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Antiviral Research (v.92, #3)

Prelim II: Editorial Board (pp. ii).

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

Nanobodies®: New ammunition to battle viruses by Peter Vanlandschoot; Catelijne Stortelers; Els Beirnaert; Lorena Itatí Ibañez; Bert Schepens; Erik Depla; Xavier Saelens (pp. 389-407).

► Heavy chain-only antibodies discovered in Camelidae. ► Biophysical and biochemical features of Nanobodies. ► Pre-clinical anti-viral therapeutic applications of Nanobodies.In 1989, a new type of antibody was identified, first in the sera of dromedaries and later also in all other species of the Camelidae family. These antibodies do not contain a light chain and also lack the first constant heavy domain. Today it is still unclear what the evolutionary advantage of such heavy chain-only antibodies could be. In sharp contrast, the broad applicability of the isolated variable antigen-binding domains (VHH) was rapidly recognized, especially for the development of therapeutic proteins, called Nanobodies®. Here we summarize first some of the unique characteristics and features of VHHs. These will next be described in the context of different experimental therapeutic applications of Nanobodies against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Next, the diagnostic application of VHHs (Vaccinia virus, Marburg virus and plant Tulip virus X), as well as an industrial application (lytic lactococcal 936 phage) will be described. In addition, the described data show that monovalent Nanobodies can possess unique characteristics not observed with conventional antibodies. The straightforward formatting into bivalent, multivalent, and/or multispecific Nanobodies allowed tailoring molecules for potency and cross-reactivity against viral targets with high sequence diversity.

Keywords: Nanobody; Camelidae; Heavy chain-only antibody; VHH; Antiviral

Brain distribution of ribavirin after intranasal administration by Gaia Colombo; Luca Lorenzini; Elisa Zironi; Viola Galligioni; Fabio Sonvico; Anna Giulia Balducci; Giampiero Pagliuca; Alessandro Giuliani; Laura Calzà; Alessandra Scagliarini (pp. 408-414).

Ribavirin has proved to be effective in vitro against several RNA viruses responsible for encephalitis in humans and animals. However, the in vivo efficacy towards the cerebral viral load seems to be limited by the blood–brain barrier. Since the nose-to-brain pathway has been indicated for delivering drugs to the brain, we investigated here the distribution of ribavirin in the central nervous system (CNS) after intranasal administration. We first tested in vitro ribavirin diffusion from an aqueous solution across a biological membrane, using Franz cells and rabbit nasal mucosa. About 35% of ribavirin permeated in 4h across the mucosa, after reaching steady-state flux in less than 30min. In the first in vivo experiment, ribavirin aqueous solution was administered intranasally to Sprague Dawley rats (10mg/kg). Animals were sacrificed at 10, 20 or 30min after administration to collect brain areas (cerebellum, olfactory bulb, cerebral cortex, basal ganglia and hippocampus) and biological fluids (cerebrospinal fluid and plasma). Ribavirin, quantified by LC–MS/MS spectrometry, was detected at each time point in all compartments with the highest concentration in olfactory bulb and decreasing in rostro-caudal direction. Two subsequent in vivo experiments compared the nasal route (ribavirin solution) with the intravenous one and the nasal administration of ribavirin solution with ribavirin powder (10mg/kg). It was found that 20min after administration, ribavirin concentration in olfactory bulb was similar after intravenous or nasal administration of the ribavirin solution, whereas the powder led to significantly higher levels. Ribavirin was also present in deeper compartments, such as basal ganglia and hippocampus.Even if the mechanisms involved in ribavirin nose-to-brain transport are not clear, these results suggest a rapid extracellular diffusive flux from the nasal epithelium to the olfactory bulb and different CNS areas.

Keywords: Ribavirin; Nasal delivery; Blood brain barrier; Nose-to-brain pathway

HIV protease inhibitors induce metabolic dysfunction in part via increased JNK1/2 pro-inflammatory signaling in L6 cells by Lindsey D. Bogachus; Lorraine P. Turcotte (pp. 415-423).

► Atazanavir sulfate+ritonavir treatment increases glucose and fatty acid metabolism. ► Atazanavir sulfate+ritonavir treatment induces a pro-inflammatory state. ► The protease inhibitor-induced pro-inflammatory state induces insulin resistance.Protease inhibitors (PIs), such as atazanavir sulfate and ritonavir, are used clinically to prevent the progression of HIV and are known to induce insulin resistance. To determine whether PI-mediated insulin resistance is induced by activation of pro-inflammatory cascades, L6 skeletal muscle cells were treated ±atazanavir sulfate, ritonavir, or atazanavir sulfate+ritonavir, and ±insulin. Treatment with atazanavir sulfate, ritonavir, or atazanavir sulfate+ritonavir for 24 or 48h significantly increased basal glucose uptake ( P<0.05) and atazanavir sulfate+ritonavir treatment increased basal glucose uptake significantly more than ritonavir or atazanavir sulfate treatment alone ( P<0.05). Atazanavir sulfate+ritonavir treatment for 48h completely prevented insulin stimulation of glucose uptake ( P>0.05). When compared to untreated cells, basal palmitate uptake and oxidation was found to be significantly higher in cells treated with PIs alone or in combination ( P<0.05). Prior PI treatment alone or in combination prevented ( P>0.05) the insulin-mediated increase in palmitate uptake and the insulin-mediated decrease in palmitate oxidation observed in the control group. Atazanavir sulfate treatment alone or in combination with ritonavir significantly increased JNK1/2 phosphorylation when compared to the control or ritonavir group ( P<0.05) and this was accompanied by a rise ( P<0.05) in AKT^Ser473 phosphorylation in the basal state. Total JNK1/2 and p38 MAPK protein content and p38 MAPK phosphorylation state were not altered in any of the treatment groups ( P>0.05). Our data indicate that, in muscle cells, PIs induce metabolic dysfunction that is not limited to insulin-sensitive metabolism and that is potentially mediated by a rise in JNK1/2 pro-inflammatory signaling.

Keywords: Glucose uptake; p38 MAPK (; M; itogen; A; ctivated; P; rotein; K; inase); Inflammation; AKT2; Fatty acid oxidation

Exploring the molecular basis of dsRNA recognition by NS1 protein of influenza A virus using molecular dynamics simulation and free energy calculation by Dabo Pan; Huijun Sun; Yulin Shen; Huanxiang Liu; Xiaojun Yao (pp. 424-433).

► This study explored NS1A–dsRNA recognition mechanism. ► By MM-GBSA calculation, the driving force for NS1A–dsRNA binding was presented. ► Seventeen hotspot residues from NS1A were identified. ► The origin of decrease of binding affinity for different NS1A mutants is different.The frequent outbreak of influenza pandemic and the limited available anti-influenza drugs highlight the urgent need for the development of new antiviral drugs. The dsRNA-binding surface of nonstructural protein 1 of influenza A virus (NS1A) is a promising target. The detailed understanding of NS1A–dsRNA interaction will be valuable for structure-based anti-influenza drug discovery. To characterize and explore the key interaction features between dsRNA and NS1A, molecular dynamics simulation combined with MM-GBSA calculations were performed. Based on the MM-GBSA calculations, we find that the intermolecular van der Waals interaction and the nonpolar solvation term provide the main driving force for the binding process. Meanwhile, 17 key residues from NS1A were identified to be responsible for the dsRNA binding. Compared with the wild type NS1A, all the studied mutants S42A, T49A, R38A, R35AR46A have obvious reduced binding free energies with dsRNA reflecting in the reduction of the polar and/or nonpolar interactions. In addition, the structural and energy analysis indicate the mutations have a small effect to the backbone structures but the loss of side chain interactions is responsible for the decrease of the binding affinity. The uncovering of NS1A–dsRNA recognition mechanism will provide some useful insights and new chances for the development of anti-influenza drugs.

Keywords: Influenza A virus; Nonstructural protein 1; Protein–RNA interaction; Molecular dynamics simulation; Molecular mechanics generalized born surface area (MM-GBSA)

Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection by Prakash Shankaran; Lenka Vlkova; Jana Liskova; Zora Melkova (pp. 434-446).

► We analyzed effects of heme arginate on Human immunodeficiency virus-1 replication and provirus reactivation. ► Heme arginate inhibited the overall replication and reverse transcription of HIV-1. ► Heme arginate synergized with PMA or TNF-α in the reactivation of HIV-1 provirus. ► The mechanism involved heme oxygenase-1 and increased redox stress. ► The results suggest a new direction to explore in treatment of HIV/AIDS disease.Human immunodeficiency virus-1 (HIV-1) successfully escapes from host immune surveillance, vaccines and antiretroviral agents. The available antiretroviral compounds can only control viremia, but it is impossible to eliminate the virus from the organism, namely because HIV-1 provirus persists in the reservoir cells from which the virus repeatedly disseminates into new cells. Current therapeutic approaches, however, do not specifically address the stage of virus reactivation.Heme has been demonstrated as very efficient in inhibiting HIV-1 reverse transcription, while its derivative hemin ameliorated HIV-1 infection via induction of heme oxygenase-1. Normosang (heme arginate; HA) is a human hemin-containing compound used to treat acute porphyria. In this work, we studied the effects of HA in HIV-1-acutely infected T-cell lines, and in cell lines harboring either a complete HIV-1 provirus (ACH-2 cells) or an HIV-1 “mini-virus” (Jurkat clones expressing EGFP under control of HIV LTR). We demonstrate that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA alone stimulated the reactivation of HIV-1 “mini-virus” and synergized with phorbol ester or TNF-α in the reactivation of HIV-1 provirus. The stimulatory effects of HA were inhibited by N-acetyl cysteine, suggesting an increased redox stress and activation of NF-κB. Further, HA induced expression of heme oxygenase-1 (HO-1) in ACH-2 cells, while HO-1 was found expressed in untreated Jurkat clones. Inhibitor of HO-1 activity, tin protoporphyrin IX, further increased HA-mediated reactivation of HIV-1 “mini-virus” in Jurkat clones, and this effect was also inhibited by N-acetyl cysteine. The stimulatory effects of HA on HIV-1 reactivation thus seem to involve HO-1 and generation of free radicals. Additionally, the effective concentrations of HA did neither affect normal T-cell activation with PMA nor induce activation of the unstimulated cells.In conclusion, HA appears to possess a combination of unique properties that could help to decrease the pool of latently infected reservoir cells, while simultaneously inhibiting HIV-1 replication in newly infected cells. Our results thus suggest a new direction to explore in treatment of HIV/AIDS disease.

Keywords: HIV; Heme arginate; Normosang; Replication inhibition; Reactivation; Heme oxygenase-1

Inhibition of viral RNA synthesis in canine distemper virus infection by proanthocyanidin A2 by Laura Gallina; Fabiana Dal Pozzo; Viola Galligioni; Ezio Bombardelli; Alessandra Scagliarini (pp. 447-452).

Canine distemper virus (CDV) is a contagious and multisystemic viral disease that affects domestic and wild canines as well as other terrestrial and aquatic carnivores. The disease in dogs is often fatal and no specific antiviral therapy is currently available.In this study, we evaluated the in vitro antiviral activity against CDV of proanthocyanidin A2 (PA2), a phenolic dimer belonging to the class of condensed tannins present in plants. Our results showed that PA2 exerted in vitro antiviral activity against CDV with a higher selectivity index compared to ribavirin, included in our study for the previously tested anti-CDV activity. The time of addition assay led us to observe that PA2 was able to decrease the viral RNA synthesis and to reduce progeny virus liberation, at different times post infection suggesting multiple mechanisms of action including inhibition of viral replicative complex and modulation of the redox milieu. These data suggest that PA2, isolated from the bark of Aesculus hippocastanum, has potential usefulness as an anti-CDV compound inhibiting viral replication.

Keywords: Canine distemper virus; Antiviral activity; Proanthocyanidin A2; Phytochemical

Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model by Abhay P.S. Rathore; Prasad N. Paradkar; Satoru Watanabe; Kah Hin Tan; Cynthia Sung; John E. Connolly; Jenny Low; Eng Eong Ooi; Subhash G. Vasudevan (pp. 453-460).

► Celgosivir is an effective nanomolar concentration inhibitor against all four dengue virus serotypes. ► Celgosivir effectively inhibits DENV-2 in ADE infected THP-1 cells. ► NS1 protein misfolds and accumulates in the ER during celgosivir treatment. ► Celgosivir treatment promotes the induction of pro-survival ER stress response genes. ► Celgosivir treatment is effective upto 48h post-virus infection in a lethal challenge mouse model.Dengue virus (DENV) infections continue to spread aggressively around the world. Here we demonstrate that celgosivir (6- O-butanoyl castanospermine), strongly inhibits all four DENV serotypes. We show by fluorescence microscopy that the antiviral mechanism of celgosivir, is in part, due to misfolding and accumulation of DENV non-structural protein 1 (NS1) in the endoplasmic reticulum. Moreover, celgosivir modulates the host’s unfolded protein response (UPR) for its antiviral action. Significantly, celgosivir is effective in lethal challenge mouse models that recapitulate primary or secondary antibody-dependent enhanced DENV infection. Celgosivir treated mice showed enhanced survival, reduced viremia and robust immune response, as reflected by serum cytokine analysis. Importantly, survival increased even after treatment was delayed till 2days post-infection. Together the present study suggests that celgosivir, which has been clinically determined to be safe in humans, may be a valuable candidate for clinical testing in dengue patients.

Keywords: Dengue virus; Celgosivir; α-Glucosidase inhibitor; Dengue NS1 protein misfolding; AG129 mouse model for dengue infection

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity by Jeffrey W. Koehler; Lesley C. Dupuy; Aura R. Garrison; Brett F. Beitzel; Michelle J. Richards; Daniel R. Ripoll; Anders Wallqvist; Shia-Yen Teh; Andrew A. Vaewhongs; Fakhrieh S. Vojdani; Hal S. Padgett; Connie S. Schmaljohn (pp. 461-469).

► Hybrid, human interferons expressed in plants by gene crossbreeding. ► Hybrid interferons identified with improved antiviral properties. ► Several hybrid interferons inhibit interferon resistant viruses. ► Structural analysis suggests improved activity is related to receptor binding.Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.

Keywords: Abbrevations; IFNs; interferons; IFNAR; interferon-α receptor; VEEV; Venezuelan equine encephalitis virus; RVFV; Rift Valley fever virus; EBOV; Ebola virus; MPXV; monkeypox virus; GRAMMR™; Genetic Reassortment by MisMatch Resolution; BSA; bovine serum albumin; GFP; green fluorescent protein; PSPP; protein structure prediction pipeline; PDB; Protein Data BankType I interferons; Ebola virus; Rift Valley fever virus; Venezuelan equine encephalitis virus; Monkeypox virus

Virucidal mechanism of action of NVC-422, a novel antimicrobial drug for the treatment of adenoviral conjunctivitis by Jungjoo Yoon; Andreas Jekle; Ramin Najafi; Francis Ruado; Meghan Zuck; Behzad Khosrovi; Bahram Memarzadeh; Dmitri Debabov; Lu Wang; Mark Anderson (pp. 470-478).

► We demonstrated the virucidal activity of NVC-422 against adenovirus type 5. ► In vitro assays show that NVC-422 has a good therapeutic index. ► The virucidal activity of NVC-422 is rapid with complete inactivation of Ad5 within 15min. ► The virucidal activity of NVC-422 is due to its reacting with key viral proteins.Human adenoviral conjunctivitis is a highly contagious eye infection affecting millions of people world-wide. If untreated, it can further develop into keratitis, corneal ulceration, scarring and possible blindness. Despite the significant patient morbidity and socio-economic costs, it is an unmet medical need with no FDA approved treatment. Here, we demonstrate the virucidal activity of NVC-422 ( N, N-dichloro-2,2-dimethyltaurine) against adenovirus type 5 (Ad5) and investigated its mechanism of action of Ad5 inactivation. NVC-422 inhibits Ad5-induced loss of cell viability in vitro with 50% inhibitory concentration (IC₅₀) ranging from 9 to 23μM. NVC-422 does not cause any cytotoxicity at concentrations as high as 250μM. Invitro, NVC-422 inactivates Ad5 but does not interfere with viral replication, indicating that NVC-422 acts on the extracellular adenovirus as a virucidal agent. NVC-422 inactivates Ad5 by oxidative inactivation of key viral proteins such as fiber and hexon as evidenced by SDS–PAGE, Western blotting and reversed-phase HPLC. These data, combined with measurements of the kinetics of the NVC-422 reactivity with selected amino acids, indicate that the changes in the viral proteins are caused by the selective oxidation of sulfur-containing amino acids. The conformational changes of the viral proteins result in the destruction of the viral morphology as shown by transmission electron microscopy. In summary, NVC-422 exhibits virucidal activity against Ad5 by the oxidative inactivation of key viral proteins, leading to the loss of viral integrity and infectivity.

Keywords: Adenovirus; Conjunctivitis; Mechanism of action; Virucidal; Antimicrobial

Compensatory mutations rescue the virus replicative capacity of VIRIP-resistant HIV-1 by Emmanuel González-Ortega; Ester Ballana; Roger Badia; Bonaventura Clotet; José A. Esté (pp. 479-483).

► Mutations conferring resistance to VIRIP/VIR-353 do not appear in the fusion peptide of gp41. ► Resistance to VIRIP induced a change in virus replicative capacity. ► Mutations in gp120 and gp41 restored virus replicative capacity maintaining the resistant phenotype. ► There is a high genetic barrier for resistance to VIRIP and its analogues.VIRIP has been identified as a highly specific natural inhibitor of HIV-1 that blocks HIV-1 gp41-dependent fusion by interacting with the gp41 fusion peptide. Two analogues of VIRIP (VIR-353 and VIR-576) with a few amino acid changes increase its antiretroviral potency by two orders of magnitude in cell culture. VIR-576 has been shown effective in a phase I/II clinical trial. Resistance to VIRIP and its analogue VIR-353 were generated after long-term passage in cell culture suggesting a high genetic barrier to resistance. Mutations conferring resistance to VIRIP and VIR-353 significantly reduced virus fitness. However, accumulation of additional mutations rescued the replication capacity of the virus while retaining resistance to VIR-353 and full sensitivity to T20. Combinations of VIR-353 and T20 had an additive effect on the inhibition of wild type HIV-1 replication, but only a single agent was active when combinations were tested against T20-resistant HIV-1, suggesting that both gp41 peptides do not interfere with each other in their binding to gp41. Our results provide additional support to the development of a new class of antiretroviral agents targeting gp41-dependent fusion.

Keywords: HIV-1; Fitness; gp41; Fusion; Resistance

The efficacy of an anti-CD4 monoclonal antibody for HIV-1 treatment by W. Jeffrey Fessel; Brooke Anderson; Stephen E. Follansbee; Mark A. Winters; Stanley T. Lewis; Steven P. Weinheimer; Christos J. Petropoulos; Robert W. Shafer (pp. 484-487).

► The monoclonal antibody ibalizumab is active in vivo vs. multidrug-resistant HIV-1. ► We show this in a subject treated with ibalizumab, enfuvirtide re-use, and etravirine. ► A missed ibalizumab infusion markedly affected HIV dynamics and drug resistance. ► We confirmed the role of V5 env glycosylation site changes in ibalizumab resistance.The availability of 24 antiretroviral (ARV) drugs within six distinct drug classes has transformed HIV-1 infection (AIDS) into a treatable chronic disease. However, the ability of HIV-1 to develop resistance to multiple classes continues to present challenges to the treatment of many ARV treatment-experienced patients. In this case report, we describe the response to ibalizumab, an investigational CD4-binding monoclonal antibody (mAb), in a patient with advanced immunodeficiency and high-level five-class antiretroviral resistance. After starting an ibalizumab-based salvage regimen, the patient had an approximately 4.0 log₁₀ reduction in viral load. An inadvertently missed infusion at week 32 led to the rapid loss of virologic response and decreased susceptibility to the remainder of the patient’s salvage therapy regimen. Following the reinstitution of ibalizumab, phenotypic and genotypic resistance to ibalizumab was detected. Nonetheless, plasma HIV-1 RNA levels stabilized at ∼2.0 log₁₀ copies/ml below pre-ibalizumab levels. Continued ARV drug development may yield additional clinical and public health benefits. This report illustrates the promise of mAbs for HIV-1 therapy in highly treatment-experienced patients. Therapeutic mAbs may also have a role in pre-exposure prophylaxis in high-risk uninfected populations and may facilitate directly observed therapy (DOT) if two or more synergistic long acting agents become available.

Keywords: HIV-1; Monoclonal antibody; Drug resistance; Ibalizumab

Rescue of HIV-1 long-time archived X4 strains to escape maraviroc by Franky Baatz; Daniel Struck; Morgane Lemaire; Sebastien De Landtsheer; Jean-Yves Servais; Vic Arendt; Jean-Claude Schmit; Danielle Perez Bercoff (pp. 488-492).

► Investigation of HIV-1 coreceptor usage in a patient failing maraviroc treatment. ► Coreceptor usage is assessed genotypically and phenotypically. ► Phylogenetic analysis is performed on V3-loop sequences. ► Recombination plays a key role in rescuing X4 strains that lead to maraviroc failure. ► Interleukin-2 has a strong impact on viral population diversity.Entry of Human Immunodeficiency Virus type 1 (HIV-1) into target cells is mediated by the CD4 receptor and a coreceptor, CCR5 or CXCR4. Maraviroc interferes with HIV entry by binding the CCR5 coreceptor. Virological failure to maraviroc-containing regimens can occur through the emergence of resistance, or through tropism evolution and broadened coreceptor usage. In the latter case, the physiological relevance of minority strains is a major concern.Here we report a retrospective analysis of coreceptor-usage and evolution based on 454-ultra-deep-sequencing of plasma and Peripheral Blood Mononuclear Cell (PBMC)-derived envelope V3-loops, accounting for coreceptor usage, from a patient who failed a maraviroc-containing regimen through the emergence of X4 strains. The X4 maraviroc-escape variant resulted from recombination between a long time archived proviral sequence from 2003 (5′-portion, including the V3-loop) and the dominant R5 strains circulating in plasma at the time of maraviroc-treatment initiation (3′-portion). Phylogenetic analyses and BEAST modeling highlighted that an early diverse viral quasispecies underwent a severe bottleneck following reinitiation of HAART and repeated IL-2 cycles between 1999 and 2001, leading to the transient outgrowth and archiving of one highly homogeneous X4 population from plasma, and to the expansion in plasma of one PBMC-derived R5 strain. Under maraviroc selective pressure, the early, no longer detectable X4 strains archived in PBMC were partially rescued to provide X4-determinants to the main circulating strain.

Keywords: HIV-1 tropism; Recombination; Maraviroc; Evolution; Interleukin-2

Protective immunity elicited by a pseudotyped baculovirus-mediated bivalent H5N1 influenza vaccine by Qunfeng Wu; Shaobo Xiao; Huiying Fan; Yang Li; Jinfang Xu; Zhen Li; Wei Lu; Xiaoyue Su; Wei Zou; Meilin Jin; Huanchun Chen; Liurong Fang (pp. 493-496).

► BV-HAs induce enhanced humoral and cellular immunity in a mouse model. ► BV-HAs conferred 100% protection against several strains of H5N1 viruses. ► The effective immunizing dose of BV-HAs was very low compared with mono-HA-based baculovirus. ► Recombinant baculovirus exhibited robust adjuvant efficiency in vivo without pre-existing antibody.The development of novel H5N1 influenza vaccines to elicit a broad immune response is a priority in veterinary and human public health. In this report, a baculovirus vector was used to construct bivalent recombinant baculovirus vaccine encoding H5N1 influenza virus hemagglutinin proteins (BV-HAs) from clade 2.3.4 and clade 9 influenza viruses. Mice immunized with 5×10⁷IFU BV-HAs developed significantly high levels of H5-specific neutralizing antibodies and cellular immunity that conferred 100% protection against infection with H5N1 influenza viruses. This study suggests that baculovirus-delivered multi-hemagglutinin proteins might serve as a candidate vaccine for the prevention of pre-pandemic and pandemic H5N1 influenza viruses.

Keywords: Influenza virus; Vaccine; Hemagglutinin; Bivalent vaccine

No reduction of HCV viral load in HIV patients co-infected with HCV genotype 1 during a 30days course of nitazoxanide monotherapy by N. Laufer; L. Abusamra; F. Bolcic; A. Gun; M.J. Rolón; H. Pérez; A. Krolewiecki; H. Salomón; J. Quarleri; P. Cahn (pp. 497-499).

► HCV RNA kinetics during treatment with nitazoxanide monotherapy was evaluated. ► Nitazoxanide was well tolerated in HIV/HCV co-infected patients. ► No changes in HCV RNA were observed in HIV/HCV genotype 1 co-infected patients.There are two new drugs approved and several in development for treatment of chronic HCV; among them nitazoxanide (NTZ). Twelve HIV/HCV genotype 1 co-infected patients were enrolled prospectively to receive a 30days course of oral NTZ 500mg bid. This therapy was well tolerated in this group of HIV patients co-infected with HCV genotype 1. Nevertheless no changes in HCV viral load were observed during treatment in none of the patients evaluated. This data suggests that despite the promising results reported for HCV genotype 4 mono-infected patients, NTZ exhibit poor activity as monotherapy in HIV/HCV co-infected patients with genotype 1.

Keywords: Nitazoxanide; HCV genotype 1; HIV/HCV co-infection; HCV treatment

Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection by Miguel Rodríguez-Pulido; Francisco Sobrino; Belén Borrego; Margarita Sáiz (pp. 500-504).

► FMDV NCRs have prophylactic and therapeutic activity against viral infection. ► FMDV IRES induces heterotypic protection against virus challenge in suckling mice. ► FMDV 5′ and 3′ NCR transcripts induce dose-dependent protection against FMDV.We have recently described the ability of in vitro-transcribed RNAs, mimicking structural domains in the 5′ and 3′ non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome, to trigger the innate immune response in porcine cultured cells and mice. In this work, the antiviral effect exerted in vivo by these small synthetic non-infectious RNA molecules was analyzed extensively. The susceptibility of transfected newborn Swiss mice to FMDV challenge was tested using a wide range of viral doses. The level of protection depended on the specific RNA inoculated and was dose-dependent. The RNA giving the best protection was the internal ribosome entry site (IRES), followed by the transcripts corresponding to the S fragment. The time course of resistance to FMDV of the RNA-transfected mice was studied. Our results show the efficacy of these RNAs to prevent viral infection as well as to contain ongoing FMDV infection in certain time intervals. Protection proved to be independent of the serotype of FMDV used for challenge. These results support the potential use of the FMDV NCR transcripts as both prophylactic and therapeutic molecules for new FMDV control strategies.

Keywords: Foot-and-mouth disease; Viral non-coding regions; RNA antiviral activity; Anti-FMDV strategies; Innate immunity

Antiviral interactions of combinations of highly potent 2,4(1H,3H)-pyrimidinedione congeners and other anti-HIV agents by Tracy L. Hartman; Lu Yang; Robert W. Buckheit Jr. (pp. 505-508).

► The PYD congeners are highly potent dual-acting inhibitors of HIV-1 and HIV-2. ► The PYDs interact in an additive to synergistic manner with other anti-HIV agents. ► Antagonism or synergistic toxicity was not observed with the PYD combinations. ► The optimal PYD combinations are distinct for subtype B and subtype C HIV. ► PYDs with cyclopropyl substitutions appear to represent the best lead compounds.Structure–activity relationship evaluation of seventy-four 2,4(1H,3H)-pyrimidinedione derivatives identified seven lead compounds based on anti-HIV-1 potency, extended range of action to include HIV-2, virus entry inhibition, reverse transcriptase inhibition, and lack of cytotoxicity to human cells. The selected pyrimidinedione congeners are highly active inhibitors of HIV-1 with EC₅₀ values ranging from 0.6 to 2nM in CEM-SS cells infected with laboratory derived viruses, 11–20nM in fresh human PBMCs infected with subtype B (HT/92/599) virus, and 2–7nM in PBMCs infected with the clinical subtype C (ZA/97/003) virus. Combination antiviral assays were performed using the laboratory adapted RF strain of HIV-1 in CEM-SS cells and with a clade B and C low passage clinical isolate in fresh human peripheral mononuclear cells and the compound interactions were analyzed using MacSynergy II. The seven pyrimidinedione compounds resulted in additive to synergistic interactions in combination with entry and fusion inhibitors, nonnucleoside and nucleoside reverse transcriptase inhibitors, and the protease inhibitors. No evidence of antagonistic antiviral activity or synergistic cytotoxicity was detected with the combinations of compounds tested. The dual mechanism of action of the pyrimidinediones resulting in inhibition of both virus entry and reverse transcription suggests excellent potential of these lead pyrimidinediones as candidates for combination therapy with other approved HIV inhibitors of varying mechanism of action.

Keywords: NNRTI; HIV-1; Combination therapy; Pyrimidinedione

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Antiviral Research (v.92, #3)