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Antiviral Research (v.74, #2)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs by Yun-xia He; Rong-hong Hua; Yan-jun Zhou; Hua-ji Qiu; Guang-zhi Tong (pp. 83-91).

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit PRRSV replication on MARC-145 cells. Four short interfering RNA (siRNA) sequences (N95, N179, N218 and N294) directed against a well-conserved region of PRRSV genome ORF7 gene were selected. Sense and antisense siRNA encode sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. Using a polymerase chain reaction (PCR)-based approach, shRNAs were generated from shRNA expression cassettes. The PCR products were cloned into pEGFP-N1 vector and shRNA expression vectors were constructed. When MARC-145 cells were transfected with shRNA expression vectors and then infected with PRRSV, N179 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by PRRSV. Western blot, indirect immunofluorescence and fluorescence quantitative PCR (FQ-PCR) confirmed that the expression of ORF7 was reduced both at protein and RNA levels comparing to controls. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of PRRS virus (approximately 681-fold reduction of viral titers) on MARC-145 cells.

Keywords: PRRSV; RNAi; shRNA; siRNA

Emodin blocks the SARS coronavirus spike protein and angiotensin-converting enzyme 2 interaction by Tin-Yun Ho; Shih-Lu Wu; Jaw-Chyun Chen; Chia-Cheng Li; Chien-Yun Hsiang (pp. 92-101).

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus (SARS-CoV). SARS-CoV spike (S) protein, a type I membrane-bound protein, is essential for the viral attachment to the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening 312 controlled Chinese medicinal herbs supervised by Committee on Chinese Medicine and Pharmacy at Taiwan, we identified that three widely used Chinese medicinal herbs of the family Polygonaceae inhibited the interaction of SARS-CoV S protein and ACE2. The IC₅₀ values for Radix et Rhizoma Rhei (the root tubers of Rheum officinale Baill.), Radix Polygoni multiflori (the root tubers of Polygonum multiflorum Thunb.), and Caulis Polygoni multiflori (the vines of P. multiflorum Thunb.) ranged from 1 to 10μg/ml. Emodin, an anthraquinone compound derived from genus Rheum and Polygonum, significantly blocked the S protein and ACE2 interaction in a dose-dependent manner. It also inhibited the infectivity of S protein-pseudotyped retrovirus to Vero E6 cells. These findings suggested that emodin may be considered as a potential lead therapeutic agent in the treatment of SARS.

Keywords: Abbreviations; SARS; severe acute respiratory syndrome; SARS-CoV; SARS coronavirus; S; spike; ACE2; angiotensin-converting enzyme 2; HIV; human immunodeficiency virus; ELISA; enzyme-linked immunosorbent assay; E. coli; Escherichia coli; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS; phosphate-buffered saline; BSA; bovine serum albumin; IFA; immunofluorescence assay; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HSV; herpes simplex virusSARS coronavirus; Spike protein; Angiotensin-converting enzyme 2; Emodin

Inhibition of Epstein Barr Virus LMP1 gene expression in B lymphocytes by antisense oligonucleotides: Uptake and efficacy of lipid-based and receptor-mediated delivery systems by Roberta Galletti; Silvia Masciarelli; Cinzia Conti; Giulia Matusali; Livia Di Renzo; Stefania Meschini; Giuseppe Arancia; Carlo Mancini; Elena Mattia (pp. 102-110).

Epstein Barr Virus (EBV), is associated with an increasing number of lymphoid and epithelial malignancies. Among the genes expressed by EBV during latency, LMP1 plays a key role for growth transformation and immortalization of B lymphocytes. We have previously shown that antisense oligonucleotides (ONs) directed to LMP1 mRNA, effectively suppressed LMP1 gene expression and substantially reduced proliferation of the infected cells.The use of antisense phosphodiester oligonucleotides as therapeutic agents is limited by inefficient cellular uptake and intracellular transport to the target mRNA. We tested the ability of three cationic carriers internalized by different pathways, to increase the delivery of anti-LMP1-ON to their site of action in EBV-infected B lymphocytes. We report here that liposomes, dendrimers or transferrin-polylysine-conjugated ON were internalized by the cells at an extent several fold higher than that of the naked oligomers. However, only the delivery system exploiting the transferrin receptor pathway of internalization, was able to vectorize biologically active antisense LMP1-ON.

Keywords: Antisense oligonucleotides; DNA vectors; LMP1; EBV; Lymphoma

A dual chamber model of female cervical mucosa for the study of HIV transmission and for the evaluation of candidate HIV microbicides by Yven Van Herrewege; Jo Michiels; Anouk Waeytens; Gitte De Boeck; Evelyne Salden; Leo Heyndrickx; Guy van den Mooter; Marie-Pierre de Béthune; Koen Andries; Paul Lewi; Marleen Praet; Guido Vanham (pp. 111-124).

A dual chamber system was established to model heterosexual HIV transmission. Cell-associated, but not cell-free HIV, added to a confluent layer of cervical epithelial cells in the apical chamber, reproducibly infected monocyte-derived dendritic cells (MO-DC) and CD4⁺ T cells in the basal compartment. Only minimal epithelial transmigration of HIV-infected mononuclear cells (HIV-PBMCs) was observed. Most evidence points to transepithelial migration of virus, released from HIV-PBMCs after their activation by epithelial cells.We used this model for evaluation of the therapeutic index of various potentially preventive antiviral compounds, including non-nucleoside reverse transcriptase inhibitors (NNRTIs, including UC781 and various diaryltriazines and diarylpyrimidines), poly-anionic entry inhibitors (including PRO2000, cellulose sulphate, dextrane sulphate 5000 and polystyrene sulphonate) and the fusion inhibitor T-20. The epithelium was pre-treated with compound and incubated with HIV-PBMCs for 24h. Afterwards the apical chamber was removed and MO-DC/CD4⁺ T cell co-cultures were further cultured without compound. NNRTIs, including a TMC120 gel, blocked infection of the sub-epithelial targets at sub-micromolar concentrations. Polyanionic entry inhibitors (up to 100μg/ml) and T-20 (up to 449μg/ml) failed to inhibit transmission. Moreover, whereas the NNRTIs used interfered with epithelial integrity with cervical epithelium only at very high concentrations, the evaluated entry inhibitors showed toxicity at concentrations that did not prevent infection.

Keywords: Microbicides; Sexual HIV transmission; NNRTI; Entry inhibitors; Dendritic cells; In vitro model

Novel CCR5 monoclonal antibodies with potent and broad-spectrum anti-HIV activities by Changhua Ji; Michael Brandt; Marianna Dioszegi; Andreas Jekle; Stephan Schwoerer; Steven Challand; Jun Zhang; Yun Chen; Lisa Zautke; Gunthar Achhammer; Monika Baehner; Sandra Kroetz; Gabrielle Heilek-Snyder; Ralf Schumacher; Nick Cammack; Surya Sankuratri (pp. 125-137).

To identify monoclonal antibodies (mAbs) with high potency and novel recognition sites, more than 25,000 of mouse hybridomas were screened and 4 novel anti-human CCR5 mAbs ROAb12, ROAb13, ROAb14, and ROAb18 showing potent activity in cell–cell fusion (CCF) assay were identified. These mAbs demonstrated potent antiviral activities in both single-cycle HIV infection (IC₅₀ range: 0.16–4.3μg/ml) and PBMC viral replication (IC₅₀ range: 0.02–0.04μg/ml) assays. These potent antiviral effects were donor-independent. All 4 mAbs were also highly potent in the PhenoSense assay against 29 HIV isolates covering clade A through G. In all antiviral assays, these mAbs showed potency superior to the previously reported mAb 2D7 in side-by-side comparison studies. All 4 mAbs were also fully active against viruses resistant to HIV fusion inhibitor enfuvirtide and CCR5 antagonist maraviroc. Although ROAb12, ROAb14, and ROAb18 inhibited RANTES, MIP1α and MIP1β binding and cell activation, the other novel mAb ROAb13 was inactive in inhibiting cell activation by these three ligands. Furthermore, highly synergistic antiviral effects were found between mAb ROAb13 and 2D7 or ROAb12. In addition, none of these mAbs showed agonist activity or caused internalization of the CCR5 receptor.

Keywords: HIV; CCR5; Monoclonal antibody; Entry inhibitor; Synergism

Cidofovir is effective against caprine herpesvirus 1 infection in goats by Maria Tempesta; Michele Camero; Anna Lucia Bellacicco; Julien Thiry; Giuseppe Crescenzo; Johan Neyts; Etienne Thiry; Canio Buonavoglia (pp. 138-141).

Caprine herpesvirus 1 (CpHV-1) is a virus able to cause genital infection leading to vulvovaginitis or balanoposthitis in adult goats. CpHV-1 shares several biological similarities with herpes simplex type 2 (HSV-2) infection in man, such as genital tropism, type and site of typical lesions and it might provide an animal model for studies on antiviral drugs for HSV-2 infection in man. In this view the efficacy of cidofovir (CDV) drug was tested in six goats intravaginally infected with BA.1 strain of CpHV-1. Three goats received an intravaginal application of 3ml of a 1% CDV preparation at 4h post infection and then every 12h for five consecutive days. Three goats were kept as untreated controls. The goats were daily examined for clinical evidence of the infection and viral shedding. CDV was able to protect against disease progression and inhibited the onset of the local lesions due to the CpHV-1 replication. Treated animals shed virus for a shorter period (3 days less) and at lower titres than the control animals. CpHV-1 infection in goats may represent an excellent animal model for the study of novel strategies for the treatment of primary genital HSV-2 infection in man.

Keywords: Cidofovir; Antiviral drug; Caprine herpesvirus 1; Goat; Animal model; Herpes simplex virus 2

Simultaneously inhibition of HIV and HBV replication through a dual small interfering RNA expression system by Kailang Wu; Yongxin Mu; Jing Hu; Lu Lu; Xue Zhang; Yongbo Yang; Yan Li; Fang Liu; Degui Song; Ying Zhu; Jianguo Wu (pp. 142-149).

Human immunodeficiency virus (HIV) is often acquired in individuals already infected with hepatitis B virus (HBV) as a result of shared routes of transmission. Since current options for the treatment of HIV and HBV infections are limited, there is an essential need for the development of effective therapies against HIV/HBV co-infections. RNA interference (RNAi) has been used as a powerful tool to silence genes in cells and animals. In this study, we developed a small interfering RNA generation system that expressed two different siRNAs to target the HBs gene of HBV and the gp120 gene of HIV in Bel-7402 and HEK293T cells, respectively. Our results demonstrated that the two siRNA molecules could simultaneously inhibit the expression of HBs and gp120 by 81% and 89%, respectively. In addition, dual siRNA molecules significantly decreased the production of HBs, and simultaneously inhibited the replication of HBV and HIV. This dual siRNA generation system not only proved to be a novel approach for studying functions of multiple genes simultaneously, but also provides a potential approach for the treatment and prevention of HIV and HBV co-infection.

Keywords: HBV; HIV; Dual siRNA; Viral replication

Therapeutic inhibition of yellow head virus multiplication in infected shrimps by YHV-protease dsRNA by Witoon Tirasophon; Supansa Yodmuang; Wanlop Chinnirunvong; Nongluk Plongthongkum; Sakol Panyim (pp. 150-155).

Yellow head virus (YHV) is an invertebrate nidovirus which causes a severe mortality in cultured Penaeus monodon. The mortality may be prevented by prior treatment of shrimps with YHV-protease dsRNA. Whether the YHV infected shrimp might be cured by the dsRNA remains to be investigated. P. monodon injected with 10⁻⁶ YHV showed a high virus replication and mortality within 2 days. Injection of 25μg YHV-protease dsRNA at 3, 6, 12 or 24h post YHV infection showed a strong inhibition of YHV replication up to 12h. Unrelated dsRNA-GFP showed no inhibition, indicating that the inhibition was nucleic acid sequence specific through RNAi pathway. Shrimp mortality could be prevented at 3h post YHV infection by the dsRNA, but not at 24h. These results demonstrate that YHV-protease dsRNA gives therapeutic effect and pave the way to develop a cure for YHV-infected shrimps.

Keywords: Yellow head virus (YHV); RNAi; antiviral; dsRNA-protease

Progressive multifocal leukoencephalopathy in a haploidentical stem cell transplant recipient: A clinical, neuroradiological and virological response after treatment with risperidone by Daniele Focosi; Rita Fazzi; Domenico Montanaro; Michele Emdin; Mario Petrini (pp. 156-158).

JC virus (JCV) is a double-stranded DNA virus belonging to family Polyomaviridae. It causes progressive multifocal leukoencephalopathy (PML), mainly in immunosuppressed people. JCV had been shown to require the serotonin 2A receptor for host cell entry. We report a case of clinical, neuroradiological and virological response of biopsy-proven PML in a 33-year-old comatose woman after treatment with the anti-psychotic drug risperidone. Since risperidone is the tightest binding of current drugs to this receptor we think this may have blocked JCV entry in our patient, allowing her immune recovery and viral clearance.

Keywords: Immunosuppression; Leukemia; Progressive multifocal leukoencephalopathy; Risperidone; Ziprasidone

Characterization of drug-resistant recombinant influenza A/H1N1 viruses selected in vitro with peramivir and zanamivir by Mariana Baz; Yacine Abed; Guy Boivin (pp. 159-162).

There is a limited information with regard to the neuraminidase (NA) mutations conferring resistance to peramivir and zanamivir in the influenza N1 background. In this study, an influenza A/WSN/33 (H1N1) recombinant virus was passaged under peramivir or zanamivir pressure. The peramivir-selected variant had a H274Y mutation in the neuraminidase (NA) gene conferring resistance to peramivir and oseltamivir but susceptibility to zanamivir. The zanamivir-selected variant had a massive deletion in the region encoding the NA active center and an A200T hemagglutinin mutation. This variant exhibited reduced susceptibility to zanamivir with a drug-dependent phenotype.

Keywords: Influenza; Neuraminidase inhibitors; Resistance

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Antiviral Research (v.74, #2)