Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase.

Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development.

The impact of the butterfly effect on human parainfluenza virus haemagglutinin-neuraminidase inhibitor design.

Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid's C-4 substituent's size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

Modified vaccinia virus ankara (MVA) as production platform for vaccines against influenza and other viral respiratory diseases.

Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses.

ISG56/IFIT1 is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis.

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

[The specific features of the cocirculation of influenza viruses in the 2010-2011 postpandemic period according to the results of activities of the D. I. Ivanovsky Research Institute of Virology, Ministry of Health and Social Development of Russia].

The paper gives the results of monitoring the circulation of influenza viruses in the 2010-2011 season, that covers the second year of circulation of pandemic A(H1N1)v virus strains, and their interaction with seasonal A (H3N2) and B strains. Unlike the previous season, the beginning of an increase in morbidity was recorded in January 2011; its peak in the most of contiguous areas was noted at 5-7 weeks of 2011, with its further decline to threshold levels at week 11 of 2011. Preschool and school children were most involved in the epidemic process. Three influenza virus strains (A(H1N1)v, A(H3N2), and B) were found to circulate. Differences were found in the level of participation of the isolated strains in individual areas of the Russian Federation. Detailed typing of the isolated strains determined the compliance of the vast majority of them with vaccine viruses. The pandemic influenza A(H1N1)v virus strains retained their susceptibility to oseltamivir and were resistant to rimantadine. The participation of non-influenza acute respiratory viral infection pathogens was estimated as follows: 11.9% for parainfluenza viruses, 5.9% for adenoviruses, and 3.5% for PC viruses, and 0.7% for pneumonia Mycoplasma, which was comparable with the previous epidemic seasons.

Human parainfluenza virus infection after hematopoietic stem cell transplantation: risk factors, management, mortality, and changes over time.

Human parainfluenza viruses (HPIVs) are uncommon, yet high-risk pathogens after hematopoietic stem cell transplant (HCT). We evaluated 5178 pediatric and adult patients undergoing HCT between 1974 and 2010 to determine the incidence, risk factors, response to treatment, and outcome of HPIV infection as well as any change in frequency or character of HPIV infection over time. HPIV was identified in 173 patients (3.3%); type 3 was most common (66%). HPIV involved upper respiratory tract infection (URTI; 57%), lower respiratory tract infection (LRTI; 9%), and both areas of the respiratory tract (34%), at a median of 62 days after transplantation. In more recent years, HPIV has occurred later after HCT, whereas the proportion with nosocomial infection and mortality decreased. Over the last decade, HPIV was more common in older patients and in those receiving reduced intensity conditioning (RIC). RIC was a significant risk factor for later (beyond day +30). HPIV infections, and this association was strongest in patients with URTI. HCT using a matched unrelated donor (MURD), mismatched related donor (MMRD), age 10 to 19 years, and graft-versus-host disease (GVHD) were all risk factors for HPIV infections. LRTI, early (<30 days), age 10 to 19 years, MMRD, steroid use, and coinfection with other pathogens were risk factors for mortality. The survival of patients with LRTI, especially very early infections, was poor regardless of ribavirin treatment. HPIV incidence remains low, but may have delayed onset associated with RIC regimens and improving survival. Effective prophylaxis and treatment for HPIV are needed.

Antiviral and antimicrobial profiles of selected isoquinoline alkaloids from Fumaria and Corydalis species.

In the current study, 33 isoquinoline alkaloids belonging to protopine-, benzylisoquinoline-, benzophenanthridine-, spirobenzylisoquinoline-, phthalideisoquinoline-, aporphine-, protoberberine-, cularine-, and isoquinolone-types as well as 7 derivatives of them obtained from some Fumaria and Corydalis species growing in Turkey have been evaluated for their in vitro antiviral and antimicrobial activities. Both DNA virus Herpes simplex (HSV) and RNA virus Parainfluenza (PI-3) were employed for antiviral assessment of the compounds using Madine-Darby bovine kidney and Vero cell lines and their maximum non-toxic concentrations (MNTC) and cytopathogenic effects (CPE) were determined using acyclovir and oseltamivir as the references. Antibacterial and antifungal activities of the alkaloids were tested against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Bacillus subtilis, and Candida albicans by the microdilution method and compared to ampicilline, ofloxacine, and ketocanazole as the references. The alkaloids did not present any notable antibacterial effect, while they had significant antifungal activity at 8 microg/ml concentration. On the other hand, the alkaloids were found to have selective inhibition against the PI-3 virus ranging between 0.5 and 64 microg/ml as minimum and maximum CPE inhibitory concentrations, whereas they were completely inactive towards HSV.

Neuraminidase inhibitors as antiviral agents.

The enzyme neuraminidase (NA) is an attractive target for antiviral strategy because of its essential role in the pathogenicity of many respiratory viruses. NA removes sialic acid from the surface of infected cells and virus particles, thereby preventing viral self-aggregation and promoting efficient viral spread; NA also plays a role in the initial penetration of the mucosal lining of the respiratory tract. Random screening for inhibitors has identified only low-affinity and nonselective viral NA inhibitors. Selective, high-affinity inhibitors of influenza virus neuraminidase, zanamivir and oseltamivir, were developed using computer-aided design techniques on the basis of the three-dimensional structure of the influenza virus NA. These drugs were highly efficient in inhibiting replication of both influenza A and B viruses in vitro and in vivo and were approved for human use in 1999. Subsequently, the same structure-based design approach was used for the rational design of inhibitors of the parainfluenza virus hemagglutinin-neuraminidase (HN). One of these compounds, BCX 2798, effectively inhibited NA activity, cell binding, and growth of parainfluenza viruses in tissue culture and in the lungs of infected mice. Clinical reports indicate high efficiency of NA inhibitors for prophylaxis and treatment of influenza virus infection, good tolerance, and a low rate of emergence of drug-resistant mutants. Future experimental and clinical studies should establish the viability of NA inhibitors for the treatment of other respiratory virus infections.

Inhibition of respiratory viruses by nasally administered siRNA.

Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are two respiratory pathogens of paramount medical significance that exert high mortality. At present, there is no reliable vaccine or antiviral drug against either virus. Using an RNA interference (RNAi) approach, we show that individual as well as joint infection by RSV and PIV can be specifically prevented and inhibited by short interfering RNAs (siRNAs), instilled intranasally in the mouse, with or without transfection reagents. The degree of protection matched the antiviral activity of the siRNA in cell culture, allowing an avenue for quick screening of an efficacious siRNA. When targeting both viruses in a joint infection, excess of one siRNA moderated the inhibitory effect of the other, suggesting competition for the RNAi machinery. Our results suggest that, if properly designed, low dosages of inhaled siRNA might offer a fast, potent and easily administrable antiviral regimen against respiratory viral diseases in humans.

[Structure and antiviral activity of adamantane-containing polymer preparation]. / Struktura i antivirusnaia aktivnost' adamantansoderzhashchikh polimernykh preparatov.

New water-soluble antiviral chemical agents, containing 10 to 30% of adamantane derivatives (amino-, aminopropyl-adamantane-, aminomethyl- and rimantadine), which were conjugated with polycarboxylic matrixes of the divinyl ether and maleic anhydride copolymers (DIVEMA), were developed. The polymeric drugs exhibited a low cytotoxicity (4 to 10 times less than rimantadine) and a wide spectrum of antiviral activity against influenza viruses, including both the remantadine-resistant strains of A/PR/8/34 (H1N1) and the B/Saint-Petersburg strain/71/77 as well as against herpes viruses of type 1, parainfluenza viruses of types 1 and 3 and RS-virus. A reduction of the viral infection titer in their reproduction in sensitive cells' cultures was more than 2.0 Ig ID50. Complete inhibition of viral-specific syntheses, registered by immune-enzyme assay (IEA) and by hemagglutination test was observed at low infection doses ranging from 1 to 100 ID50. The efficiency of the antiviral effect depends on a drug's molecular weight and a structure of chemical bonds between the adamantane nucleus and the polymeric matrix.

Substituted benzimidazoles with nanomolar activity against respiratory syncytial virus.

A cell-based assay was used to discover compounds inhibiting respiratory syncytial virus (RSV)-induced fusion in HeLa/M cells. A lead compound was identified and subsequent synthesis of >300 analogues led to the identification of JNJ 2408068 (R170591), a low molecular weight (MW 395) benzimidazole derivative with an EC(50) (0.16 nM) against some lab strains almost 100,000 times better than that of ribavirin (15 microM). Antiviral activity was confirmed for subgroup A and B clinical isolates of human RSV and for a bovine RSV isolate. The compound did not inhibit the growth of representative viruses from other Paramyxovirus genera, i.e. HPIV2 and Mumps Virus (genus Rubulavirus), HPIV3 (genus Respirovirus), Measles virus (genus Morbillivirus) and hMPV. Efficacy in cytopathic effect inhibition assays correlated well with efficacy in virus yield reduction assays. A concentration of 10nM reduced RSV production 1000-fold in multi-cycle experiments, irrespective of the multiplicity of infection. Time of addition studies pointed to a dual mode of action: inhibition of virus-cell fusion early in the infection cycle and inhibition of cell-cell fusion at the end of the replication cycle. Two resistant mutants were raised and shown to have single point mutations in the F-gene (S398L and D486N). JNJ 2408068 was also shown to inhibit the release of proinflammatory cytokines IL-6, IL-8 and Rantes from RSV-infected A549 cells.

[Experimental study on effect of recombined decoction on mumps virus].

OBJECTIVE: To evaluate possible inactivating effect of recombined decoction in on mumps virus. METHODS: By adopting tissue cell culturing technology, a group of viruses including the mumps virus, herpes simplex virus (type I, II), rubella virus, cytomegalovirus (CMV), herpes zoster virus, influenza virus, parainfluenza virus, adeno viruses, respiratory syneytial virus (RSV) were cultured. The cells infected with the viruses were treated with the decoction. RESULTS: The decoction showed remarkable inhibitory and killing effects on the mumps virus while had no obvious inhibitory and killing effects on host's cells, herpes simplex virus (type I, II), rubella virus, cytomegalovirus (CMV), herpes zoster virus, influenza virus, parainfluenza virus, adeno viruses, respiratory syneytial virus (RSV). CONCLUSIONS: The decoction had obvious inhibitory and killing effects on mumps virus during single layer cells culture.

Y2, the smallest of the Sendai virus C proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral RNA synthesis.

An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C', C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-alpha/beta) and IFN-gamma and to inhibit viral transcription. The assay protocols included monitoring of IFN-alpha/beta-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-alpha/beta and IFN-gamma and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(-) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

Antiviral substance from silkworm faeces: characterization of its antiviral activity.

The antiviral activity of a substance (L4-1) purified from silkworm faeces was examined in an HVJ (Sendai virus)-LLC-MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4-1 is associated with active oxygen species produced from the substance. SDS-polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4-1.

Versatility of the accessory C proteins of Sendai virus: contribution to virus assembly as an additional role.

The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.

[Immunostimulating activity of a saponin-containing extract of Saponaria officinalis]. / Izuchenie immunostimuliruiushchei aktivnosti saponinsoderzhashchego ékstrakta Saponaria officinalis.

The immunostimulating activity of saponin-containing extract of Saponaria officinalis has been studied. Use of an S. officinalis extract in a concentration close to the critical concentration of saponin micella formation increased the immunogenicity of viral glycoproteins. The immunogenicity of glycoprotein complexes with S. officinalis was higher than the immunogenicity of intact virus and micellae of purified glycoproteins and was comparable to that of glycoprotein complexes with Quil A glycoside.

Impaired antiviral response in human hepatoma cells.

Hepatitis B, C, and D viruses can infect liver cells and in some individuals establish a chronic phase of infection. Presently, relatively little information is available on the antiviral mechanisms in liver cells. Because no good in vitro model infection systems for hepatitis viruses are available, we have used influenza A, Sendai, and vesicular stomatitis (VSV) viruses to characterize interferon (IFN) responses and IFN-induced antiviral mechanisms in human hepatoma cell lines. HepG2 or HuH7 cells did not show any detectable IFN-alpha/beta production in response to influenza A or Sendai virus infections. Treatment of cells with IFN-alpha resulted in upregulation of IFN-alpha-inducible Mx, 2',5'-oligoadenylate synthetase (OAS) and HLA class I gene expression but only with exceptionally high levels of IFN-alpha (>/=100 IU/ml). Accordingly, high pretreatment levels of IFN-alpha, 1000 IU/ml for influenza A and VSV and 100 IU/ml for Sendai virus, were required before any detectable antiviral activity against these viruses was seen. IFN-gamma had some antiviral effect against influenza A virus but appeared to be ineffective against VSV and Sendai virus. IFN-gamma upregulated HLA class I protein expression, whereas Mx or OAS expression levels were not increased. There was a modest upregulation of HLA class I expression during Sendai virus infection, whereas influenza A virus infection resulted, after an initial weak upregulation, in a clear decrease in HLA class I expression at late times of infection. The results suggest that hepatoma cells may have intrinsically poor ability to produce and respond to type I IFNs, which may contribute to their inability to efficiently resist viral infections.

Selective lysis of virus-infected cells by cobra snake cytotoxins: A sendai virus, human erythrocytes, and cytotoxin model.

By using a Sendai virus-human erythrocyte model, this work found that virus-infected cells were 10-fold more susceptible to lysis in two of five examined cobra venoms. Four cytotoxins were isolated from the venom of the cobra Naja nigricollis that also showed 10-fold higher cytotoxicity toward virus-infected cells than to untreated cells. As selective destruction of virus-infected cells is of immense importance in clinical practice, this work demonstrates the potential of cobra cytotoxins to serve as leading compounds for the generation of derivatives or fractions with high cytotoxic specificity toward virus-infected cells.

Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon-alpha/beta-mediated responses.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.

[Profile and effectiveness of a phytogenic combination preparation for treatment of sinusitis]. / Wirkprofil und Wirksamkeit eines pflanzlichen Kombinationspräparates zur Behandlung der Sinusitis.

Complex combinations of botanical drugs are usually regarded as non-rational medicines. In the case of Sinupret, a combination of 5 botanical drugs, the pharmacological profile was investigated thoroughly with respect to activities on secretion of the respiratory epithelium, with respect to anti-inflammatory activities and to effects against a murine Sendai-virus infection model (including in-vitro antiviral activities), after a randomised, placebo-controlled study of the ENT-clinic of the University of Freiburg had shown superiority of the verum in patients with chronic sinusitis. For patients with acute sinusitis, the additional treatment with Sinupret improved the response rate of the patients, who were basically treated with antibiotics and nasal decongestants, significantly compared to placebo. In a series of randomised trials of the combination versus active controls, the botanical combination was shown to be at least as effective and to have a low prevalence of adverse side effects. The individual ingredients contribute to the overall pharmacological profile of the combination with secretolytic, anti-inflammatory, immunomodulating and anti-viral effects.