Mitigating the risk of Zika virus contamination of raw materials and cell lines in the manufacture of biologicals.

Ensuring the virological safety of biologicals is challenging due to the risk of viral contamination of raw materials and cell banks, and exposure during in-process handling to known and/or emerging viral pathogens. Viruses may contaminate raw materials and biologicals intended for human or veterinary use and remain undetected until appropriate testing measures are employed. The outbreak and expansive spread of the mosquito-borne flavivirus Zika virus (ZIKV) poses challenges to screening human- and animal -derived products used in the manufacture of biologicals. Here, we report the results of an in vitro study where detector cell lines were challenged with African and Asian lineages of ZIKV. We demonstrate that this pathogen is robustly detectable by in vitro assay, thereby providing assurance of detection of ZIKV, and in turn underpinning the robustness of in vitro virology assays in safety testing of biologicals.

Schmallenberg virus, an emerging viral pathogen of cattle and sheep and a potential contaminant of raw materials, is detectable by classical in-vitro adventitious virus assays.

Emerging viruses, as potential contaminants of raw materials used in the manufacture of biologicals represent a challenge in the safety testing of biopharmaceutical products intended for human or veterinary use. Here, we report the challenge of an in vitro adventitious virus platform used in safety testing of biologicals, where a broad panel of detector cell lines was challenged to provide evidence that Schmallenberg virus is detectable by a classical reporting endpoint of cytopathic effect with Vero, BHK-21 and CHO-K1 detector cells, within typical in vitro assay timescales. We conclude that Schmallenberg virus is robustly detectable by classical in vitro viral biosafety assays.

TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase. In vitro combination studies with an HCV NS3/4A protease inhibitor demonstrated potent suppression of HCV RNA replication, confirming the potential for combination of these two classes in the treatment of chronic HCV infection. TMC647055 is a potent nonnucleoside NS5B polymerase inhibitor of HCV replication with a promising in vitro biochemical, kinetic, and virological profile that is currently undergoing clinical evaluation.

Herpes simplex virus type 1 penetrates the basement membrane in human nasal respiratory mucosa.

BACKGROUND: Herpes simplex virus infections are highly prevalent in humans. However, the current therapeutics suffer important drawbacks such as limited results in neonates, increasing occurrence of resistance and impeded treatment of stromal infections. Remarkably, interactions of herpesviruses with human mucosa, the locus of infection, remain poorly understood and the underlying mechanisms in stromal infection remain controversial. METHODOLOGY/PRINCIPAL FINDINGS: A human model consisting of nasal respiratory mucosa explants was characterised. Viability and integrity were examined during 96 h of cultivation. HSV1-mucosa interactions were analysed. In particular, we investigated whether HSV1 is able to reach the stroma. Explant viability and integrity remained preserved. HSV1 induced rounding up and loosening of epithelial cells with very few apoptotic and necrotic cells observed. Following 16-24 h of infection, HSV1 penetrated the basement membrane and replicated in the underlying lamina propria. CONCLUSIONS/SIGNIFICANCE: This human explant model can be used to study virus-mucosa interactions and viral mucosal invasion mechanisms. Using this model, our results provide a novel insight into the HSV1 stromal invasion mechanism and for the first time directly demonstrate that HSV1 can penetrate the basement membrane.

Resistance to raltegravir highlights integrase mutations at codon 148 in conferring cross-resistance to a second-generation HIV-1 integrase inhibitor.

Raltegravir is the first integrase strand-transfer inhibitor (INSTI) approved for use in highly active antiretroviral therapy (HAART) for the management of HIV infection. Resistance to antiretrovirals can compromise the efficacy of HAART regimens. Therefore it is important to understand the emergence of resistance to RAL and cross-resistance to other INSTIs including potential second-generation INSTIs such as MK-2048. We have now studied the question of whether in vitro resistance selection (IVRS) with RAL initiated with viruses derived from clinical isolates would result in selection of resistance mutations consistent with those arising during treatment regimens with HAART containing RAL. Some correlation was observed between the primary mutations selected in vitro and during therapy, initiated with viruses with identical IN sequences. Additionally, phenotypic cross-resistance conferred by specific mutations to RAL and MK-2048 was quantified. N155H, a RAL-associated primary resistance mutation, was selected after IVRS with MK-2048, suggesting similar mechanisms of resistance to RAL and MK-2048. This was confirmed by phenotypic analysis of 766 clonal viruses harboring IN sequences isolated at the point of virological failure from 106 patients on HAART (including RAL), where mutation Q148H/K/R together with additional secondary mutations conferred reduced susceptibility to both RAL and MK-2048. A homology model of full length HIV-1 integrase complexed with viral DNA and RAL or MK-2048, based on an X-ray structure of the prototype foamy virus integrase-DNA complex, was used to explain resistance to RAL and cross-resistance to MK-2048. These findings will be important for the further discovery and profiling of next-generation INSTIs.

Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with first- and second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTI-bound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/A mutations in drug-resistant viral strains.

Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha.

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

Liver cell lines for the study of hepatocyte functions and immunological response.

BACKGROUND: Liver cell lines closely resembling primary hepatocyte are essential for research on hepatitis viruses and hepatocyte function. Currently used cell lines are derived from hepatic tumours and have altered gene expression. AIMS: The generation and characterisation of novel human hepatocyte lines (HHLs) derived from healthy human liver, retaining the primary hepatocyte phenotype. RESULTS: Primary hepatocytes were immortalised with Moloney's mouse leukaemia virus expressing E6 and E7 proteins of human papillomavirus, and cultures propagated long-term. All HHLs contained markers of hepatocyte and biliary phenotype (cytokeratins 7, 8, 18 and 19), Cytochrome P450 and albumin. The HHLs did not express high levels of p53 or alpha-fetoprotein. When grown in a collagen sandwich culture, or at the air-liquid interface, HHLs were maintained as monolayer whereas Huh-7 and HepG2 formed thick layers. All HHLs showed increased capacity to bind recombinant hepatitis C virus-like particles in comparison with Huh-7 and HepG2. We also demonstrate that HHLs contained active gap junctions, and that the cells respond to stimulation with IFN-alpha by upregulation of major histocompatibility complex (MHC)-I and -II. CONCLUSIONS: These HHLs retain primary hepatocyte phenotype and should be useful for investigating mechanisms of entry and replication of hepatotropic viruses, and should also be valuable in the study of hepatocyte biology and pathology.

Bunyamwera virus nonstructural protein NSs counteracts interferon regulatory factor 3-mediated induction of early cell death.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.

Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles.

Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2(660)) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2(660) with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2(660) but not VLPs or FL E1E2 complex. The antigenic differences between E2(660,) FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2.

Structure-function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2(660)) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein-CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396-407, 412-423 and 528-535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432-443, 436-443 and 436-447 inhibited the interaction of VLPs, but not of E2(660) or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure-function analysis.

Evidence for structural differences in the S domain of L in comparison with S protein of hepatitis B virus.

The structures of the large (L), middle (M) and small (S) versions of the envelope proteins of hepatitis B virus remain poorly characterized due to the complex nature of their conformations. Several groups have proposed transmembrane topological models depicting the lumenally and cytosolically disposed regions of these proteins. Recently, post-translational topological changes in L have been described. However, no overall differences in the topology of the S domains of the L or M, to the S protein are predicted. In this report, we investigated a previously uncharacterized anti-S monoclonal antibody (MAb), 6B1, which recognizes a conformation-sensitive epitope in S. Unlike other anti-S MAbs tested, this MAb did not recognize its epitope in the S domain of L protein. Interestingly, however, the M protein was efficiently recognized. This unique characteristic of MAb 6B1 has allowed us to study the intracellular distribution of L and S proteins. In cells expressing both L and S, L re-localized from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to the membrane-associated distribution of S protein indicating that L and S interact with each other. This was confirmed by immunoprecipitation assays, which also showed that the interaction between L and S results in the secretion of L protein from cells. Overall, the ability of MAb 6B1 to selectively recognize S and M, but not L, strongly points to the existence of significant topological differences in the S domain of L. The availability of this important reagent should help further our understanding of the structure of HBV surface antigens.