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Antiviral Research (v.93, #3)

Inside Back Cover: Notes to Authors (pp. ibc).

Prelim II: Editorial Board (pp. ii).

Current and future applications of dried blood spots in viral disease management by Ingrid J.M. Snijdewind; Jeroen J.A. van Kampen; Pieter L.A. Fraaij; Marchina E. van der Ende; Albert D.M.E. Osterhaus; Rob A. Gruters (pp. 309-321).

Almost five decades after their first application in diagnostics, dried blood spot (DBS) cards remain to be of key interest in many research areas and clinical applications. The advantages of sample stability during transport and storage, can now be combined with the high sensitivity of novel diagnostic techniques for the measurement and analysis of nucleic acids, proteins and small molecules which may overcome the limitations of the small samples sizes in DBS cards. Here we present a survey of the literature on the use of DBS cards for diagnosis, monitoring and epidemiological studies of virus infections other than HIV, including CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles-, rubella- and dengue-virus. The minimal invasiveness of sampling and the relative ease of handling and storing DBS cards is expected to offer additional opportunities to measure and analyze biomarkers of viral disease in resource poor settings or when limited amount of blood can be obtained. Large retrospective studies of virus infections in newborns using stored DBS cards have already been undertaken for screening of congenital infections. In addition, DBS cards have been used prospectively for prevalence studies, outbreak surveillance, mass screening for viral infections, follow-up of chronic infection and its treatment in resource-limited areas. We do not expect that current wet sampling techniques of plasma or serum will be replaced by DBS sampling but it allows extension of sampling in persons and settings that are currently difficult to access or that lack suitable storage facilities. In conclusion, DBS card sampling and storage will aid adequate outbreak management of existing and emerging viral diseases.

Keywords: DBS; PCR; ELISA; Guthrie cards; Genotyping; Virus

Susceptibility of avian influenza viruses of the N6 subtype to the neuraminidase inhibitor oseltamivir by Terri D. Stoner; Scott Krauss; Jasmine C.M. Turner; Patrick Seiler; Nicholas J. Negovetich; David E. Stallknecht; Sharon Frase; Elena A. Govorkova; Robert G. Webster (pp. 322-329).

► First report antiviral susceptibility of avian influenza viruses of N6 NA subtype. ► Isolates with molecular marker for oseltamivir resistance moderately susceptible. ► Avian influenza viruses of N6 NA subtype highly susceptible to the NAI oseltamivir. ► Natural resistance is rare among the N6 subtype.Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC₅₀ values ranging from <0.01 to 5.0nM; 10% were from 5.1 to 50.0nM; and none were >50.0nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades ( P⩽0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC₅₀, 30.0 and 40.0nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.

Keywords: Avian N6 subtype; Natural reservoir; Neuraminidase inhibitors; Resistance; Oseltamivir

Characterization of RNA aptamers directed against the nucleocapsid protein of Rift Valley fever virus by Mary Ellenbecker; Leila Sears; Ping Li; Jean-Marc Lanchy; J. Stephen Lodmell (pp. 330-339).

► In this study, we use SELEX to derive high affinity RNA ligands to Rift Valley fever virus nucleocapsid protein. ► The sequences of the RNA aptamers inform about the RNA sequence and structural preferences of nucleocapsid protein. ► We constructed a fluorescent biosensor that reports on nucleocapsid-RNA interactions. ► This biosensor can be used as a molecular target assay for potential new antiviral drug screening.Nucleocapsid protein (N) is an essential RNA binding protein in many RNA viruses. During replication, N protein encapsidates viral genomic and antigenomic RNA, but not viral mRNA or other cellular RNAs. To discriminate between different species of RNA in a host cell, it is likely that N interacts with specific sequences and/or secondary structures on its target RNA. In this study, we explore the RNA binding properties of N using both natural and artificially selected RNAs as ligands. We found that N binds to RNAs that resemble the terminal panhandle structures of RVFV genomic and antigenomic RNA. Furthermore, we used SELEX to isolate RNA aptamers that bound N with high affinity and determined that N specifically recognizes and binds to GAUU and pyrimidine/guanine motifs. Interestingly, BLAST analysis revealed the presence of these motifs within the coding region of the viral genome, suggesting that N may interact with non-terminal viral RNA sequences during replication. Finally, the aptamer RNAs were used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies.

Keywords: Rift Valley fever virus; Nucleocapsid; RNA aptamer; SELEX

Lactoferricin but not lactoferrin inhibit herpes simplex virus type 2 infection in mice by Andrey Shestakov; Håvard Jenssen; Inger Nordström; Kristina Eriksson (pp. 340-345).

► Genital co-administration of lactoferricin with HSV-2 is blocking the infection. ► Lactoferrin and Lactoferricin induce local vaginal production of CCL5. ► Lactoferricin, given therapeutically delay the onset of the HSV-2 infection.We have evaluated the potential of bovine lactoferrin and lactoferricin for their ability to prevent and/or treat genital HSV-2 infection in mice. We confirm previous data showing that both lactoferrin and lactoferricin have antiviral properties in vitro and can inhibit HSV-2 infection of GMK cells in a dose-dependent manner. When tested in vivo, lactoferricin but not lactoferrin was also a potent inhibitor of HSV-2 infection. When admixed with virus prior to inoculation, lactoferricin inhibited disease development and significantly reduced the viral load in a genital model of HSV-2 infection in mice. Lactoferrin and lactoferricin were also tested for their ability to stimulate the production of chemokines. Neither of the compounds induced the production of CCL3, CCL5, CXCL1 or CXCL2 by mouse splenocytes in vitro. However, when tested in vivo, both lactoferrin and lactoferricin were able to induce local vaginal production of CCL5. Lactoferrin also induced CXCL2 production. The prophylactic and/or therapeutic effects of lactoferrin or lactoferricin were also tested. But none of the compounds were efficient in blocking HSV-2 infection when given 24h prior to HSV-2 infection. Lactoferricin however showed promising results as a therapeutic agent and delayed both disease onset by 3days as well as reducing the viral load almost 15-fold when given as a single dose 24h post-infection. These data show that lactoferricin can block genital herpes infection in mice, and perhaps also be used for post-infection treatment.

Keywords: HSV-2; Lactoferrin; Lactoferricin; Antiviral activity; Mouse

Protective efficacy of an H1N1 cold-adapted live vaccine against the 2009 pandemic H1N1, seasonal H1N1, and H5N1 influenza viruses in mice by Jianzhong Shi; Zhiyuan Wen; Jing Guo; Ying Zhang; Guohua Deng; Yuelong Shu; Dayan Wang; Yongping Jiang; Yoshihiro Kawaoka; Zhigao Bu; Hualan Chen (pp. 346-353).

► Vaccination is a key strategy for preventing influenza virus infections. ► An H1N1 cold-adapted live attenuated vaccine was generated and evaluated. ► The vaccine induced complete protection against 2009 pandemic virus challenge. ► The vaccine induced significant CD4+ and CD8+ T cell responses. ► The vaccine provided protection against heterologous H1N1 and H5N1 virus challenge.Vaccination is a key strategy for preventing influenza virus infections. Here, we generated a reassortant virus (SC/AA ca) containing the hemagglutinin and neuraminidase genes from a 2009 pandemic influenza virus A/Sichuan/1/2009 (H1N1) (SC/09) and six internal genes from the cold-adapted virus A/Ann Arbor/6/60 (H2N2) (AA ca). The SC/AA ca reassortant induced a sound humoral immune response and complete protection against homologous SC/09 virus challenge in mice after intranasal administration of an at least 10⁶ 50% egg infectious dose (EID₅₀) of SC/AA ca. SC/AA ca inoculation also induced significant CD4+ and CD8+ T cell responses and provided solid protection against heterologous H1N1 and H5N1 virus challenge. Our results suggest that this 2009 H1N1 live vaccine will provide protection against both 2009 pandemic and seasonal H1N1 virus infection and might reduce the severity of H5N1 virus infection in humans. The induction of cross-reactive virus-specific T cell responses may be an effective approach to develop universal influenza vaccines.

Keywords: Influenza; Live attenuated vaccine; H1N1; H5N1

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35 by Luca Zinzula; Francesca Esposito; Daniela Pala; Enzo Tramontano (pp. 354-363).

► We established a novel in vitro magnetic pull down assay to measure Ebola full length recombinant VP35–dsRNA binding. ► We determined the full length rVP35 K_d value for dsRNA to be 2.8nM. ► We measured the IFN-inhibiting defective R305A, K309A and R312A rVP35 mutants dsRNA binding K_d values. ► We identified ATA as the first rVP35–dsRNA binding inhibitor.The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)–dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC₅₀ value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K_d values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35–dsRNA binding functions.

Keywords: Ebola virus; VP35; dsRNA binding; Biochemical screening assay

RNA interference in vitro and in vivo using DsiRNA targeting the nucleocapsid N mRNA of human metapneumovirus by Magali Darniot; Verena Schildgen; Oliver Schildgen; Brian Sproat; Michael Kleines; Vanessa Ditt; Cécile Pitoiset; Pierre Pothier; Catherine Manoha (pp. 364-373).

► First RNA silencing against metapneumovirus in an animal model (mouse). ► DsiRNA reduced in vivo viral replication. ► DsiRNA did not stimulate host immunity. ► Distribution of the DsiRNA evaluated by whole-animal imaging. ► Predicted cleavage of the mRNA confirmed by RACE analysis.Human metapneumovirus causes respiratory diseases with outcomes that can be severe in children, the immunocompromised, and the elderly. Synthetic small interfering RNAs (siRNAs) that silence targeted genes can be used as therapeutic agents. Currently, there is no specific therapy for hMPV. In this study, we designed Dicer-substrate siRNAs (DsiRNAs) that target metapneumovirus sequences on the mRNAs of the N, P, and L genes. In vitro, six DsiRNAs were shown to inhibit virus replication using cell proliferation tests. Of those, the DsiRNA that targets the most conserved mRNA sequence was then resynthesized in Evader™ format with heavy 2′- O-methyl modification of the guide strand. In a murine model, the prophylactic administration of this Evader™ DsiRNA was effective at partially inhibiting viral replication of hMPV (13×10³ vs. 29×10³PFU/g of lung; p<0.01), which was not the case for the control, a mismatched DsiRNA. Inhibition was achieved without inducing cytokines or off-target effects. Moreover, the specificity of the siRNA mechanism of action was demonstrated in vitro and in vivo using 5′-RACE methodology. This in vivo approach of using a DsiRNA against hMPV is an important step in the development of synthetic siRNA as a therapeutic agent for this virus.

Keywords: Abbreviations; hMPV; human metapneumovirus; N; nucleoprotein; RNAi; RNA interference; D; dicer; siRNA; small interfering RNA; RISC; RNA-induced silencing complex; TLR; Toll-like receptor; IFN-α; interferon-α; IL-6; interleukin-6; TNF-α; tumor necrosis factor-α; i.n.; intranasal(ly); BAL; bronchoalveolar lavage; AO; airway obstruction; HRP; horseradish peroxidase; AEC; 3-amino-9-ethyl-carbazole; CCID; tissue culture infectious doseRNA interference; Metapneumovirus; siRNA; RACE; Mice

H1N1 influenza A virus neuraminidase modulates infectivity in mice by Olivier Ferraris; Vanessa Escuret; Maude Bouscambert; Jean-Sébastien Casalegno; Frédéric Jacquot; Hervé Raoul; Valérie Caro; Martine Valette; Bruno Lina; Michèle Ottmann (pp. 374-380).

► Infectivity of neuraminidase H1N1 reassortants is compared in murine model. ► Genetic origin of reassortants is determined for each segment. ► Infectivity of oseltamivir-resistant NA viruses is higher than the sensitive virus.In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.

Keywords: Influenza A virus; Pandemic 2009 H1N1 virus; Neuraminidase; Oseltamivir; Reassortant virus; Mouse model

Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States by Ha T. Nguyen; Alma A. Trujillo; Tiffany G. Sheu; Marnie Levine; Vasiliy P. Mishin; Michael Shaw; Edwin W. Ades; Alexander I. Klimov; Alicia M. Fry; Larisa V. Gubareva (pp. 381-386).

► Utility of pyrosequencing for quickly detecting resistant influenza viruses. ► Necessity of phenotypic assays to detect novel resistance mutations. ► Correlation of H275Y pyrosequencing and IC₅₀ values by functional assays. ► Prevalence of H275Y (pure or mixed populations) in patients examined (∼38%). ► Detection of additional synergistic mutations at NA 223 (I/R-K) in 2 patients.During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, peramivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wildtype-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrosequencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates containing H275Y variant at⩾50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38–52- and 33–97-fold enhancement in oseltamivir- and peramivir-resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions.

Keywords: Pandemic H1N1; Neuraminidase inhibitor; Pyrosequencing; Neuraminidase inhibition assay; H275Y; I223K; I223R

Myxomatosis in Australia and Europe: A model for emerging infectious diseases by Peter J Kerr (pp. 387-415).

► Myxomatosis is the classic example of virus/host coevolution. ► Myxoma virus was introduced into Australia as a control for the European rabbit. ► The subsequent coevolution of the virus and its new host was studied in real time.Myxoma virus is a poxvirus naturally found in two American leporid (rabbit) species ( Sylvilagus brasiliensis and Sylvilagus bachmani) in which it causes an innocuous localised cutaneous fibroma. However, in European rabbits ( Oryctolagus cuniculus) the same virus causes the lethal disseminated disease myxomatosis. The introduction of myxoma virus into the European rabbit population in Australia in 1950 initiated the best known example of what happens when a novel pathogen jumps into a completely naïve new mammalian host species. The short generation time of the rabbit and their vast numbers in Australia meant evolution could be studied in real time. The carefully documented emergence of attenuated strains of virus that were more effectively transmitted by the mosquito vector and the subsequent selection of rabbits with genetic resistance to myxomatosis is the paradigm for pathogen virulence and host–pathogen coevolution. This natural experiment was repeated with the release of a separate strain of myxoma virus in France in 1952. The subsequent spread of the virus throughout Europe and its coevolution with the rabbit essentially paralleled what occurred in Australia. Detailed molecular studies on myxoma virus have dissected the role of virulence genes in the pathogenesis of myxomatosis and when combined with genomic data and reverse genetics should in future enable the understanding of the molecular evolution of the virus as it adapted to its new host. This review describes the natural history and evolution of myxoma virus together with the molecular biology and experimental pathogenesis studies that are informing our understanding of evolution of emerging diseases.

Keywords: Myxoma virus; Myxomatosis; Poxvirus; Rabbit; Coevolution; Virulence

The role of antigen-presenting cells in filoviral hemorrhagic fever: Gaps in current knowledge by Osvaldo Martinez; Lawrence W. Leung; Christopher F. Basler (pp. 416-428).

► Antigen-presenting cells (APCs) are important targets of filovirus infection in vivo. ► APCs are postulated to be major sites of virus replication and to promote inflammation. ► Understanding of filovirus–APC interactions will suggest novel therapeutic approaches. ► This review summarizes our current knowledge of filovirus–APC interactions.The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), are highly lethal zoonotic agents of concern as emerging pathogens and potential bioweapons. Antigen-presenting cells (APCs), particularly macrophages and dendritic cells, are targets of filovirus infection in vivo. Infection of these cell types has been proposed to contribute to the inflammation, activation of coagulation cascades and ineffective immune responses characteristic of filovirus hemorrhagic fever. However, many aspects of filovirus–APC interactions remain to be clarified. Among the unanswered questions: What determines the ability of filoviruses to replicate in different APC subsets? What are the cellular signaling pathways that sense infection and lead to production of copious quantities of cytokines, chemokines and tissue factor? What are the mechanisms by which innate antiviral responses are disabled by these viruses, and how may these mechanisms contribute to inadequate adaptive immunity? A better understanding of these issues will clarify the pathogenesis of filoviral hemorrhagic fever and provide new avenues for development of therapeutics.

Keywords: Filovirus; Ebola virus; Marburg virus; Antigen presenting cell; Macrophage; Dendritic cell; Interferon

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Antiviral Research (v.93, #3)