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Antiviral Research (v.79, #2)

Notes to Authors (pp. co3).

Editorial Board (pp. ii).

Adamantane resistance in circulating human influenza A viruses from Alberta, Canada (1970–2007) by Kanti Pabbaraju; Kevin C.Y. Ho; Sallene Wong; Sandy Shokoples; Xiao-Li Pang; Kevin Fonseca; Julie D. Fox (pp. 81-86).

Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leads to resistance against the adamantane class of anti-influenza drugs. To investigate the emergence and prevalence of adamantane resistance in Alberta, Canada (between 1970 and 2007), 381 influenza A positive samples (original patient specimens) or isolates (virus cultured from patient specimens) were analyzed for changes in these critical amino acid residues. Our results show a significant increase in adamantane resistance in circulating H3N2 viruses in Alberta from 2005 and 2006 when compared with those from 2004 ( p<0.001). Adamantane resistance peaked at 74% in 2006 and then decreased (to 38%) in 2007 ( p=0.001). All resistant H3N2 viruses contained the substitution Ser to Asn at amino acid position 31 of the M2 protein with two viruses having an additional Ala to Val substitution at position 30. Resistance was not observed in the H1N1 viruses tested. Results presented here are concordant with, and extend, previous reports of increased resistance to adamantanes in Asia and North America in recent years. It is important to continue studies to evaluate circulating influenza A viruses for antiviral resistance markers to ensure their optimal use for prophylaxis and treatment of influenza.

Keywords: Influenza; Adamantane; Amantadine; Resistance; M2 protein; Antiviral

Effective inhibition of infectious bursal disease virus replication in vitro by DNA vector-based RNA interference by Yulong Gao; Wei Liu; Honglei Gao; Xiaole Qi; Huan Lin; Xiaomei Wang; Rongxian Shen (pp. 87-94).

Infectious bursal disease (IBD) leads to considerable economic losses for the poultry industry by inducing severe immunosuppression and high mortality in chickens. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit IBDV replication in vitro. We selected 3 short interfering RNA (siRNA) sequences (siVP1⁶¹⁸, siVP1¹¹¹⁵, and siVP1²⁵⁷¹) based on conserved regions in the vp1 gene of the infectious bursal disease virus (IBDV). When the Vero cells were transfected with siRNA, synthesized via in vitro transcription, and then infected with IBDV, siVP1²⁵⁷¹ was discovered to be the most effective site for inhibiting IBDV replication. For long-term expression of siRNA and due to its suitability for large-scale preparation, the mouse U6 promoter was amplified using primers designed according to the siVP1²⁵⁷¹ sequence. The resulting products were then subcloned into pEGFP-C1 to construct the shRNA expression vector pEC2571-shRNA. The shRNA-transfected Vero cells were then infected with IBDV. As compared to the control, the inhibitory rate in the pEC2571-shRNA-transfected group was 87.4%. Indirect immunofluorescence and real-time polymerase chain reaction (PCR) confirmed that VP1 expression decreased at both the protein and RNA levels as compared to that in the controls. The results presented here indicate that DNA vector-based RNAi could effectively inhibit IBDV replication in vitro.

Keywords: RNAi; siRNA; shRNA; Infectious bursal disease virus (IBDV); Virus replication

Specific small interfering RNAs-mediated inhibition of replication of porcine encephalomyocarditis virus in BHK-21 cells by Hong Jia; Xinna Ge; Xin Guo; Hanchun Yang; Kangzhen Yu; Zhenhai Chen; Yanhong Chen; Zhenlin Cha (pp. 95-104).

Encephalomyocarditis virus (EMCV) is recognized as a pathogen inducing acute myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. In this study, eight specific small interfering RNA (siRNA) duplexes targeting different genomic regions of EMCV BJC3 were designed and their ability to inhibit virus replication in BHK-21 cells was investigated. The results showed that BHK-21 cells transfected with siRNA duplexes to 2C gene (JH-4666, BJC-1739), 2B gene (BJC-807), 3C gene (BJC-2363) and 3D gene (BJC-3269) were specifically resistant to EMCV infection when exposed to 500 times the 50% cell culture infective dose (CCID₅₀) of EMCV. The levels of the 3D gene in the transfected cells were obviously decreased. IFA and Western blotting analysis confirmed that the expression of VP1 protein in cell culture transfected with the siRNAs was apparently reduced. Of the five siRNAs, JH-4666, BJC-2363 and BJC-3269 were the most effective. Combination of the siRNA duplexes enhanced the inhibition of EMCV replication. Our data indicated that specific siRNAs are able to inhibit the replication of porcine encephalomyocarditis virus in BHK-21 cells, suggesting that RNAi might provide a new approach to prevent EMCV infection.

Keywords: Encephalomyocarditis virus (EMCV); RNA interference (RNAi); Small interfering RNA (siRNA); EMCV BJC3 isolate; BHK-21 cells; Inhibition; Replication

Is the anti-psychotic, 10-(3-(dimethylamino)propyl)phenothiazine (promazine), a potential drug with which to treat SARS infections? by Dale L. Barnard; Craig W. Day; Kevin Bailey; Matthew Heiner; Robert Montgomery; Larry Lauridsen; Kie-Hoon Jung; Joseph K.-K. Li; Paul K.S. Chan; Robert W. Sidwell (pp. 105-113).

Phenothiazine and derivatives were tested for inhibition of SARS-CoV replication. Phenothiazine slightly inhibited SARS-CoV replication in a neutral red (NR) uptake assay. Adding a propylamino group to give promazine reduced virus yields (VYR assay) with an EC₉₀=8.3±2.8μM, but without selectivity. Various substitutions in the basic phenothiazine structure did not promote efficacy. Phenazine ethosulfate was the most potent compound by VYR assay (EC₉₀=6.1±4.3μM). All compounds were toxic (IC₅₀=6.6–74.5μM) except for phenoxathiin (IC₅₀=858±208μM) and 10-(alpha-diethylamino-propionyl) phenothiazine·HCl (IC₅₀=195±71.2μM). Consequently, none were selective inhibitors of SARS-CoV replication (SI values <1–3.3μM). These data portended the poor efficacy of promazine in a SARS-CoV mouse lung replication model. Intraperitoneal treatment with promazine using a prophylactic (−4h)/therapeutic regimen of 1, 10, or 50mg/(kgday) did not reduce virus lung titers at day 3, yet prolonged virus replication to 14 days. Similar therapeutic promazine doses were not efficacious. Thus, promazine did not affect SARS-CoV replication in vitro or in vivo, nor were any other phenothiazines efficacious in reducing virus replication. Therefore, treating SARS infections with compounds like promazine is not warranted.

Keywords: Promazine; Anti-psychotic; SARS-CoV; Coronavirus; Mouse model; Phenothiazines

In vitro and in vivo anti-hepatitis B virus activities of a plant extract from Geranium carolinianum L by Jiyang Li; Hai Huang; Meiqing Feng; Wei Zhou; Xunlong Shi; Pei Zhou (pp. 114-120).

Natural products provide a large reservoir of potentially active agents with anti-hepatitis B virus (HBV) activity. We examined the effect of the polyphenolic extract from Geranium carolinianum L. (PPGC) on HBV replication both in vitro and in vivo. In the human HBV-transfected liver cell line HepG₂ 2.2.15, PPGC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC₅₀ values of 46.85μg/ml for HBsAg and 65.60μg/ml for HBeAg at day 9. Consistent with the HBV antigen reduction, PPGC (100μg/ml) also reduced HBV DNA level by 35.9%. In the duck hepatitis B virus (DHBV) infected ducks, after PPGC was dosed intragastricly (i.g.) once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED₅₀ value of 47.54mg/kg. In addition, Southern blot analysis confirmed the in vivo anti-HBV effect of PPGC in ducks and PPGC also reduced the plasma and the liver DHBV DNA level in a dose-dependent manner. Furthermore, significant improvement of the liver was observed after PPGC treatment, as evaluated by the histopathological analysis.

Keywords: Geranium carolinianum; L.; Polyphenolic extract; Hepatitis B virus (HBV); Duck hepatitis B virus (DHBV)

Evaluation of orally delivered ST-246 as postexposure prophylactic and antiviral therapeutic in an aerosolized rabbitpox rabbit model by Aysegul Nalca; Josh M. Hatkin; Nicole L. Garza; Donald K. Nichols; Sarah W. Norris; Dennis E. Hruby; Robert Jordan (pp. 121-127).

Orthopoxviruses, such as variola and monkeypox viruses, can cause severe disease in humans when delivered by the aerosol route, and thus represent significant threats to both military and civilian populations. Currently, there are no antiviral therapies approved by the U.S. Food and Drug Administration (FDA) to treat smallpox or monkeypox infection. In this study, we showed that administration of the antiviral compound ST-246 to rabbits by oral gavage, once daily for 14 days beginning 1h postexposure (p.e.), resulted in 100% survival in a lethal aerosolized rabbitpox model used as a surrogate for smallpox. Furthermore, efficacy of delayed treatment with ST-246 was evaluated by beginning treatment on days 1, 2, 3, and 4 p.e. Although a limited number of rabbits showed less severe signs of the rabbitpox disease from the day 1 and day 2 p.e. treatment groups, their illness resolved very quickly, and the survival rates for these group of rabbits were 88% and 100%, respectively. But when the treatment was started on days 3 or 4 p.e., survival was 67% and 33%, respectively. This work suggests that ST-246 is a very potent antiviral compound against aerosolized rabbitpox in rabbits and should be investigated for further development for all orthopoxvirus diseases.

Keywords: Orthopoxvirus; Rabbitpox virus; Aerosol; Smallpox; Rabbit; ST-246

Substrate specificity of feline and canine herpesvirus thymidine kinase by N. Solaroli; M. Johansson; L. Persoons; J. Balzarini; A. Karlsson (pp. 128-132).

The thymidine kinases from feline herpesvirus (FHV TK) and canine herpesvirus (CHV TK) were cloned and characterized. The two proteins are closely sequence-related to each other and also to the herpes simplex virus type 1 thymidine kinase (HSV-1 TK). Although FHV TK and CHV TK have a level of identity of 31 and 35%, respectively, with HSV-1 TK, and a general amino acid similarity of ≈54% with HSV-1 TK, they do not recognize the same broad range of substrates as HSV-1 TK does. Instead the substrate recognition is restricted to dThd and pyrimidine analogs such as 1-β-d-arabinofuranosylthymine (araT), 3′-azido-2′,3′-dideoxythymidine (AZT) and ( E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU). FHV TK and CHV TK differ in substrate recognition from mammalian cytosolic thymidine kinase 1 (TK1) in that TK1 does not phosphorylate BVDU and they also differ from mammalian mitochondrial thymidine kinase 2 (TK2), which, in addition to thymidine and thymidine analogs also phosphorylates dCyd. Although the nucleoside analog BVDU was a good substrate for FHV and CHV TK, the compound was poorly inhibitory to virus-induced cytopathic effect in FHV- and CHV-infected cells. The reason is likely the poor, if any, thymidylate kinase activity of FHV and CHV TK, which in HSV-1 TK-expressing cells convert BVDU-MP to its 5′-diphosphate derivative.

Keywords: Herpesviruses; Thymidine kinase; Gene therapy; Suicide gene; Nucleoside kinases

Effect of oral treatment with ( S)-HPMPA, HDP-( S)-HPMPA or ODE-( S)-HPMPA on replication of murine cytomegalovirus (MCMV) or human cytomegalovirus (HCMV) in animal models by Debra C. Quenelle; Deborah J. Collins; Latisha R. Pettway; Caroll B. Hartline; James R. Beadle; W. Brad Wan; Karl Y. Hostetler; Earl R. Kern (pp. 133-135).

We utilized BALB/c mice infected with murine CMV (MCMV) or severe combined immunodeficient (SCID) mice implanted with human fetal tissue and infected with HCMV to determine the efficacy of ( S)-9-[3-hydroxy-2-(phophonomethoxy)propyl]adenine (( S)-HPMPA), hexadecyloxypropyl-( S)-HPMPA (HDP-( S)-HPMPA) or octadecyloxyethyl-( S)-HPMPA (ODE-( S)-HPMPA). In MCMV-infected BALB/c mice, oral HDP-( S)-HPMPA at 30mg/kg significantly reduced mortality when started 24–48h post inoculation. In the experimental HCMV infection, oral administration of vehicle or 10mg/kg of ( S)-HPMPA, HDP-( S)-HPMPA or ODE-( S)-HPMPA was initiated 24h after infection and continued for 28 consecutive days. Cidofovir (CDV), at 20mg/kg given i.p., was used as a positive control. HDP-( S)-HPMPA or ODE-( S)-HPMPA significantly reduced viral replication compared to vehicle-treated mice, while oral ( S)-HPMPA was ineffective.

Keywords: Cytomegalovirus; Antiviral; Animal models; Phosphonate nucleotides

Creation of a bi-directional protein transduction system for suppression of HIV-1 expression by p27SJ by Nune Darbinian; Yuri Popov; Kamel Khalili; Shohreh Amini (pp. 136-141).

p27SJ is a novel protein from a callus culture of St. John's wort that modulates transcription of the HIV-1 promoter in several mammalian cells [Darbinian-Sarkissian, N., Darbinyan, A., Otte, J., Radhakrishnan, S., Sawaya, B.E., Arzumanyan, A., Chipitsyna, G., Popov, Y., Rappaport, J., Amini, S., Khalili, K., 2006. p27(SJ), a novel protein from St. John's wort, that suppresses expression of HIV-1 genome. Gene Ther. 13, 288–295]. Here, we armed p27SJ with signals from Ig-kappa light chain that allow its efficient excretion from the cells, and from HIV-1 Tat that facilitates its uptake by other cells for its utilization by a protein transduction method. We demonstrate that treatment of cells containing the HIV-1 LTR with conditioned media from cells expressing the armed p27SJ (_excp27SJ_upt) results in suppression of the viral activation by the C/EBPβ transcription factor. Once imported into the cells,_excp27SJ_upt impacts the nuclear localization of C/EBPβ and by retaining the protein in the cytoplasm affects its DNA binding and hence transcriptional activity. The armed p27SJ also inhibits Tat-induced activation of the LTR and decreases the level of viral replication in promonocytic cells including U-937 and T-lymphocytic cells. Our observations introduce a new bi-directional protein transduction system with a broad spectrum of applications for manufacturing therapeutic peptides by a specific group of cells called donor, and delivery to the target cells named recipient. Furthermore, our results support the utility of soluble p27SJ in suppressing transcription and replication of HIV-1 by interfering with the function of cellular proteins such as C/EBPβ and viral activators including Tat.

Keywords: HIV-1; St. John's wort

Erratum to “Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs” [Antiv. Res. 73 (2007) 169–174] by A. Scagliarini; C.J. McInnes; L. Gallina; F. Dal Pozzo; L. Scagliarini; R. Snoeck; S. Prosperi; J. Sales; J.A. Gilray; P.F. Nettleton (pp. 142-142).

( S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide^®) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

Keywords: Orf virus; Contagious ecthyma; Cidofovir; HPMPC; Topical treatment; Formulation

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Antiviral Research (v.79, #2)