Virus Infectivity Assays Research Articles

Investigation of the molecular characteristics of bisindole inhibitors as HIV-1 glycoprotein-41 fusion inhibitors

In previous work, we described 6-6‘-bisindole compounds targeting a hydrophobic pocket on the N-heptad repeat region of viral glycoprotein-41 as effective inhibitors of HIV-1 fusion. Two promising compounds with sub-micromolar IC50's contained a benzoic acid group and a benzoic acid ester attached at the two indole nitrogens. Here we have conducted a thorough structure-activity relationship (SAR) study evaluating the contribution of each of the ring systems and various substituents to compound potency. Hydrophobicity, polarity and charge were varied to produce 35 new compounds that were evaluated in binding, cell-cell fusion and viral infectivity assays. We found that (a) activity based solely on increasing hydrophobic content plateaued at ∼ 200 nM; (b) the bisindole scaffold surpassed other heterocyclic ring systems in efficacy; (c) a polar interaction possibly involving Gln575 in the pocket could supplant less specific hydrophobic interactions; and (d) the benzoic acid ester moiety did not appear to form specific contacts with the pocket. The importance of this hydrophobic group to compound potency suggests a mechanism whereby it might interact with a tertiary component during fusion, such as membrane. A promising small molecule 10b with sub-μM activity was discovered with molecular weight <500 da and reduced logP compared to earlier compounds. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion.

A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to cyprinid herpesvirus 2 infection with the induction of apoptosis

Herpesviral haematopoietic necrosis induced by cyprinid herpesvirus 2 (CyHV-2) in Carassius auratus gibelio has caused huge economic losses in aquaculture. To better characterize this pathogen at molecular and cellular levels, it is necessary to study its pathogenesis in a sustainable cell line. In this study, a new cell line (GiCF) from the caudal fin of Carassius auratus gibelio (C. a. gibelio) was established and characterised. Typical cytopathic effect represented by characteristic morphological changes was induced in the cells at the 7th day post infection with CyHV-2, which was isolated from diseased C. a. gibeli. The susceptibility of GiCF to CyHV-2 was further demonstrated by the presence of viral assembling factory in the cytoplasm unveiled by transmission electron microscopy, and viral protein synthesis through immunofluorescence and Western blot assays. Sustainable propagation of CyHV-2 in GiCF had been demonstrated by virus infection and titration assays in a consecutive manner. The virus titer reached 104.9±0.22 TCID50/ml, and infectious CyHV-2 was produced from the GiCF cells over 30 subcultures from the established cell lines. In addition, apoptosis was observed in the GiCF cells upon CyHV-2 infection by DAPI staining, TUNEL and Annexin V staining, and quantitative expression patterns of caspase genes suggested that caspase activation might involve in CyHV-2 induced apoptosis. In summary, a new cell line, GiCF, susceptible to CyHV-2 infection was established from caudal fin tissue of C. a. gibelio, and it should serve as an ideal infection platform to characterize CyHV-2 and its pathogenic effect including apoptosis.

Identification of an RNA silencing suppressor encoded by citrus vein enation virus

In order to know if any protein encoded by citrus vein enation virus (CVEV) might act as an RNA silencing suppressor (RSS), five open reading frames (ORF) of CVEV, including the coat protein (CP) gene, were individually cloned into the binary expression vector pBI121. The silencing suppression ability of the five ORFs was tested by ‘viral minireplicon infectivity assays’ based on a beet yellows virus (BYV) minireplicon carrying a reporter gene (BYVminiR-GFP). GFP imaging and microscopic analyses were used to observe whether RNA silencing suppression occurred or not. In addition, real-time quantitative RT-PCR was used to quantify the mRNA expression of GFP in the injected region. The fluorescence intensity of leaves with CP expression was stronger than that of the empty vector negative control, but it was weaker than that of the positive control HC-Pro of tobacco etch virus (TEV), indicating that the CP of CVEV partly inhibited the GFP gene silencing. Co-expression of each of the other four ORFs with BYVminiR-GFP showed an almost complete quenching of GFP fluorescence in infiltrated areas, similar to that of the negative control. The amount of GFP mRNA in the leaves co-expressed with CP was nearly twice as much as that of either the negative control or other ORFs. These results suggest that CP of CVEV is a weak gene silencing suppressor.

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays

Many biological assays are employed in virology to quantify parameters of interest. Two such classes of assays, virus quantification assays (VQAs) and infectivity assays (IAs), aim to estimate the number of viruses present in a solution and the ability of a viral strain to successfully infect a host cell, respectively. VQAs operate at extremely dilute concentrations, and results can be subject to stochastic variability in virus-cell interactions. At the other extreme, high viral-particle concentrations are used in IAs, resulting in large numbers of viruses infecting each cell, enough for measurable change in total transcription activity. Furthermore, host cells can be infected at any concentration regime by multiple particles, resulting in a statistical multiplicity of infection and yielding potentially significant variability in the assay signal and parameter estimates. We develop probabilistic models for statistical multiplicity of infection at low and high viral-particle-concentration limits and apply them to the plaque (VQA), endpoint dilution (VQA), and luciferase reporter (IA) assays. A web-based tool implementing our models and analysis is also developed and presented. We test our proposed new methods for inferring experimental parameters from data using numerical simulations and show improvement on existing procedures in all limits.

Binding of Escherichia coli Does Not Protect Tulane Virus from Heat-Inactivation Regardless the Expression of HBGA-Like Molecules.

Histo-blood group antigens (HBGAs) are considered as receptors/co-receptors for human norovirus (HuNoV). It has been reported that binding of HuNoV-derived virus-like particles (VLPs) to HBGA-like molecules-expressing bacteria increased the stability of VLPs to heat-denaturation (HD). In this study, we tested for HBGA-like-binding-conveyed protection against HD on viral replication using Tulane virus (TV) and Escherichia coli O86:H2 (O86:H2), with E. coli K-12 (K-12) used as a control. Expression of HBGA type B was confirmed by ELISA in O86:H2 but not in K-12. Binding of TV was confirmed by ELISA in O86:H2 (P/N = 2.23) but not in K-12 (P/N = 1.90). Pre-incubation of TV with free HBGA could completely inhibit its ability to bind to O86:H2 (p = 0.004), while producing no significant change in its ability to bind K-12 (p = 0.635). We utilized a bacterial-capture-RT-qPCR procedure to confirm that both bacterial strains were capable of binding TV, and that O86:H2 exhibited fivefold greater binding capacity than K-12. Pre-incubation of TV with free HBGA would partially inhibit the binding of TV to O86:H2 (p = 0.047). In contrast, not only did pre-incubation of TV with free HBGA not inhibit the binding of TV to K-12, binding was slightly enhanced (p = 0.13). The viral infectivity assay allowed us to conduct a direct evaluation of the ability of HBGA-like-bound bacteria to confer HD protection to TV. Prior to inoculate to LLC-MK2 cells, TV was incubated with each bacterial strain at ratios of 1:0, 1:1 and 100:1, then both partially and fully HD. The viral amplification was quantitated by RT-qPCR 48 h later. The binding of bacteria to TV reduced viral replication in a dose-dependent matter. We found that neither bound O86:H2 nor K-12 conferred protection of TV against partial or full HD conditions. Partial HD reduction of viral replication was not significantly impacted by the binding of either bacterial strain, with infectivity losses of 99.03, 99.42, 96.32, 96.10, and 98.88% for TV w/o bacteria, TV w/O86:H2 (1:1), TV w/O86:H2 (100:1), TV w/K-12 (1:1), and TV w/K-12 (100:1), respectively. Full HD reduction of viral replication was not impacted by the binding of either bacterial strain, as full loss of infectivity was observed in all cases.

Plasmonic Enhancement of Selective Photonic Virus Inactivation

Femtosecond (fs) pulsed laser irradiation techniques have attracted interest as a photonic approach for the selective inactivation of virus contaminations in biological samples. Conventional pulsed laser approaches require, however, relatively long irradiation times to achieve a significant inactivation of virus. In this study, we investigate the enhancement of the photonic inactivation of Murine Leukemia Virus (MLV) via 805 nm femtosecond pulses through gold nanorods whose localized surface plasmon resonance overlaps with the excitation laser. We report a plasmonically enhanced virus inactivation, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers ≥0.3 W. Importantly, the fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. Based on the observations that physical contact between nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation, a model of virus inactivation based on plasmon enhanced shockwave generation is proposed.

Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China

ObjectiveNew rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China. MethodsThe antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells. ResultsWe found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67. ConclusionHydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.

Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells.

Exosomes, 30–200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies.

Viral dynamics of persistent hepatitis C virus infection in high-sensitive reporter cells resemble patient's viremia.

Hepatitis C virus (HCV) infection has a high persistence rate in patients. Although immune cells play a central role in determining the outcomes of HCV infection, the liver is crucial in controlling HCV activity from acute to chronic stages. This investigation grew HCV from a long-term cell culture, and provided an experimental model for studies on HCV persistence in hepatocytes. Huh7.5 cells implanted with the NS3/4 protease-based secreted alkaline phosphatase (SEAP) reporter were infected with JFH-1 HCV (moiety of infection=0.01) and incubated for over 130 days. The viral activity was obtained by sampling supernatant continuously for SEAP activity measurement. Combined with extracellular and intracellular HCV-RNAs and viral infectivity assays, the experimental results exhibited invitro viral dynamics resembling the patients' viremia pattern from acute to chronic infections. The HCV in acute infection comprised exponential accumulation (week 1), plateau (week 2), declining production (weeks 3-4) and silencing (weeks 5-14) phases, and were then reactivated at the onset of chronic infection (after week 15). The HCV-infected cells grew more slowly than the mock controls, and exhibited a prominent decrease of cell growth rate and increase of early apoptosis in the declining-to-silencing phase transition, suggesting that fitness selection might occur as the infected cells moved across the boundary of active to occult viral activity. Cultivated HCV in the highly sensitive NS3/4-based SEAP reporter cells could establish persistence, which might mimic the viral dynamics from acute to chronic infections in hepatitis C patients.

HIF-1α binding to AEG-1 promoter induced upregulated AEG-1 expression associated with metastasis in ovarian cancer.

Ovarian cancer with the highest mortality rate among gynecological malignancies is one of common cancers among female cancer patients. As reported in recent years, AEG‐1 was associated with the occurrence, development, and metastasis of ovarian cancer, but the mechanisms remain unclear. In the current study, invasion capabilities of ovarian cancer OVCAR3 cells were measured by viral infection and transwell assay. Western blot analysis was used to evaluate the expression levels of β‐catenin, E‐cadherin, MMP2, and MMP9. With qRT‐PCR analysis, AEG‐1 and HIF‐1α gene expression were detected. We used luciferase reporter gene to measure AEG‐1 promoter activity under normoxia/hypoxia in OVCAR3 cells. Our work demonstrated that AEG‐1 significantly enhanced invasion capabilities of OVCAR3 cells and the expression levels of β‐catenin, E‐cadherin, MMP2, and MMP9 associated with invasion capabilities of OVCAR3 cells were upregulated. Furthermore, hypoxia enhanced invasion capabilities of OVCAR3 cells and induced AEG‐1 high gene expression, which was reversed by AEG‐1 knockdown lentivirus. HIF‐1α expression upregulation was induced in OVCAR3 cells after hypoxia. HIF‐1α knockdown lentivirus induced downregulated expression of AEG‐1 and invasion capabilities of OVCAR3 cells were also inhibited. Wild‐type AEG‐1 promoter activity under hypoxic conditions was significantly higher than that AEG‐1 mutation under normoxic conditions in the absence of hypoxia response. Our results suggested that HIF‐1α binds to AEG‐1 promoter to upregulate its expression, which was correlated with metastasis in ovarian cancer by inducing the expression of MMP2 and MMP9 as well as inhibiting expression of E‐cadherin and β‐catenin.

Antiviral activity of self-assembled glycodendro[60]fullerene monoadducts.

A series of amphiphilic glycodendro[60]fullerene monoadducts were efficiently synthesized using the CuAAC "click chemistry" approach. These glycodendrofullerenes can self-assemble in aqueous media, in a process favoured through π-π interactions between the [60]fullerene moieties. This aggregation process leads to big and well-defined compact micelles with a uniform size and spherical-shape. The supramolecular aggregates were characterized using electronic microscopy (SEM and TEM), light scattering methods (DLS) and X-ray methodologies (SAXS and XRD). The antiviral efficiency of these aggregates has been tested in an experimental infection assay using Ebola virus glycoprotein (EboGP) pseudotyped viral particles on Jurkat cells overexpressing DC-SIGN and an improvement in the IC50 value with respect to other systems endowed with a higher number of carbohydrate ligands is observed.

Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases

Porcine epidemic diarrhea coronavirus (PEDV) is currently devastating the United States pork industry by causing an 80–100% fatality rate in infected piglets. Coronavirus spike proteins mediate virus entry into cells, a process that requires the spike proteins to be proteolytically activated. It has been a conundrum which proteases activate PEDV entry. Here we systematically investigated the roles of different proteases in PEDV entry using pseudovirus entry, biochemical, and live virus infection assays. We found that the PEDV spike is activated by lysosomal cysteine proteases but not proprotein convertases or cell surface serine proteases. Extracellular trypsin activates PEDV entry when lysosomal cysteine proteases are inhibited. We further pinpointed cathepsin L and cathepsin B as the lysosomal cysteine proteases that activate the PEDV spike. These results advance our understanding of the molecular mechanism for PEDV entry and identify potential antiviral targets for curbing the spread of PEDV.

Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity

IntroductionPrimary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. MethodsWe isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. ResultsWe found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. DiscussionWhen compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.

Bat Severe Acute Respiratory Syndrome-Like Coronavirus WIV1 Encodes an Extra Accessory Protein, ORFX, Involved in Modulation of the Host Immune Response.

Bats harbor severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) from which the causative agent of the 2002-2003 SARS pandemic is thought to have originated. However, despite the fact that a large number of genetically diverse SL-CoV sequences have been detected in bats, only two strains (named WIV1 and WIV16) have been successfully cultured in vitro These two strains differ from SARS-CoV only in containing an extra open reading frame (ORF) (named ORFX), between ORF6 and ORF7, which has no homology to any known protein sequences. In this study, we constructed a full-length cDNA clone of SL-CoV WIV1 (rWIV1), an ORFX deletion mutant (rWIV1-ΔX), and a green fluorescent protein (GFP)-expressing mutant (rWIV1-GFP-ΔX). Northern blotting and fluorescence microscopy indicate that ORFX was expressed during WIV1 infection. A virus infection assay showed that rWIV1-ΔX replicated as efficiently as rWIV1 in Vero E6, Calu-3, and HeLa-hACE2 cells. Further study showed that ORFX could inhibit interferon production and activate NF-κB. Our results demonstrate for the first time that the unique ORFX in the WIV1 strain is a functional gene involving modulation of the host immune response but is not essential for in vitro viral replication. Bats harbor genetically diverse SARS-like coronaviruses (SL-CoVs), and some of them have the potential for interspecies transmission. A unique open reading frame (ORFX) was identified in the genomes of two recently isolated bat SL-CoV strains (WIV1 and -16). It will therefore be critical to clarify whether and how this protein contributes to virulence during viral infection. Here we revealed that the unique ORFX is a functional gene that is involved in the modulation of the host immune response but is not essential for in vitro viral replication. Our results provide important information for further exploration of the ORFX function in the future. Moreover, the reverse genetics system we constructed will be helpful for study of the pathogenesis of this group of viruses and to develop therapeutics for future control of emerging SARS-like infections.

Identifying possible sites for antibody neutralization escape: Implications for unique functional properties of the C-terminal tail of Human Immunodeficiency Virus Type 1 gp41

A previous amino acid sequence analyses from our laboratory reported nine potential sites in gp41 glycoprotein of HIV-1 that may contribute to virus escape from antibody neutralization. Besides four sites found outside the membrane of HIV-1 virus, five located in the C-terminal tail of gp41 specifically in the lentivirus lytic peptides motifs (LLPs). To further study the bioinformatical results, the virus infectivity assay and the standard neutralization assay were conducted on conservatively mutated virus. Two sites in the LLP3 domain stood out with the ability to alter the resistance of HIV-1 virus to certain broadly neutralizing antibodies (bNAbs). While the glycoprotein incorporation on the viral membrane and the interaction of the LLP3 domain with the lipid membrane remained unaltered, the increase in neutralization resistance of the mutant virus was associated with the changes on Env conformation. Our findings demonstrate different sensibility of bNAbs to mutations in the C-terminal tail and indicate an unrecognized potential role for even minor sequence variation in the C-terminal tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

Epithelial cell lines of the cotton rat (Sigmodon hispidus) are highly susceptible in vitro models to zoonotic Bunya-, Rhabdo-, and Flaviviruses.

BackgroundSmall mammals such as bats and rodents have been increasingly recognized as reservoirs of novel potentially zoonotic pathogens. However, few in vitro model systems to date allow assessment of zoonotic viruses in a relevant host context. The cotton rat (Sigmodon hispidus) is a New World rodent species that has a long-standing history as an experimental animal model due to its unique susceptibility to human viruses. Furthermore, wild cotton rats are associated with a large variety of known or potentially zoonotic pathogens.MethodsA method for the isolation and culture of airway epithelial cell lines recently developed for bats was applied for the generation of rodent airway and renal epithelial cell lines from the cotton rat. Continuous cell lines were characterized for their epithelial properties as well as for their interferon competence. Susceptibility to members of zoonotic Bunya-, Rhabdo-, and Flaviviridae, in particular Rift Valley fever virus (RVFV), vesicular stomatitis virus (VSV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV) was tested. Furthermore, novel arthropod-derived viruses belonging to the families Bunya-, Rhabdo-, and Mesoniviridae were tested.ResultsWe successfully established airway and kidney epithelial cell lines from the cotton rat, and characterized their epithelial properties. Cells were shown to be interferon-competent. Viral infection assays showed high-titre viral replication of RVFV, VSV, WNV, and TBEV, as well as production of infectious virus particles. No viral replication was observed for novel arthropod-derived members of the Bunya-, Rhabdo-, and Mesoniviridae families in these cell lines.ConclusionIn the current study, we showed that newly established cell lines from the cotton rat can serve as host-specific in vitro models for viral infection experiments. These cell lines may also serve as novel tools for virus isolation, as well as for the investigation of virus-host interactions in a relevant host species.

Immunological and biochemical characterizations of coxsackievirus A6 and A10 viral particles

Childhood exanthema caused by different serotypes of coxsackievirus (CV-A) and enterovirus A71 (EV-A71) has become a serious global health problem; it is commonly known as hand, foot, and mouth disease (HFMD). Current EV-A71 vaccine clinical trials have demonstrated that human antibody responses generated by EV-A71 vaccinations do not cross-neutralize coxsackievirus A16 (CV-A16). An effective multivalent HFMD vaccine is urgently needed. From molecular epidemiological studies in Southeast Asia, CV-A6 and CV-A10 are commonly found in HFMD outbreaks. In this study, CV-A6 and CV-A10 were individually cultured in rhabdomyosarcoma (RD) cells grown in medium containing serum, harvested and concentrated. In viral downstream purification, two viral fractions were separated by sucrose gradient zonal ultracentrifugation and detected using a SDS-PAGE analysis and a virus infectivity assay. These two viral fractions were formalin-inactivated, and only the infectious particle fraction was found to be capable of inducing CV-A serotype-specific neutralizing antibody responses in animal immunogenicity studies. These mouse and rabbit antisera also failed to cross-neutralize EV-A71 and CV-A16 infections. Only a combination of formalin-inactivated EV-A71, CV-A6, CV-A10 and CV-A16 multivalent vaccine candidates elicited cross-neutralizing antibody responses in both mouse and rabbit immunogenicity studies. The current results certainly provide important information for multivalent HFMD vaccine development.

New Approach for Inhibition of HIV Entry: Modifying CD4 Binding Sites by Thiolated Pyrimidine Derivatives.

Thiolated pyrimidine derivatives have been synthetized and their antiretroviral effect against human immunodeficiency virus type 1 (HIV-1IIIB) and HIV-1 chimeric pseudovirions have been quantitatively determined in cell-based viral infectivity assays including syncytium inhibition assay as well as a single-cycle viral infection assay on HeLaCD4-LTR/ß-gal cells. Pseudotype virions prepared bearing HIV-1 envelope preference for CCR5 coreceptor, CXCR4 coreceptor or for both, respectively, with a HIV-1 core containing luciferase reporter gene were able to infect susceptible cells but are replication defective so unable to replicate in the cells . Data indicate that thiolated pyrimidine derivatives inhibited effectively virally induced cell fusion in vitro as well as infectivity of primary HIV-1IIIB strain and HIV-1 pseudovirions using chemokine receptors CCR5 or CXCR4 or both for virus entry a dose dependent manner. Inhibition was selective, depended on the pseudovirus coreceptor preference. Our results suggest that some of these sulfur containing pyrimidines interact with redoxactive -SH groups required for successful HIV entry, including a redox active disulfide in the CD4 molecule as well as -SH groups in HIV viral envelope gp120. This mode of action is unique representing a new class of potential HIV entry inhibitors.

Effective concentration, recovery, and detection of infectious adenoviruses from environmental waters

Current application of fecal indicator bacteria (FIB) as an indicator of sewage contamination has its limitation and other alternative indicator systems are needed for enhanced monitoring of recreational water quality. To explore the potential use of human enteric viruses (HEV) as a potential indicator, human adenovirus type 7 (HAdV-7) was tested in this study as a model virus. Through spiked HAdV-7 into the sample seawater, different procedures for viral concentration and elution were comparatively analyzed in a side-by-side fashion. Nearly 80% infectious viruses were recovered from the spiked seawater with the use of a magnetic stirring method and 1.0mM NaOH as elution buffer. A viral plaque assay was established with optimized conditions for the detection and quantification of infectious HAdV and used to determine the stability of HAdV-7 in seawater and a possible correlation for HAdV detection between the viral infectivity assay and PCR. Findings from this study suggest it is possible to concentrate and recover infectious HAdV from environmental waters effectively with optimized laboratory conditions, which warrants the future test and establishment of using HEV as an alternative indicator of water quality.

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution.