species tested showed high infection rate at the beginning but then significant gradual decrease in infection rate during the experiment. Conclusions/Significance: We present the first description of arboviral infection of a dipteran vector using sugar meal. In all seven sand fly species tested, MASV was detected for two weeks post-infection. Our results showed that MASV can be transmitted between P. perniciosus either through co-feeding or via an infected sugar source such as plant sap. These newly described routes of horizontal transmission may play an important role in the circulation of phleboviruses in nature.

A neutralization assay with a severe fever with thrombocytopenia syndrome virus strain that makes plaques in inoculated cells.

Severe fever with thrombocytopenia syndrome (SFTS) is a recently-discovered, potentially fatal infectious disease caused by SFTS virus (SFTSV). Due to the inability of SFTSV to make clear cytopathic effects (CPE) in cell culture, titration and neutralization assays of the virus require immunostaining of inoculated cells; consequently, the assays are time-consuming and expensive. In this report, we demonstrate the use of a highly-passaged SFTSV strain, p50-2, in a neutralization assay, which made clear plaques in inoculated Vero cells under neutral red staining. Furthermore, we performed molecular analyses to determine the characteristics of the strain. The results suggested that a single amino acid mutation within the viral glycoprotein conferred the ability to make clear plaques to SFTSV.

Isolation, characterization, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province, China.

Hubei Province is a major epidemic area of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) in China. However, to date, a few SFTSV strains have been isolated from Hubei Province, preventing effective studies of epidemic outbreaks. Here, we report three confirmed patients (2015-2016) with typical symptoms of severe fever with thrombocytopenia syndrome disease (SFTS) who were farmers resident in different regions in Hubei Province. Three new SFTSV strains were isolated from the serum samples of each patient. Characterization of viral growth properties showed that there were no significant differences in virus production. All strains were completely sequenced, and phylogenetic analysis showed that unlike the other strains from Hubei province, which belonged to the SFTSV C3 genotype, one of the three strains belonged to the SFTSV C2 genotype. These results suggested that multiple SFTSV genotypes have been circulating in Hubei Province, providing insights into SFTSV evolution and improving our understanding of SFTSV prevalence in Hubei Province.

Experimental infections of mosquitoes with severe fever with thrombocytopenia syndrome virus.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified emerging infectious disease, which is caused by a novel bunyavirus (termed SFTSV) in Asia. Although mosquitoes have not been identified as the primary vectors, as revealed by epidemiological surveys, their role in transmitting this SFTSV as a suspicious vector has not been validated. FINDINGS: In this study, we conducted experimental infections of mosquitoes with SFTSV to examine the role of mosquitoes in the transmission of the virus. We did not detect viral replication in Culex pipiens pallens, Aedes aegyptis and Anopheles sinensis as revealed by qRT-PCR assay. In addition, we failed to isolate SFTSV from the Vero cells cultured with suspensions of SFTSV-infected mosquitoes. CONCLUSION: The results of the present study demonstrate little possibility that mosquitoes act as vectors for the emerging pathogen SFTSV.

18F-FDG PET imaging for identifying the dynamics of intestinal disease caused by SFTSV infection in a mouse model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that causes fever, enteritis, thrombocytopenia, and leucopenia and can be fatal in up to 30% of cases. However, the mechanism of severe disease is not fully understood. Molecular imaging approaches, such as positron-emission tomography (PET), are functional in vivo imaging techniques that provide real-time dynamics of disease progression, assessments of pharmacokinetics, and diagnoses for disease progression. Molecular imaging also potentially provides useful approaches to explore the pathogenesis of viral infections. Thus, the purpose of this study was to image the pathological features of SFTSV infection in vivo by PET imaging. In a mouse model, we showed that 18F-FDG accumulations clearly identified the intestinal tract site as a pathological site. We also demonstrated that 18F-FDG PET imaging can assess disease progression and response to antiserum therapy within the same individual. This is the first report demonstrating a molecular imaging strategy for SFTSV infection. Our results provide potentially useful information for preclinical studies such as the elucidation of the mechanism of SFTSV infection in vivo and the assessment of drugs for SFTS treatment.

Apodemus agrarius is a potential natural host of severe fever with thrombocytopenia syndrome (SFTS)-causing novel bunyavirus.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne, novel bunyavirus-caused emerging infectious disease. It becomes a threat to public health due to its high fatality rate. OBJECTIVE: To clarify the epidemiological characteristics of SFTS and natural host(s) of SFTS-causing virus (SFTSV) in East China. STUDY DESIGN: Serum antibody against SFTSV in 496 healthy villagers was examined by ELISA. SFTSV in acute sera of SFTS cases and lung tissues of house and field mouse/rats were identified using quantitative RT-PCR, cell culture, and sequencing. RESULTS: A total of 22 laboratory-confirmed SFTS cases were diagnosed between 2012 and 2014, of which 5 (22.7%) reported a history of tick-bites. The seroprevalance of SFTSV antibody was 10.5% in healthy villagers. SFTSV genomic RNAs were identified in 2 of 8 Apodemus agrarius but not in Rattus norvegicus (n=40) and Rattus losea (n=4). The 3 segments of SFTSV from 11 SFTS cases and 2 A. agrarius were successfully sequenced. Phylogenetic analyses indicated that at least 3 different SFTSV strains (inland-type, Ningbo-native-type, and their reassortant-type) were present in Ningbo. The 3 segments of the 2 SFTSV isolates from A. agrarius shared great sequence homologies to those isolated from the patients living in nearby villages. CONCLUSION: The present study indicated that A. agrarius might be a natural host of SFTSV in East China.

Studies on the biology of phleboviruses in sand flies (Diptera: Psychodidae). I. Experimental infection of the vector.

This paper describes a series of experiments which were done to determine the behavior of 14 different phleboviruses in laboratory-reared sand flies (Phlebotomus papatasi, P. perniciosus and Lutzomyia longipalpis) after oral and parenteral infection. Most of the viruses replicated in the sand flies after intrathoracic inoculation; however, the insects were quite refractory to oral infection. Six of 11 phleboviruses tested were transovarially transmitted in one or more sand fly species. The percentage of infected F1 offspring produced by parenterally infected female parents ranged from 1.5-60%, depending on the virus type used. These data support the hypothesis that some of the phleboviruses are maintained in sand flies by transovarial transmission.

Experimental studies on the maintenance of Toscana and Arbia viruses (Bunyaviridae: Phlebovirus).

Transovarial transmission (TOT) of Toscana (TOS) and Arbia (ARB) viruses in a laboratory colony of Phlebotomus perniciosus is reported. Toscana and ARB viruses were maintained in P. perniciosus females, initially infected by intrathoracic inoculation, for 2 and 3 consecutive generations respectively. TOT was demonstrated in F1 (75%) and F2 (67%) generation adults for TOS and F1 (47%), F2 (37%), and F3 (34%) generation adults for ARB virus. The progressive decline of virus infection rates in each generation suggests that these agents cannot be maintained indefinitely by TOT. No infection was observed in F1 progeny after female parents were fed through membranes with either virus. Transovarially infected females were able to transmit TOS virus by bite to a susceptible vertebrate. Venereal infection of P. perniciosus females mated to males transovarially infected with TOS virus was seen.

Experimental studies of Rio Grande virus in rodent hosts.

Rio Grande (RG) virus, a new member of the Phlebotomus fever serogroup, was inoculated into wild wood rats (Neotoma micropus) and laboratory-reared cotton rats (Sigmodon hispidus) to determine if these potential hosts could be experimentally infected. Nine of 14 (64%) wood rats became viremic, with titers of circulating virus ranging from 10(2.3) to 10(5.3) plaque-forming units (PFU)/ml and a geometric mean titer of 10(3.7) PFU/ml. Virus was not detected in urine specimens from inoculated wood rats but was found in a single saliva specimen. RG virus was detected in the blood of 1 of 12 (8%) cotton rats. Neutralizing (N) antibody developed in 8 of 9 inoculated wood rats which survived for 30 days postinoculation and in 11 of 12 cotton rats. N antibody was still detectable in 4 of 7 wood rats which survived for 1 year, and all 7 were resistant to rechallenge with the virus, as were 3 wood rats with naturally-acquired antibody. High mortality (36%) occurred in inoculated wood rats; whereas low mortality (8%) occurred in cotton rats. The specific cause of death of the rats was not determined. Modes of transmission of the virus in nature are discussed.

Dimethyl sulfoxide enhancement of phlebotomus fever virus plaque formation.

Dimethyl sulfoxide (DMSO)incorporated into an agar overlay containing DEAE-dextran enhanced plaque formation in Vero cells by Naples sandfly fever virus passaged in mouse brain or Vero cell cultures. No plaques were visible when DMSO was used without the DEAE-dextran, some plaques were rarely visible (less than 0.5mm) when DEAE-dextran was used without the DMSO, and up to 10-fold more plaques were clearly visible (0.5-1.5 mm) when both chemicals were used. The combined enhancing effect of DMSO and DEAE-dextran was also observed with mouse brain passaged, but not Vero passaged Sicilian sandfly fever virus. Other Phlebotomus group viruses produced a bit plaques (3-5 mm) and did not require DMSO for plaque formation, although an increase in plaque clarity was obtained with DMSO for some of them. Plaque reduction neutralization tests were assayed successfully under agar containing DMSO. The alphavirus Sindbis produced slightly larger plaques under agar containing DMSO, but there was no effect on clarity or size of plaques produced by the flavivirus dengue-2.

First detection of heartland virus (Bunyaviridae: Phlebovirus) from field collected arthropods.

Heartland virus (HRTV), the first pathogenic Phlebovirus (Family: Bunyaviridae) discovered in the United States, was recently described from two Missouri farmers. In 2012, we collected 56,428 ticks representing three species at 12 sites including both patients' farms. Amblyomma americanum and Dermacentor variabilis accounted for nearly all ticks collected. Ten pools composed of deplete nymphs of A. americanum collected at a patient farm and a nearby conservation area were reverse transcription-polymerase chain reaction positive, and eight pools yielded viable viruses. Sequence data from the nonstructural protein of the Small segment indicates that tick strains and human strains are very similar, ≥ 97.6% sequence identity. This is the first study to isolate HRTV from field-collected arthropods and to implicate ticks as potential vectors. Amblyomma americanum likely becomes infected by feeding on viremic hosts during the larval stage, and transmission to humans occurs during the spring and early summer when nymphs are abundant and actively host seeking.

Host genetic variation in susceptibility to Punta Toro virus.

Infection of small laboratory animals by Punta Toro virus (PTV), family Bunyaviridae, genus Phlebovirus, is a model for the study of the human pathogen Rift Valley fever virus (RVFV). We have identified inbred mouse strains with significant differences in host response to the Adames strain of PTV. Nine inbred strains of mice representing major branches in the Mus musculus phylogeny were inoculated subcutaneously with a high dose of PTV in survival experiments. Two inbred strains of mice, NZW/LacJ and 129S1/SvImJ, died ~4 days after PTV infection, whereas 7 other strains survived the challenge and showed no clinical signs of disease. Histologically, 129S1/SvImJ mice showed massive hepatocellular necrosis and had additional lesions in lung, brain, and spleen, whereas NZW/LacJ mice had mild piecemeal hepatocellular necrosis. PTV viral loads in the livers of infected mice were determined by reverse transcriptase quantitative PCR. Inbred mice from strains that showed clinical signs and succumbed to PTV infection had higher liver viral loads than did mice of resistant strains. Hybrid F1 mice were generated by crossing susceptible 129S1 and resistant FVB/N mice and tested for susceptibility. The hybrid F1 mice showed significantly higher viral loads in the liver than the resistant parental FVB/N mice, suggesting that susceptibility is dominant. These findings will enable an unbiased genetic approach to identify host genes mediating susceptibility to PTV.

Punta Toro virus (Bunyaviridae, Phlebovirus) infection in mice: strain differences in pathogenesis and host interferon response.

The Adames strain of Punta Toro virus (PTV-A, Bunyaviridae, Phlebovirus) causes an acute lethal disease in hamsters and mice. The Balliet strain of the virus (PTV-B) is generally considered to be avirulent. The difference in hamster susceptibility is likely due to the ability of PTV-A to suppress interferon (IFN)-beta similarly to that described for Rift Valley fever virus. Here we investigated strain differences in PTV pathogenesis and the IFN response in mice. Although PTV-B infection in mice did not induce systemic IFN-beta release, primary macrophages produced dramatically higher levels when exposed to the virus in culture. The importance of IFN in resistance to PTV infection was borne out in studies employing STAT-1 knock-out mice. Also, a number of genes specific to IFN response pathways were upregulated in PTV-B-infected macrophages. Our findings provide new insights into the type I IFN response during PTV infection in the mouse model of phleboviral disease.

Inhibition of sandfly fever Sicilian virus (Phlebovirus) replication in vitro by antiviral compounds.

Sandfly fever Sicilian virus (SFSV) was used in our laboratory to screen antiviral substances active toward viruses of the Bunyaviridae family. Antiviral activity was estimated by the reduction of the cytopathic effect of SFSV on infected Vero cells. Cytotoxicity was evaluated by determining the inhibition of Trypan blue exclusion. The specificity of action of each tested compound was estimated by the selectivity index (CD50/ED50). Selectivity indices of human recombinant interferon-alpha (IFN alpha) (Roferon and Introna), iota-, kappa- and lambda- carrageenans, fucoidan and 6-azauridine were much higher than that of ribavirin, the only antiviral substance which has been previously investigated for its inhibitory effects on Phlebovirus infections. Other compounds showed significant antiviral activity: glycyrrhizin, suramin sodium, dextran sulphate and pentosan polysulphate. All these compounds caused a concentration-dependent reduction in the virus yield. Ribavirin, 6-azauridine and IFN alpha have been shown to inhibit a late step of the virus replicative cycle, whereas glycyrrhizin and suramin sodium were active at an early step and the sulphated polysaccharides inhibited adsorption of SFSV on the cells. The antiviral compounds selected in this study as specific inhibitors of in vitro replication of SFSV are promising candidates for the chemotherapy of haemorrhagic fevers caused by viruses of the Bunyaviridae family. The combination of IFN alpha and ribavirin, which showed a synergistic antiviral effect, should be evaluated for the treatment of these infections.