Avian Influenza A viruses present a unique challenge to public health as a result of their rapid evolution. Since the beginning of the 20th century, there have been five IAV pandemics, including the 1918 pandemic, which is estimated to have been responsible for 50-100 million deaths worldwide (Taubenberger & Morens, 2006). Each major influenza pandemic has been associated with the appearance of a new subtype circulating in humans. Additionally, particularly virulent pandemic strains and in particular the 1918 strain tend to attenuate during the course of the pandemic, likely a result of adaptation to humans (Taubenberger & Morens, 2006). However, the precise mechanism for this is poorly defined (Qi et al., 2012). A number of specific mutations
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PB1 is the viral RNA-dependent RNA polymerase, while PB2 and PA participate in cap-snatching (Taft et al., 2014). One mutation, PB2 E627K, has been demonstrated as sufficient to adapt avian H5N1 to mammals, and has been identified in multiple clinical isolates (Linster et al, 2014). Intriguingly, adaptive mutations are often associated with changes in virulence. While polymerase mutations that facilitate more efficient replication in mammals may increase viral titers and therefore increase virulence, this cannot explain every aspect of increased virulence associated with certain mutations, and ignores entirely the reality that adaptive mutations are just as, if not more, often correlated with decreased virulence. Therefore, this represents a poorly understood aspect of IAV biology, and given the recent avian H7N9 outbreak in China, and the continuing threat posed by avian H5N1, one of considerable importance to public health. Here we propose research aimed at defining the links between adaptive mutations and virulence by utilizing an original approach that combines the techniques of targeted mutagenesis and serial passage with a novel ultradeep RNA sequencing …show more content…
To date, work examining host adaptation has focused largely on the adaptive mutations themselves, and has to some extent ignored the implications the adaptive mutations observed may have on virulence. In addition, the two major tools used to study host adaptation in IAVs, serial passage in animal models and targeted mutation, have previously been used largely independently. This research is innovative because it utilizes both of these approaches, and examines an aspect of IAV biology that has been poorly studied. We have previously identified a number of adaptive polymerase mutations, and this proposal expands this work to demonstrate a link between adaptive mutations and changes in virulence. Additionally, this proposal expands on the serial passage approach to IAV host adaptation by pairing it with circular RNA sequencing technology, a novel approach to deep sequencing of RNAs that allows for the detection of extremely low frequency mutants by drastically reducing noise from PCR-induced mutations (Acevedo et al., 2014). Essentially, circularized RNA templates are used to generate concatameric DNAs which are used for library preparation and next generation sequencing. This methodology reduces the error probability of Illumina sequencing to a level that is considerably below the error rate of any RNA virus polymerase, from 10-4 per base for conventional Illumina RNA-seq, to
PB1 is the viral RNA-dependent RNA polymerase, while PB2 and PA participate in cap-snatching (Taft et al., 2014). One mutation, PB2 E627K, has been demonstrated as sufficient to adapt avian H5N1 to mammals, and has been identified in multiple clinical isolates (Linster et al, 2014). Intriguingly, adaptive mutations are often associated with changes in virulence. While polymerase mutations that facilitate more efficient replication in mammals may increase viral titers and therefore increase virulence, this cannot explain every aspect of increased virulence associated with certain mutations, and ignores entirely the reality that adaptive mutations are just as, if not more, often correlated with decreased virulence. Therefore, this represents a poorly understood aspect of IAV biology, and given the recent avian H7N9 outbreak in China, and the continuing threat posed by avian H5N1, one of considerable importance to public health. Here we propose research aimed at defining the links between adaptive mutations and virulence by utilizing an original approach that combines the techniques of targeted mutagenesis and serial passage with a novel ultradeep RNA sequencing …show more content…
To date, work examining host adaptation has focused largely on the adaptive mutations themselves, and has to some extent ignored the implications the adaptive mutations observed may have on virulence. In addition, the two major tools used to study host adaptation in IAVs, serial passage in animal models and targeted mutation, have previously been used largely independently. This research is innovative because it utilizes both of these approaches, and examines an aspect of IAV biology that has been poorly studied. We have previously identified a number of adaptive polymerase mutations, and this proposal expands this work to demonstrate a link between adaptive mutations and changes in virulence. Additionally, this proposal expands on the serial passage approach to IAV host adaptation by pairing it with circular RNA sequencing technology, a novel approach to deep sequencing of RNAs that allows for the detection of extremely low frequency mutants by drastically reducing noise from PCR-induced mutations (Acevedo et al., 2014). Essentially, circularized RNA templates are used to generate concatameric DNAs which are used for library preparation and next generation sequencing. This methodology reduces the error probability of Illumina sequencing to a level that is considerably below the error rate of any RNA virus polymerase, from 10-4 per base for conventional Illumina RNA-seq, to