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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 February 2012, Vol.109(9), pp.3341-6
    Description: Viruses have an extraordinary ability to diversify and evolve. For segmented viruses, reassortment can introduce drastic genomic and phenotypic changes by allowing a direct exchange of genetic material between coinfecting strains. For instance, multiple influenza pandemics were caused by reassortments of viruses typically found in separate hosts. What is unclear, however, are the underlying mechanisms driving these events and the level of intrinsic bias in the diversity of strains that emerge from coinfection. To address this problem, previous experiments looked for correlations between segments of strains that coinfect cells in vitro. Here, we present an information theory approach as the natural mathematical framework for this question. We study, for influenza and other segmented viruses, the extent to which a virus's segments can communicate strain information across an infection and among one another. Our approach goes beyond previous association studies and quantifies how much the diversity of emerging strains is altered by patterns in reassortment, whether biases are consistent across multiple strains and cell types, and if significant information is shared among more than two segments. We apply our approach to a new experiment that examines reassortment patterns between the 2009 H1N1 pandemic and seasonal H1N1 strains, contextualizing its segmental information sharing by comparison with previously reported strain reassortments. We find evolutionary patterns across classes of experiments and previously unobserved higher-level structures. Finally, we show how this approach can be combined with virulence potentials to assess pandemic threats.
    Keywords: Models, Biological ; Influenza A Virus, H1n1 Subtype -- Genetics ; Influenza A Virus, H3n2 Subtype -- Genetics ; Influenza A Virus, H7n7 Subtype -- Genetics ; Reassortant Viruses -- Genetics ; Recombination, Genetic -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(10), p.e25964
    Description: A highly conserved RNA-motif of yet unknown function, called stem-loop-2-like motif (s2m), has been identified in the 3′ end of the genomes of viruses belonging to different RNA virus families which infect a broad range of mammal and bird species, including Astroviridae , Picornaviridae , Coronaviridae and Caliciviridae . Since s2m is such an extremely conserved motif, it is an ideal target for screening for viruses harbouring it. In this study, we have detected and characterized novel viruses harbouring this motif in pigeons by using a s2m-specific amplification. 84% and 67% of the samples from feral pigeons and wood pigeons, respectively, were found to contain a virus harbouring s2m. Four novel viruses were identified and characterized. Two of the new viruses belong to the genus Avastrovirus in the Astroviridae family. We propose two novel species to be included in this genus, Feral pigeon astrovirus and Wood pigeon astrovirus . Two other novel viruses, Pigeon picornavirus A and Pigeon picornavirus B , belong to the Picornaviridae family, presumably to the genus Sapelovirus . Both of the novel picornaviruses harboured two adjacent s2m, called (s2m) 2 , suggesting a possible increased functional effect of s2m when present in two copies.
    Keywords: Research Article ; Biology ; Virology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PLoS ONE, 2011, Vol.6(4), p.e19075
    Description: Driven by the impact of influenza A viruses on human and animal health, much research is conducted on this pathogen. To support this research, we designed an all influenza A-embracing reverse transcription-PCR (RT-PCR) for the generation of DNA from influenza A virus negative strand RNA genome segments for full-length genome deep sequencing on a Genome Sequencer FLX instrument. For high reliability, the RT-PCRs are designed such that every genome segment is divided into two amplicons and for the most variable segments redundancy is included. Moreover, to minimize the risk of contamination of diagnostic real-time PCRs by sequencing amplicons, RT-PCR does not generate amplicons that are amenable to RT-qPCR detection. With the presented protocol we were able to generate virtually all amplicons (99.3% success rate) from isolates representing all so far known 16 hemagglutinin and 9 neuraminidase subtypes and from an additional 2009 pandemic influenza A H1N1 virus. Three isolates were sequenced to analyze the suitability of the DNA for sequencing. Moreover, we provide a short R script that disambiguates the sequences of the primers used. We show that using unambiguous primer sequences for read trimming prior to assembly with the genome sequencer assembler software results in higher quality of the final genome sequences. Using the disambiguated primer sequences, high quality full-length sequences for the three isolates used for sequencing trials could be established from the raw data in de novo assemblies.
    Keywords: Research Article ; Biology ; Medicine ; Veterinary Science ; Genetics And Genomics ; Virology ; Infectious Diseases
    E-ISSN: 1932-6203
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  • 4
    Language: English
    In: Science (New York, N.Y.), 12 August 2011, Vol.333(6044), pp.843-50
    Description: Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of V(H)1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the V(H)1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.
    Keywords: Antibodies, Monoclonal -- Immunology ; Antibodies, Neutralizing -- Immunology ; Antibodies, Viral -- Immunology ; Antigens, Viral -- Immunology ; Hemagglutinin Glycoproteins, Influenza Virus -- Immunology ; Influenza A Virus -- Immunology
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 5
    Language: English
    In: PLoS ONE, 2011, Vol.6(12), p.e28647
    Description: Astroviruses are a known cause of human diarrhea. Recently the highly divergent astrovirus MLB1 (MLB1) was identified in a stool sample from a patient with diarrhea. It has subsequently been detected in stool from individuals with and without diarrhea. To determine whether MLB1 is associated with diarrhea, we conducted a case control study of MLB1. In parallel, the prevalence of the classic human astroviruses (HAstVs) was also determined in the same case control cohort. 400 cases and 400 paired controls from a longitudinal birth cohort in Vellore, India were analyzed by RT-PCR. While HAstVs were associated with diarrhea (p = 0.029) in this cohort, MLB1 was not; 14 of the controls and 4 cases were positive for MLB1. Furthermore, MLB1 viral load did not differ significantly between the cases and controls. The role of MLB1 in human health still remains unknown and future studies are needed.
    Keywords: Research Article ; Biology ; Medicine ; Virology ; Infectious Diseases ; Pediatrics And Child Health ; Gastroenterology And Hepatology
    E-ISSN: 1932-6203
    Source: PLoS
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  • 6
    Language: English
    In: Science (New York, N.Y.), 14 September 2012, Vol.337(6100), pp.1343-8
    Description: Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.
    Keywords: Antibodies, Monoclonal -- Immunology ; Hemagglutinin Glycoproteins, Influenza Virus -- Immunology ; Immunodominant Epitopes -- Immunology ; Influenza B Virus -- Immunology ; Influenza Vaccines -- Immunology ; Orthomyxoviridae Infections -- Prevention & Control
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 7
    Language: English
    In: PLoS ONE, 2011, Vol.6(11), p.e27228
    Description: Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL pro ), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. ; Here, we profiled the substrate specificities of 3CL from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19×8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL prefers P4-Pro and SARS-CoV 3CL prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences ‘VARLQ↓SGF’ that can be cleaved efficiently by all 3CL with relative activity of 1.7 to 3.2, and ‘VPRLQ↓SGF’ that can be cleaved specifically by IBV 3CL with relative activity of 4.3. ; The comprehensive substrate specificities of 3CL from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.
    Keywords: Research Article ; Biology ; Medicine ; Virology ; Infectious Diseases ; Respiratory Medicine ; Biochemistry
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: PLoS ONE, 2011, Vol.6(9), p.e24384
    Description: Although the 2009 (H1N1) influenza pandemic officially ended in August 2010, the virus will probably circulate in future years. Several types of H1N1 vaccines have been tested including various dosages and adjuvants, and meta-analysis is needed to identify the best formulation. ; We searched MEDLINE, EMBASE, and nine clinical trial registries to April 2011, in any language for randomized clinical trials (RCTs) on healthy children, adolescents, adults and the elderly. Primary outcome was the seroconversion rate according to hemagglutinination-inhibition (HI); secondary outcomes were adverse events. For the primary outcome, we used head-to-head meta-analysis and multiple-treatments meta-analysis. ; Eighteen RCTs could be included in all primary analyses, for a total of 76 arms (16,725 subjects). After 2 doses, all 2009 H1N1 split/subunit inactivated vaccines were highly immunogenic and overcome CPMP seroconversion criteria. After 1 dose only, all split/subunit vaccines induced a satisfactory immunogenicity (〉 = 70%) in adults and adolescents, while only some formulations showed acceptable results for children and elderly (non-adjuvanted at high-doses and oil-in-water adjuvanted vaccines). Vaccines with oil-in-water adjuvants were more immunogenic than both nonadjuvanted and aluminum-adjuvanted vaccines at equal doses and their immunogenicity at doses 〈 = 6 µg (even with as little as 1.875 µg of hemagglutinin antigen) was not significantly lower than that achieved after higher doses. Finally, the rate of serious vaccine-related adverse events was low for all 2009 H1N1 vaccines (3 cases, resolved in 10 days, out of 22826 vaccinated subjects). However, mild to moderate adverse reactions were more (and very) frequent for oil-in-water adjuvanted vaccines. ; Several one-dose formulations might be valid for future vaccines, but 2 doses may be needed for children, especially if a low-dose non-adjuvanted vaccine is used. Given that 15 RCTs were sponsored by vaccine manufacturers, future trials sponsored by non-industry agencies and comparing vaccines using different types of adjuvants are needed.
    Keywords: Research Article ; Medicine ; Public Health And Epidemiology ; Infectious Diseases ; Pharmacology
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: PLoS ONE, 2012, Vol.7(3), p.e32858
    Description: Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro .
    Keywords: Research Article ; Biology ; Medicine ; Veterinary Science ; Genetics And Genomics ; Infectious Diseases
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: PLoS ONE, 2012, Vol.7(3), p.e33166
    Description: During the Northern Hemisphere winter of 2003–2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal “control” cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003–2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection.
    Keywords: Research Article ; Biology ; Medicine ; Virology ; Public Health And Epidemiology ; Infectious Diseases ; Pediatrics And Child Health
    E-ISSN: 1932-6203
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