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  • Darragh Hartley posted an update 1 week, 1 day ago

    Cell-based vaccines, in general have been produced using egg grown candidate viruses. It has been reported that it would be a major advantage to dispose of candidate vaccine viruses that are grown in cell culture (Zhang et al., 2011; Reed, 1938). This is because the use of egg grown candidate viruses could be a constraint if the subsequent vaccine manufacture occurs in cell culture and, on the other hand, currently circulating H3N2 viruses are progressively more difficult to grow in eggs (Reed, 1938). MDCK and Vero chir99021 Supplier are promising platforms to grow vaccine candidate viruses. The experimental procedures in this study have been performed in MDCK-SIAT1 since previous research by other authors have placed this cell line as a preferred candidate for cell-based vaccine manufacture. A(H1N1) viruses, including PR8, have been proven to have similar growth characteristics in MDCIK-SIAT1 cells when compared to MDCK at lower MOI, which makes the process more cost-effective (Abdoli et al., 2016). Additionally, MDCK-SIAT1has been shown to induce less mutations to the HA since the higher receptor levels results in a higher avidity of the interaction receptor-ligand, making this a preferred cell line for laboratory research (YP L, 2012).Conflict of interest statementAcknowledgmentsThe authors would like to thank the Centre for Disease Control and Prevention (CDC) – Atlanta, for the training on Reverse genetics for the construction of viral reassortants, which was applied for the design of the experimental strategy. Also, the authors would like to thank the CDC – Atlanta for kindly providing the cDNA clones of A/PuertoRico/8/34 and the pCIPolISapIT transcription plasmid.This research has been funded by the Fundação para a Ciência e Tecnologia Grants PTDC/SAU-MIC/122780/2010, SFRH/BD/65211/2009, SFRH/BD/62676/2009 and SFRH/BD/48532/2008.IntroductionEnterovirus 71 (EV71), a common pathogenic agent of hand-foot-and-mouth disease (HFMD), can cause severe complications including herpangina, aseptic meningitis, encephalitis, cardiorespiratory failure, poliomyelitis-like syndrome or even fatal disease (Chan et al., 2000; McMinn et al., 2001). EV71 predominantly affects children under 5 years old, and causes severe diseases with high morbidity and mortality (Sato et al., 2006). Since it was first isolated in 1969, EV71 outbreaks have occurred frequently in western Pacific region countries, including China (Lee et al., 2010; Samuda et al., 1987), Japan (Fujimoto et al., 2002), Malaysia (Chan et al., 2000), and Singapore (Singh et al., 2002). The World Health Organization has estimated the occurrence of one million new HFMD cases in China alone between 2011 and 2014 (World Health Organization, 2015). To date, no effective vaccines and antiviral drugs have been available to prevent or treat EV71 infection (Wu et al., 2010; Yee and Poh, 2015). Therefore, it is necessary to identify the host factors involved in the replication of EV71 to control the development and complications of HFMD.EV71 is a non-enveloped virus with a single-stranded, positive-sense genomic RNA of approximately 7.4kb nucleotides. Protein 3A, a nonstructural viral protein of 87 amino acids in length, is required for enterovirus RNA replication (Teoule et al., 2013). 3A proteins play important role in enterovirus replication through the formation (with the 3CD protein) of replication organelles via remodeling of the internal cell membrane. Analyses of enterovirus 3A proteins have led to the identification of several cellular partners of this protein. The 3A proteins of PV and of the related coxsackievirus B3 (CV-B3) can interact with the cellular Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1). By interacting with GBF1, 3A inhibits the cellular secretory pathway. The membrane-anchored 3A protein modulates GBF1/Arf1 activity, resulting in the preferential recruitment of PI4KIIIβ to sites of viral RNA replication. PI4KIIIβ recruitment leads to the enrichment of virus-induced membranous organelles in PI4P, which has been shown to facilitate viral RNA replication. (Belov et al., 2005; Belov and van Kuppeveld, 2012; Dorobantu et al., 2015; Greninger, 2015; Hsu et al., 2010; van der Schaar et al., 2013). However, the function of 3A in the RNA replication of EV71 has been rarely reported. So, it is necessary to detect the mechanism of protein 3A and further explore the host factors involved in EV71 infection. Thus, to identify the host factors that could interact with 3A protein of EV71 may be a potential target for the therapy of HFMD.