The K526R substitution in viral protein ​PB2 enhances the effects of E627K on influenza virus replication

Host-adaptive strategies, such as the E627K substitution in the ​PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation ​PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing ​PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. ​PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. ​PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.published_or_final_versio

Large-scale plasma proteomic profiling identifies a high-performance biomarker panel for Alzheimer's disease screening and staging

INTRODUCTION: Blood proteins are emerging as candidate biomarkers for Alzheimer's disease (AD). We systematically profiled the plasma proteome to identify novel AD blood biomarkers and develop a high-performance, blood-based test for AD. METHODS: We quantified 1160 plasma proteins in a Hong Kong Chinese cohort by high-throughput proximity extension assay and validated the results in an independent cohort. In subgroup analyses, plasma biomarkers for amyloid, tau, phosphorylated tau, and neurodegeneration were used as endophenotypes of AD. RESULTS: We identified 429 proteins that were dysregulated in AD plasma. We selected 19 “hub proteins” representative of the AD plasma protein profile, which formed the basis of a scoring system that accurately classified clinical AD (area under the curve = 0.9690–0.9816) and associated endophenotypes. Moreover, specific hub proteins exhibit disease stage-dependent dysregulation, which can delineate AD stages. DISCUSSION: This study comprehensively profiled the AD plasma proteome and serves as a foundation for a high-performance, blood-based test for clinical AD screening and staging

Mammalian adaptation markers in avian-origin H7N9 virus, a comprehensive investigation in isolates and clinical specimens from the H7N9 influenza affected area

Oral PresentationsBackground: An avian-origin H7N9 virus emerged in eastern China in February 2013 and has since caused 133 confirmed human infections (Gao R, et al. N Engl J Med. 2013;368:1888-1897). We have compared human isolates and avian viruses isolated from epidemiologically linked poultry markets and confirmed that the human infections were caused by direct transmission from a poultry source (Chen Y, et al. Lancet. 2013;381:1916-1985). In addition to the Q226L substitution in the HA, which may provide virus with some ability to bind to human-type receptors, what other adaptations has this virus gained to make it different from other avian influenza viruses? Materials and Methods: We characterized H7N9 and other related H7 and N9 subtype viruses isolated in April 2013 from local poultry markets that were associated with human infections. Genetic mutations, polymerases activity, growth kinetic in mammalian and avian cells and replication ability in mice were determined using reverse genetic versions of H7N9 virus. Results: Replication ability and growth kinetics of the avian H7 subtype influenza viruses were compared in avian and mammalian cell lines. Our data suggest that the reassortant H7N9 virus has adapted to, and may have become established in, land-based poultry. It is currently not clear if this virus may still be circulating in some poultry populations, continuing to evolve and posing a threat for further human infections. We studied the virus genome for mammalian adaptation markers by performing sequence analysis on virus isolates and RT-PCR products derived from samples obtained from 46 patients hospitalized in the First Affiliated Hospital of Zhejiang University Medical School, Hangzhou, China. Virus adaptation markers in the HA, NA and PB2 genes were analyzed in sequential samples. Multiple adaptation markers were identified in these genes of clinical isolates and serial respiratory samples. Our data showed that the avian-origin H7N9 has attempted to adapt to replicate in human cells using various mechanisms already displayed by other viruses. An in vitro assay showed that viruses with substitutions at these positions exhibit enhanced RNP polymerase activity, and a study of growth kinetics demonstrated that isolates carrying these adaptation markers replicate to a higher titer in mammalian cells. Replication efficiency of these clinical variants was also evaluated in mice. As neuraminidase inhibitors are used as the first line of antiviral drugs for the treatment of H7N9 infection, we also analyzed oseltamivir resistance-associated mutations in the NA genes of viruses shed in either serial nasal swab or sputum specimens obtained from 40 of the hospitalized patients. Conclusions: This study provides a comprehensive analysis of avian-origin H7N9 virus from poultry and in human infections. The novel H7N9 virus attempted multiple adaptive strategies for efficient replication in humans. Further characterization of this H7N9 avian influenza virus for understanding the mechanism of replication adaptation and the role in efficient human transmission is necessary.published_or_final_versio

Mammalian adaptation markers in avian-origin H7N9 virus, a comprehensive investigation in isolates and clinical specimens from the H7N9 influenza affected area

Oral PresentationsBackground: An avian-origin H7N9 virus emerged in eastern China in February 2013 and has since caused 133 confirmed human infections (Gao R, et al. N Engl J Med. 2013;368:1888-1897). We have compared human isolates and avian viruses isolated from epidemiologically linked poultry markets and confirmed that the human infections were caused by direct transmission from a poultry source (Chen Y, et al. Lancet. 2013;381:1916-1985). In addition to the Q226L substitution in the HA, which may provide virus with some ability to bind to human-type receptors, what other adaptations has this virus gained to make it different from other avian influenza viruses? Materials and Methods: We characterized H7N9 and other related H7 and N9 subtype viruses isolated in April 2013 from local poultry markets that were associated with human infections. Genetic mutations, polymerases activity, growth kinetic in mammalian and avian cells and replication ability in mice were determined using reverse genetic versions of H7N9 virus. Results: Replication ability and growth kinetics of the avian H7 subtype influenza viruses were compared in avian and mammalian cell lines. Our data suggest that the reassortant H7N9 virus has adapted to, and may have become established in, land-based poultry. It is currently not clear if this virus may still be circulating in some poultry populations, continuing to evolve and posing a threat for further human infections. We studied the virus genome for mammalian adaptation markers by performing sequence analysis on virus isolates and RT-PCR products derived from samples obtained from 46 patients hospitalized in the First Affiliated Hospital of Zhejiang University Medical School, Hangzhou, China. Virus adaptation markers in the HA, NA and PB2 genes were analyzed in sequential samples. Multiple adaptation markers were identified in these genes of clinical isolates and serial respiratory samples. Our data showed that the avian-origin H7N9 has attempted to adapt to replicate in human cells using various mechanisms already displayed by other viruses. An in vitro assay showed that viruses with substitutions at these positions exhibit enhanced RNP polymerase activity, and a study of growth kinetics demonstrated that isolates carrying these adaptation markers replicate to a higher titer in mammalian cells. Replication efficiency of these clinical variants was also evaluated in mice. As neuraminidase inhibitors are used as the first line of antiviral drugs for the treatment of H7N9 infection, we also analyzed oseltamivir resistance-associated mutations in the NA genes of viruses shed in either serial nasal swab or sputum specimens obtained from 40 of the hospitalized patients. Conclusions: This study provides a comprehensive analysis of avian-origin H7N9 virus from poultry and in human infections. The novel H7N9 virus attempted multiple adaptive strategies for efficient replication in humans. Further characterization of this H7N9 avian influenza virus for understanding the mechanism of replication adaptation and the role in efficient human transmission is necessary.published_or_final_versio

Nuclear Dicer mediates IFN-b production in response to RNA virus infection

Dicer plays an essential role in the RNA-interference (RNAi) pathway. The structure of Dicer is highly conserved across eukaryotic organisms. While the significance of Dicer in regulation of antiviral RNAi in lower eukaryotes and genesis of microRNAs in mammals have been established, it remains largely unclear whether Dicer could also be involved in other innate antiviral pathways, particularly in the nucleus. In this study, we explored the link between nuclear Dicer and transcription initiation of interferon (IFN) regulatory factors in cells infected with nuclear-replicating influenza A virus. Infection of RNA viruses is initially recognized by host pattern recognition receptors (PRRS) which induce antiviral responses, such as production of type I IFN. Many RNA viruses have evolved with antagonistic mechanisms to counteract host antiviral response and it is difficult to study some host antiviral functions using wild type viruses. We examined the role of Dicer under infection by using various RNA viruses defective in antagonizing IFN-β production. Our data showed that the depletion of Dicer has no effect on IRF3 activation but down regulates IFN-β production. Furthermore, over-expression of the constitutively active CARDs from RIG-I in Dicer knockdown cells failed to activate the IFN-b promoter. Using an influenza A virus infection model, we demonstrated that Dicer exerts anti-viral function through modulation of host IFN expressions in the nucleus. It appears that only nuclear-replicating influenza virus, but not the cytoplsmic-replicating Sendai virus, can promote translocation of Dicer to the nucleus. Taken together, our findings suggested that Dicer could induce host antiviral response independent of RIG-I and microRNA pathways. Molecular details for this mechanism will be discussed

Epstein-Barr virus BART noncoding RNAs modulate host gene expression for virus latency leading to oncogenesis in epithelial cells

Epstein-Barr virus (EBV) efficiently establishes and maintains a state of latency in resting B cells, with persistent asymptomatic infections occurring in more than 95% of the human population worldwide. Besides causing lifelong infections in resting B cells in the majority of people, EBV has been found to associate with several human cancers occurring in immune competent individuals, in which the virus expresses very few viral proteins in EBV infected cancer cells through a distinct viral latency program. It is believed that this type of mechanism has been adopted by EBV to allow the virus to evade host immune surveillance. Nasopharyngeal carcinoma (NPC) is prevalent in southern China, Southeast Asian countries and some African countries but uncommon in rest of the world. In NPC 100% of cancer cells are infected with EBV, and EBV is recognized one of the major etiological factors. However, the details of how EBV maintains latency and contributes to the oncogenesis of NPC remain mostly elusive. Notably, EBV abundantly expresses a family of non-coding RNAs derived from the BamHI A region of the viral genome (BARTs). It is hypothesized that this family of viral non-coding RNAs is key to maintaining viral latency, evading immune surveillance and triggering oncogenesis in EBV associated tumors. We have shown a mechanism for the regulation of the abundant expression of BARTs in NPC which involves the constitutive activation of NF-B signaling typically observed in NPC cells and have also identified a regulatory loop through which BARTs support EBV latency in NPC. Using high throughput RNA-seq analysis we further demonstrated that BART RNAs modulate expression of cellular genes associated with anti-inflammatory and immune-modulating properties, oxidoreductase activity, and cell migration and invasion in NPC cells. BART non-coding RNAs are predominantly localized in the nucleus and associate with the CBP/p300 complex. Our data showed that EBV BARTs modulate genes responsive to the mitochondria-associated adapter molecule (MAVS) through halting cellular Pol II in the promoter region and downregulating gene expression. In the nucleus, BART non-coding RNAs modulate chromatin acetylation through interaction with CBP/p300. We have generated a working model with evidence showing that Epstein-Barr virus shuts off most of the viral proteins to evade immune surveillance, but through expression of high levels of BART non-coding RNAs can modulate cellular gene expression to provide an optimal environment for maintenance of viral latency in cancer cells. Alteration of cellular gene expression by EBV BART non-coding RNAs for establishment and maintenance of EBV latency in epithelial cells can therefore lead to the oncogenic process for EBV associated tumors

Characterization of mutations associated with cold adapted properties in DelNS1 influenza viruses; potential for new live attenuated influenza vaccine development

Poster Sessions: no. P-619BACKGROUND: Our previous study identified an A14U substitution in the M segment non-coding region that supports replication of NS1-deleted (DelNS1) virus derived from the A/WSN/33 strain. Construction of DelNS1 versions of other influenza strains revealed that different strains require different adaptive mutations to restore virus replication when NS1 is deleted. METHOD: Using a similar strategy, a panel of DelNS1 viruses derived from WSN H1N1, 2009 H1N1, H5N1 and H7N9 were constructed and characterized. These DelNS1 viruses are able to replicate in both MDCK cells and eggs, are avirulent in mice, and can provide cross protection against lethal challenge with heterosubtypic viruses in animals. In the DelNS1 virus derived from 2009 H1N1 virus, we also identified substitutions which facilitate virus replication at lower temperatures (30-33oC) but limit virus replication at higher temperatures (37-39oC). We found this cold adapted DelNS1 2009 H1N1 virus to be able to replicate to comparable titers to the wild type virus in MDCK cells and embryonated chicken eggs, but to cause no disease symptoms in mice, even at the highest dose tested. Furthermore, mice receiving nasal immunization with cold adapted DelNS1 2009 H1N1 virus are protected from lethal challenges with H1N1, H5N1 and H7N9 viruses. The DelNS1 2009 H1N1 virus appears to provide better protection than the currently available cold adapted live attenuated H1N1 virus vaccine strain in animal experiments, suggesting DelNS1 may confer an advantage augment the effectiveness of live attenuated flu vaccines , promoting the induction of stronger and broader immunity to influenza virus infection. Finally, we extended the scope of our DelNS1 system to construct a recombinant virus containing the RBD domain of MERS-CoV in the place of NS1, and found that it is able to protect against infection with MERS-CoV in the DPP4-transgenic mouse model. CONCLUSION: This study characterizes the substitutions required to support virus replication at lower temperature in the absence of NS1 protein in various influenza virus strains, and demonstrates that the combination of DelNS1 and cold adapted properties may have potential as a strategy to develop better and safer live attenuated influenza vaccines