- Volume 95, Issue 9, 2014

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-β cytokines.

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. Here, we report a recombinant PR8 influenza virus with the RSV fusion (F) protein epitopes of the subgroup A gene inserted into the influenza virus non-structural (NS) gene (rFlu/RSV/F) that was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F was also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line and CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific haemagglutination inhibition antibody responses against the PR8 influenza virus and RSV neutralization test proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited T helper type 1-dominant cytokine profiles against the RSV strain A2 virus. Taken together, our findings suggested that rFlu/RSV/F was immunogenic in vivo and warrants further development as a promising candidate vaccine.

Virologic surveillance is a critical component of measles management. One of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles is yet to be eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very active in China, providing a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a timespan of nearly two decades. Phylogenetic analysis performed on the 450 nt coding sequence for the C-terminal 150 amino acids of the nucleoprotein (N-450), fusion (F) gene and haemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence of selective pressure for the H protein was found. The substitution rates ranged from 0.75×10−3 substitutions site−1 year−1 for H to 1.65×10−3 substitutions site−1 year−1 for N-450. The time of most recent common ancestor (TMRCA) for genotype H1 was estimated as approximately 1985 (95 % highest probability density, 1979–1989). Finally, the overall diversity of measles sequences from China decreased from 2005 to 2012, coincident with a substantial decrease in measles cases. The results suggest that detailed evolutionary analyses should facilitate the documentation of eventual measles elimination in China. Moreover, the molecular approaches used in this study can be applied in other countries approaching measles elimination.

Cholesterol is a critical component of the hepatitis C virus (HCV) life cycle, as demonstrated by its accumulation within infected hepatocytes and lipoviral particles. To cope with excess cholesterol, hepatic enzymes ACAT1 and ACAT2 produce cholesteryl esters (CEs), which are destined for storage in lipid droplets or for secretion as apolipoproteins. Here we demonstrate in vitro that cholesterol accumulation following HCV infection induces upregulation of the ACAT genes and increases CE synthesis. Analysis of human liver biopsy tissue showed increased ACAT2 mRNA expression in liver infected with HCV genotype 3, compared with genotype 1. Inhibiting cholesterol esterification using the potent ACAT inhibitor TMP-153 significantly reduced production of infectious virus, but did not inhibit virus RNA replication. Density gradient analysis showed that TMP-153 treatment caused a significant increase in lipoviral particle density, suggesting reduced lipidation. These data suggest that cholesterol accumulation following HCV infection stimulates the production of CE, a major component of lipoviral particles. Inhibition of CE synthesis reduces HCV particle density and infectivity, suggesting that CEs are required for optimal infection of hepatocytes.

To initiate infections, many coronaviruses use sialic acids, either as receptor determinants or as attachment factors helping the virus find its receptor underneath the heavily glycosylated mucus layer. In the present study, the role of sialic acids in serotype I feline enteric coronavirus (FECV) infections was studied in feline intestinal epithelial cell cultures. Treatment of cells with neuraminidase (NA) enhanced infection efficiency, showing that terminal sialic acid residues on the cell surface were not receptor determinants and even hampered efficient virus–receptor engagement. Knowing that NA treatment of coronaviruses can unmask viral sialic acid binding activity, replication of untreated and NA-treated viruses was compared, showing that NA treatment of the virus enhanced infectivity in untreated cells, but was detrimental in NA-treated cells. By using sialylated compounds as competitive inhibitors, it was demonstrated that sialyllactose (2,6-α-linked over 2,3-α-linked) notably reduced infectivity of NA-treated viruses, whereas bovine submaxillary mucin inhibited both treated and untreated viruses. In desialylated cells, however, viruses were less prone to competitive inhibition with sialylated compounds. In conclusion, this study demonstrated that FECV had a sialic acid binding capacity, which was partially masked by virus-associated sialic acids, and that attachment to sialylated compounds could facilitate enterocyte infections. However, sialic acid binding was not a prerequisite for the initiation of infection and virus–receptor engagement was even more efficient after desialylation of cells, indicating that FECV requires sialidases for efficient enterocyte infections.

White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20–29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

While chickens are an important reservoir for emerging pathogens such as avian influenza viruses, little is known about the diversity of picornaviruses in poultry. We discovered a previously unknown diversity of picornaviruses in chickens in Hong Kong. Picornaviruses were detected in 87 cloacal and 7 tracheal samples from 93 of 900 chickens by reverse transcription-PCR, with their partial 3Dpol gene sequences forming five distinct clades (I to V) among known picornaviruses. Analysis of eight genomes from different clades revealed seven different picornaviruses, including six novel picornavirus species (ChPV1 from clade I, ChPV2 and ChPV3 from clade II, ChPV4 and ChPV5 from clade III, ChGV1 from clade IV) and one existing species (Avian encephalomyelitis virus from clade V). The six novel chicken picornavirus genomes exhibited distinct phylogenetic positions and genome features different from related picornaviruses, supporting their classification as separate species. Moreover, ChPV1 may potentially belong to a novel genus, with low sequence homologies to related picornaviruses, especially in the P1 and P2 regions, including the predicted L and 2A proteins. Nevertheless, these novel picornaviruses were most closely related to picornaviruses of other avian species (ChPV1 related to Passerivirus A, ChPV2 and ChPV3 to Avisivirus A and Duck hepatitis A virus, ChPV4 and ChPV5 to Melegrivirus A, ChGV1 to Gallivirus A). Furthermore, ChPV5 represented a potential recombinant picornavirus, with its P2 and P3 regions possibly originating from Melegrivirus A. Chickens are an important reservoir for diverse picornaviruses that may cross avian species barriers through mutation or recombination.

Human cardioviruses or Saffold viruses (SAFVs) of the family Picornaviridae are newly emerging viruses whose genetic and phenotypic diversity are poorly understood. We report here the full genome sequence of 11 SAFV genotypes from Pakistan and Afghanistan, along with a re-evaluation of their genetic diversity and recombination. We detected 88 SAFV from stool samples of 943 acute flaccid paralysis cases using reverse transcriptase-PCR targeting the 5′ untranslated region (UTR). Further characterization based on complete VP1 analysis revealed 71 SAFVs belonging to 11 genotypes, including three previously unidentified genotypes. SAFV showed high genetic diversity and recombination based on phylogenetic, pairwise distance distributions and recombination mapping analyses performed herein. Phylogenies based on non-structural and UTRs were highly incongruent indicating frequent recombination events among SAFVs. We improved the SAFV genotyping classification criteria by determining new VP1 thresholds based on the principles used for the classification of enteroviruses. For genotype assignment, we propose a threshold of 23 and 10 % divergence for VP1 nucleotide and amino acid sequences, respectively. Other members of the species Theilovirus, such as Thera virus and Theiler’s murine encephalomyelitis virus, are difficult to classify in the same species as SAFV, because they are genetically distinct from SAFV, with 41–56 % aa pairwise distances. The new genetic information obtained in this study will improve our understanding of the evolution and classification of SAFV.

Here, we report the isolation and functional characterization of mAbs against two murine norovirus (MNV) strains, MNV-1 and WU20, which were isolated following oral infection of mice. The mAbs were screened for reactivity against the respective homologous and heterologous MNV strain by ELISA. Selected mAbs were of IgA, IgG1, IgG2a or IgG2b isotype and showed a range of Western blot reactivities from non-binding to strong binding, suggesting recognition of conformational and linear epitopes. Some of the anti-MNV-1 antibodies neutralized both MNV-1 and WU20 infections in culture and in mice, but none of the anti-WU20 mAbs neutralized either virus. The non-neutralizing anti-MNV-1 IgG2b antibody 5C4.10 was mapped to the S domain of the MNV-1 capsid, whilst the epitopes of the neutralizing anti-MNV-1 IgA antibodies 2D3.7 and 4F9.4 were mapped to the P domain. Generation of neutralization escape viruses showed that two mutations (V339I and D348E) in the C′D′ loop of the MNV-1 P domain mediated escape from mAb 2D3.7 and 4F9.4 neutralization. These findings broaden the known neutralizing epitopes of MNV to the main surface-exposed loops of the P domain. In addition, the current panel of antibodies provides valuable reagents for studying norovirus biology and development of diagnostic tools.

The genus Flavivirus includes some of the most important human viral pathogens, and its members are found in all parts of the populated world. The temporal origin of diversification of the genus has long been debated due to the inherent problems with dating deep RNA virus evolution. A generally accepted hypothesis suggests that Flavivirus emerged within the last 10 000 years. However, it has been argued that the tick-borne Powassan flavivirus was introduced into North America some time between the opening and closing of the Beringian land bridge that connected Asia and North America 15 000–11 000 years ago, indicating an even older origin for Flavivirus. To determine the temporal origin of Flavivirus, we performed Bayesian relaxed molecular clock dating on a dataset with high coverage of the presently available Flavivirus diversity by combining tip date calibrations and internal node calibration, based on the Powassan virus and Beringian land bridge biogeographical event. Our analysis suggested that Flavivirus originated ~85 000 (64 000–110 000) or 120 000 (87 000–159 000) years ago, depending on the circumscription of the genus. This is significantly older than estimated previously. In light of our results, we propose that it is likely that modern humans came in contact with several members of the genus Flavivirus much earlier than suggested previously, and that it is possible that the spread of several flaviviruses coincided with, and was facilitated by, the migration and population expansion of modern humans out of Africa.

We demonstrated previously that immunization with a DNA vaccine expressing the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection through a poorly neutralizing antibody response. Here, we further investigated the role of the IgG subclass in this antibody-dependent protection using cytokine co-immunization and cytokine-deficient mice. A significant difference in IgG2a/c but not IgG1 was observed between mice that survived or died following a lethal challenge. Correspondingly, the IgG2a/c response and protection increased in IL-4-deficient mice but decreased in IFN-γ-deficient mice, highlighting the importance of IgG2a/c. In addition, the restoration of protection and E-specific IgG2a/c production in IFN-γ-deficient mice by a T helper (Th) type 1-biased intramuscular immunization suggested that IgG2a/c but not IFN-γ was the major component for protection. The failure of protection against a direct intracranial challenge indicated that IgG2a/c-mediated protection was restricted to outside the central nervous system. Consistent with this conclusion, passive transfer of E-specific antisera conferred protection only pre-exposure to JEV. Therefore, our data provided evidence that the IgG subclass plays an important role in protection against JEV, particular in poorly neutralizing E-specific antibodies, and Th1-biased IgG2a/c confers better protection than Th2-biased IgG1 against JEV.

The molecular basis for the increased resistance of astrocytes to a non-neuropathogenic strain of West Nile virus (WNV), WNV-MAD78, compared with the neuropathogenic strain WNV-NY remains unclear. Here, we demonstrated that the reduced susceptibility of astrocytes to WNV-MAD78 is due to a combination of both cellular activities as well as viral determinants. Analyses of the viral particle indicated that astrocyte-derived WNV-MAD78 particles were less infectious than those of WNV-NY. Additionally, inhibition of cellular furin-like proteases increased WNV-MAD78 infectious particle production in astrocytes, suggesting that high levels of furin-like protease activity within these cells acted in a cell- and strain-specific manner to inhibit WNV-MAD78 replication. Moreover, analysis of recombinant viruses indicated that the structural proteins of WNV-MAD78 were responsible for decreased particle infectivity and the corresponding reduction in infectious particle production compared with WNV-NY. Thus, the composition of the WNV virion was also a major determinant for viral fitness within astrocytes and may contribute to WNV propagation within the central nervous system. Whether the WNV-MAD78 structural genes reduce virus replication and particle infectivity through the same mechanism as the cellular furin-like protease activity or whether these two determinants function through distinct pathways remains to be determined.

Low-level hepatitis C virus (HCV) RNA may persist in PBMCs after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved a sustained virological response (SVR) after pegylated IFN and ribavirin therapy were analysed 52–66 months after the SVR. HCV RNA was detected in PBMCs from 18 patients (47.4 %), and PBMCs in two patients stained positive for non-structural protein 3 (NS3). Quantification of various cytokine and chemokine transcripts in PBMCs revealed that levels of IL-6, IL-8, IL-12, TNF-α and macrophage inflammatory protein 1β were significantly higher in HCV-positive patients than in HCV-negative individuals. In conclusion, persistence of HCV RNA in PBMCs of patients with a SVR appears to be associated with immune activation.

Coxsackievirus B3 (CVB3) infection can trigger myocarditis and can ultimately lead to dilated cardiomyopathy. It is known that CVB3-induced T-cell infiltration into cardiac tissues is one of the pathological factors causing cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T-cell infiltration by two types of CVB3: the H3 WT strain and the YYFF attenuated strain. T-cell activation was confirmed by changes in the distribution of lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 could infect and activate T-cells in vivo and in vitro, and activated T-cells were detected in CVB3-infected mouse hearts. LFA-1 expressed on the surface of these T-cells had been activated through the cAMP/Rap1 pathway. Recombinant lentiviruses expressing VP2 of CVB3 could also induce LFA-1 activation via an increase in cAMP, whilst VP2 of YYFF did not. These results indicated that CVB3 infection increased cAMP levels and then activated Rap1 in T-cells. In particular, VP2, among the CVB3 proteins, might be critical for this activation. This VP2–cAMP–Rap1–LFA-1 axis could be a potential therapeutic target for treating CVB3-induced myocarditis.

Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.

Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel, and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibodies. A widely used preclinical cottontail rabbit papillomavirus (CRPV)/rabbit model has been a surrogate model for high-risk human papillomavirus (HPV) infections. New Zealand white rabbits, either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix), were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were ~10-fold lower than those from the corresponding serum samples, but background noise was lower in tear samples. The isotypes of antibodies in tears were predominantly IgA and IgG. These findings showed clearly that tears could be a surrogate for serum samples for monitoring antibody responses. As collecting tears causes no discomfort and poses no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiological studies as well as for use in clinical practice.

Ubiquitylation is a covalent post-translational modification that regulates protein stability and is involved in many biological functions. Proteins may be modified with mono-ubiquitin or ubiquitin chains. Viruses have evolved multiple mechanisms to perturb the cell ubiquitin system and manipulate it to their own benefit. Here, we report ubiquitylation of vaccinia virus (VACV) protein N1. N1 is an inhibitor of the nuclear factor NF-κB and apoptosis that contributes to virulence, has a Bcl-2-like fold, and is highly conserved amongst orthopoxviruses. The interaction between N1 and ubiquitin occurs at endogenous protein levels during VACV infection and following ectopic expression of N1. Biochemical analysis demonstrated that N1 is covalently ubiquitylated, and heterodimers of ubiquitylated and non-ubiquitylated N1 monomers were identified, suggesting that ubiquitylation does not inhibit N1 dimerization. Studies with other VACV Bcl-2 proteins, such as C6 or B14, revealed that although these proteins also interact with ubiquitin, these interactions are non-covalent. Finally, mutagenesis of N1 showed that ubiquitylation occurs in a conventional lysine-dependent manner at multiple acceptor sites because only an N1 allele devoid of lysine residues remained unmodified. Taken together, we described a previously uncharacterized modification of the VACV protein N1 that provided a new layer of complexity to the biology of this virulence factor, and provided another example of the intricate interplay between poxviruses and the host ubiquitin system.

The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CA–NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50 % of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CA–NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.