Re: Sis 2011b Keygen Download No Virus
Tacito Fingerut
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Many viruses reorganise cellular membrane compartments and the cytoskeleton to generate subcellular microenvironments called virus factories or 'viroplasm'. These create a platform to concentrate replicase proteins, virus genomes and host proteins required for replication and also protect against antiviral defences. There is growing interest in understanding how viruses induce such large changes in cellular organisation, and recent studies are beginning to reveal the relationship between virus factories and viroplasm and the cellular structures that house them. In this review, we discuss how three supergroups of (+)RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses.
Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.
Influenza C viruses infect most humans during childhood. Unlike influenza A viruses, influenza C viruses exhibit little genetic variability and evolve at a comparably slower rate. Influenza A viruses exist as multiple subtypes and cause disease in numerous mammals. In contrast, influenza C viruses are comprised of a single subtype in its primary human host. Here we characterize a novel swine influenza virus, C/swine/Oklahoma/1334/2011 (C/OK), having only modest genetic similarity to human influenza C viruses. No cross-reaction was observed between C/OK and human influenza C viruses. Antibodies that cross react with C/OK were identified in a significant number of swine but not human sera samples, suggesting that C/OK circulates in pigs. Additionally, we show that C/OK is capable of infecting and transmitting by direct contact in both pigs and ferrets. These results suggest that C/OK represents a new subtype of influenza C viruses. This is significant, as co-circulation of multiple subtypes of influenza allows for rapid viral evolution through antigenic shift, a property previously only shown for influenza A viruses. The ability of C/OK to infect ferrets along with the absence of antibodies to C/OK in humans, suggests that such viruses may become a potential threat to human health.
Copyright: 2013 Hause et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by Newport Laboratories, SDSU AES 3AH-203 fund and the South Dakota 2010 Research Center, BCAPP (Biological Control and Analysis of Applied Photonics), and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under Contract No. HHSN266200700005C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal's policy and have the following conflicts: BMH, EAC and RRS are employed by Newport Laboratories, a company that produces swine influenza virus vaccines. This does not alter the authors' adherence to all the PLoS Journal policies on sharing data and materials.
Aside from humans, influenza C virus has been isolated only from swine in China (in 1981) [11]. Genetic analysis showed a close relation between Japanese human and Chinese swine influenza C isolates [12], [13]. Serological surveys in Japan and the United Kingdom found 9.9% and 19% of swine, respectively, to have positive HI antibody titers to human influenza C viruses, suggesting that the virus is not uncommon in swine [14], [15]. Swine inoculated with influenza C virus had mild respiratory disease and transmitted the virus to naive swine by direct contact [11]. Here we characterize an orthomyxovirus isolated from a clinically ill pig and show that the virus is distantly related to human influenza C virus and readily infects and is transmissible in both ferrets and pigs. Genetic and antigenic analysis suggest that this virus represents a new subtype of influenza C virus, raising the possibility of reassortment and antigenic shift as mechanisms for influenza C virus evolution which could pose a potential threat to human health.
Maximum-likelihood analysis in combination with 1000 bootstrap replicates was used to derive trees based on the nucleotide sequences encoding respective proteins. A scale representing the number of nucleotide changes is shown in each panel. The trees are shown for PB2, PB1, P3, HE, NP, P42, and NS segments. Bootstrap values are shown above branches to the left of major nodes.
An HI assay was performed to determine the antigenic cross-reactivity and seroprevalence of C/OK virus in humans and swine. The assay included reference strains of influenza A, B, and C genera and their matched antisera (Table S2). No cross-reactivity was observed between C/OK virus and heterologous antisera. For the human cohort, we used a set of 316 serum samples.
We compared in vitro cellular tropism between C/OK and human influenza C viruses by rt-RT-PCR in several cell lines, including ST, adenocarcinomic human alveolar basal epithelial (A549), Madin-Darby canine kidney (MDCK), Green African monkey kidney (Marc-145), human rectal tumor (HRT-18G), baby hamster kidney (BHK-21) and porcine kidney (PK-15) cells. C/OK replicated, in order of highest replication, in ST, MDCK, Marc-145, HRT-18G and A549 cells (Fig. S2A). Minimal replication was observed in BHK-21 and PK-15 cells. In marked contrast, the human influenza C virus C/Taylor showed poor growth and replicated only in ST and HRT-18G and not in other cells tested (Fig. S2B). It should be noted however that cultivation of influenza C/Taylor virus was performed at 33C, the optimal temperature that is typically used to propagate human influenza C virus [34]. This virus failed to replicate at 37C in our hands which differs from the C/OK virus that can replicate efficiently at this temperature (data not shown). These results suggest that C/OK has a broader cellular tropism than C/Taylor and is also not restricted at elevated temperatures for replication.
The influenza C virus utilizes the 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as the primary receptor for attachment to the cell surface to initiate infection [35]. The receptor binding specificity and affinity are mainly determined by the 9-O-acetyl group of the Neu5,9Ac2 [36]. As a result, the virus encodes a sialate-O-acetylesterase, not neuraminidase, in order to release virions from infected cells by cleavage of the 9-O-acetyl group [37]. To provide structural insights to the observed different tropism, we conducted structural modeling of the C/OK HE protein in complex with the receptor based on the solved X-ray crystallographic structure of a human influenza C virus (C/Johannesburg/1/66) HE protein [36]. The overall 53% sequence identity between the two HE proteins allows us to predict important structural features such as the receptor-binding pocket and the enzymatic active site of the C/OK HE protein. Based on the assumption that the HE protein uses similar sites for function, our structural modeling analysis identified a conserved enzymatic active site but revealed a variable receptor-binding pocket between two HE proteins (Figs. 4 and S3). These results are consistent with the observed difference in cellular tropism. For example, both HE proteins possess an identical catalytic triad: S71/H369/D365 for C/Johannesburg and S73/H375/D372 for C/OK (Fig. 4A). The other two substrate-interacting residues for optimal enzymatic function are also completely conserved in the two HE proteins (G99/N131 for C/Johannesburg and G101/N133 for C/OK). In addition, both HE proteins utilize two conserved arginine residues for substrate binding (R72/R332 for C/Johannesburg and R74/R342 for C/OK). The conserved enzymatic site between C/Johannesburg and C/OK suggests that C/OK utilizes 9-O-acetyl sialic acid as the cellular receptor for infection.
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