Anan Jongkaewwattana

Enhancement of reverse genetics-derived swine-origin H1N1 influenza virus seed vaccine growth by inclusion of indigenous polymerase PB1 protein.

The current pandemic of a novel swine-origin H1N1 influenza virus (S-OIV) highlighted the need to urgently develop vaccines that can be used in a rapid response against the pathogen. Reverse genetics has been employed as an alternative means for the generation of influenza seed vaccines. However, reassortant viruses containing 6 internal genes from A/PR/8/34 and the hemagglutinin (HA) and neuraminidase (NA) genes from S-OIV showed very slow growth characteristics, hampering the speed of vaccine production. Here, we showed that the reverse genetics-derived H1N1 could be rescued with sensible viral titer by replacing PB1 of A/PR/8/34 with that of S-OIV for plasmid transfection. The 5+3 reassortant viruses have shown higher growth rate after transfection compared to that of 6+2 reassortant. The difference between PB1 of S-OIV and that of A/PR/8/34 in terms of the enhancement of virus growth was possibly due to the augmentation of viral polymerase activity, but not the lack of functional PB1-F2. Furthermore, it was found that growth enhancement by PB1 was specific for reassortant harboring HA of S-OIV, suggesting that the slow growth property of S-OIV reassortant virus is possibly due to restrictions imposed by the HA gene.

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses.

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP(FluB)) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP(FluB) suggest that functional NP(FluB) is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP(FluB). Further analysis of NP(FluB) indicated that it does not affect nuclear import of NP(FluA). Taken together, these findings suggest a novel role of NP(FluB) in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.

Atypical characteristics of nucleoprotein of pandemic influenza virus H1N1 and their roles in reassortment restriction

Sequence analysis of the nucleoprotein (NP) of swine-origin influenza virus H1N1 (S-OIV) reveals a number of atypical characteristics including an early start codon and a highly conserved, non-aromatic residue at position 313. Using an in vitro viral polymerase reconstitution assay, we found that the polymerase complex containing the NP of S-OIV (NPS-OIV) yielded substantially lower activity than those assayed with NP derived from other influenza virus strains. Moreover, alteration of the early start codon or introduction of an aromatic residue at position 313 (V313Y) did not increase but instead exacerbated the poor polymerase activity. Interestingly, when NPS-OIV was allowed to compete with that of a mouse-adapted influenza virus (A/PR/8/34) to form progeny virions, only progeny bearing NPS-OIV were produced, despite the low polymerase activity associated with NPS-OIV. Our results indicated that NPS-OIV requires both the early start codon and the V313 residue for its optimal function. These characteristics are required for a strong compatibility between the S-OIV polymerase subunits and its indigenous NP over that of other strains, which might explain why productive reassortment between S-OIV and seasonal influenza viruses has yet to occur in nature.

Nuclear import of influenza B virus nucleoprotein: involvement of an N-terminal nuclear localization signal and a cleavage-protection motif.

The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the hosts nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70-GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44-47 as a putative NLS. In addition, we demonstrated that residues 3-15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70-GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization.

The N terminus of PA polymerase of swine-origin influenza virus H1N1 determines its compatibility with PB2 and PB1 subunits through a strain-specific amino acid serine 186.

Despite several lines of evidence suggesting possible mechanisms by which the influenza virus polymerase complex, comprising PB2, PB1 and PA, work in concert during virus replication, exactly how they function is not entirely understood. The N terminal region of the PA subunit has been shown to play a key role in various functions through a number of conserved amino acid residues. However, little is known about the role of amino acids reported to be unique for a virus strain. Here, we investigated the functional implication of an amino acid (S186) present uniquely in the N terminus of the PA subunit of the pandemic H1N1 influenza virus and determined the effect of its mutation in terms of polymerase activity as well as virus growth. Using chimeric constructs of PA derived from A/PR/8/34 (H1N1) (PR8) and the swine-origin influenza virus (S-OIV) H1N1, we found that, when complexed with PB2 and PB1 of PR8, the chimeric PA protein containing the N terminus of S-OIV (1-213) with the remaining region from PR8 showed significantly reduced polymerase activity. Recombinant viruses harboring the chimeric PA also grew poorly in MDCK cells and embryonated eggs. Likewise, the chimeric PA in which the N terminus of PA of PR8 (1-213) was assembled with the remaining region of PA of S-OIV showed a similar phenotype when complexed with PB2 and PB1 of S-OIV. Interestingly, when S186 in the N terminus was altered to the residue common in most strains of influenza virus (G186), the chimeric as well as wild-type PA of S-OIV showed severely impaired polymerase activity when assayed with PB2 and PB1 of S-OIV. Collectively, this finding suggests that S186 at the N terminal region of PA of S-OIV is necessary for the protein to function optimally.

Evaluation of In Vitro Cross-Reactivity to Avian H5N1 and Pandemic H1N1 2009 Influenza Following Prime Boost Regimens of Seasonal Influenza Vaccination in Healthy Human Subjects: A Randomised Trial

Introduction Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses. Methods In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose. Results Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated. Conclusion Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed. Trial Registration ClinicalTrials.gov NCT01044095

Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments

n Abstractn n Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.n n

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid

ABSTRACT Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.

Pre-Existing Cross-Reactive Antibodies to Avian Influenza H5N1 and 2009 Pandemic H1N1 in US Military Personnel

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.

A computational tool for the design of live attenuated virus vaccine based on microRNA-mediated gene silencing

BackgroundThe microRNA-based gene-silencing machinery has been recognized as a promising approach to control viral replication and used for improving safety for the live attenuated virus vaccines. The effective host microRNA response elements (MREs) have been incorporated into a virus sequence mainly based on the experimental trials for identifying both microRNA binding sites and effective mutations. The design of MREs for viral genomes or with multiple host microRNAs of interest, then, will be time and cost consuming.ResultsIn this paper, we introduced a computational flow that could be used to design MREs of human microRNAs within Influenza A H1N1 virus gene segments. The main steps of the flow includes locating possible binding sites; MREs, of human microRNAs within the viral sequences using a miRNA target prediction tool (miranda), performing various mutations among mismatched binding positions, calculating the binding energy, score, identity, and the effects of changed physical properties of amino acids according to the changed bases in RNA level, and prioritizing the mutated binding sites. The top ranked MREs of human microRNA hsa-miR-93 is consistent with previous literature while other results waited to be experimentally verified. To make the computational flow easily accessible by virologists, we also developed MicroLive, a web server version of the MRE design flow together with the database of miranda-predicted MREs within gene sequences of seven RNA viruses including Influenza A, dengue, hepatitis C, measles, mumps, poliovirus, and rabies. Users may design MREs of specific human microRNAs for their input viral sequences using MRE design tool or optimize the miranda-predicted MREs of seven viruses available on the system. Also, users could design varied number of MREs for multiple human microRNAs to modulate the degree of live vaccine attenuation and reduce the likelihood of escape mutants.ConclusionsThe computational design of MREs helps reduce time and cost for experimental trials. While the flow was demonstrated using human microRNAs and Influenza A H1N1 virus, it could be flexibly applied to other hosts (e.g., animals) and viruses of interest for constructing host-specific live attenuated vaccines. Also, it could be deployed for engineering tissue-specific oncolytic viruses in cancer virotherapeutics. The MicroLive web server is freely accessible at http://www.biotec.or.th/isl/microlive.