Triple reassortment increases compatibility among viral ribonucleoprotein genes of contemporary avian and human influenza A viruses

Compatibility among the influenza A virus (IAV) ribonucleoprotein (RNP) genes affects viral replication efficiency and can limit the emergence of novel reassortants, including those with potential pandemic risks. In this study, we determined the polymerase activities of 2,451 RNP reassortants among three seasonal and eight enzootic IAVs by using a minigenome assay. Results showed that the 2009 H1N1 RNP are more compatible with the tested enzootic RNP than seasonal H3N2 RNP and that triple reassortment increased such compatibility. The RNP reassortants among 2009 H1N1, canine H3N8, and avian H4N6 IAVs had the highest polymerase activities. Residues in the RNA binding motifs and the contact regions among RNP proteins affected polymerase activities. Our data indicates that compatibility among seasonal and enzootic RNPs are selective, and enzoosis of multiple strains in the animal-human interface can facilitate emergence of an RNP with increased replication efficiency in mammals, including humans.

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs

ABSTRACT Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations. IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1EGY and H9N2EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2EGY, expressed individually and in combination in the genetic background of H5N1EGY, were genetically compatible with the other H5N1EGY segments. At 37 °C the replication efficiencies of H5N1EGY reassortants expressing the H9N2EGY polymerase subunits PB2 and PA (H5N1PB2-H9N2EGY, H5N1PA-H9N2EGY) were higher than the wild-type H5N1EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1PB2-H9N2EGY, but reduced for H5N1PA-H9N2EGY. At 33 °C and 39 °C, H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY replicated to higher levels than the wild-type H5N1EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1EGY. Genetic analysis of the polymerase-encoding segments revealed that the PAH9N2EGY and PB2H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1EGY enabled the mutant virus H5N1PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

Evolution of H5-Type Avian Influenza A Virus Towards Mammalian Tropism in Egypt, 2014 to 2015

Highly pathogenic avian influenza viruses (HPAIV) of the H5-subtype have circulated continuously in Egypt since 2006, resulting in numerous poultry outbreaks and considerable sporadic human infections. The extensive circulation and wide spread of these viruses in domestic poultry have resulted in various evolutionary changes with a dramatic impact on viral transmission ability to contact mammals including humans. The transmitted viruses are either (1) adapted well enough in their avian hosts to readily infect mammals, or (2) adapted in the new mammalian hosts to improve their fitness. In both cases, avian influenza viruses (AIVs) acquire various host-specific adaptations. These adaptive variations are not all well-known or thoroughly characterized. In this study, a phylogenetic algorithm based on the informational spectrum method, designated hereafter as ISM, was applied to analyze the affinity of H5-type HA proteins of Egyptian AIV isolates (2006–2015) towards human-type cell receptors. To characterize AIV H5-HA proteins displaying high ISM values reflecting an increased tendency of the HA towards human-type receptors, recombinant IV expressing monobasic, low pathogenic (LP) H5-HA versions in the background of the human influenza virus A/PR/8/1934(H1N1) (LP 7+1), were generated. These viruses were compared with a LP 7+1 expressing a monobasic H5-HA from a human origin virus isolate (human LP-7271), for their receptor binding specificity (ISM), in vitro replication efficiency and in vivo pathogenicity in mammals. Interestingly, using ISM analysis, we identified a LP 7+1 virus (LP-S10739C) expressing the monobasic H5-HA of AIV A/Chicken/Egypt/S10739C/2015(H5N1) that showed high affinity towards human-type receptors. This in silico prediction was reflected by a higher in vitro replication efficiency in mammalian cell cultures and a higher virulence in mice as compared with LP-7271. Sequence comparison between the LP-S10739C and the LP-7271 H5-HA, revealed distinct amino acid changes. Their contribution to the increased mammalian receptor propensity of LP-S10739C demands further investigation to better deduce the molecular determinant behind the reported high morbidity of 2014 to 2015 HPAI H5N1 virus in humans in Egypt. This study provides insights into the evolution of Egyptian H5 HPAIVs and highlights the need to identify the viral evolution in order to recognize emerging AIV with the potential to threaten human and animal populations.

Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

Influenza Virus Polymerase Mutation Stabilizes a Foreign Gene Inserted into the Virus Genome by Enhancing the Transcription/Replication Efficiency of the Modified Segment

ABSTRACT We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venus-PR8, we discovered that the PB2-E712D mutation stabilizes the Venus gene. Here, we explored the mechanisms by which Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Deep sequencing analysis revealed that PB2-E712D does not cause an appreciable change in the mutation rate, suggesting that the stability of the Venus gene is not affected by polymerase fidelity. We found by using quantitative real-time PCR that WT-Venus-PR8 induces high-level interferon beta (IFN-β) expression. The induction of IFN-β expression seemed to result from the reduced transcription/replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription/replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. Loss of the Venus gene in WT-Venus-PR8 appeared to be caused by internal deletions in the NS segment. Moreover, to further our understanding of the Venus stabilization mechanisms, we identified additional amino acid mutations in the virus polymerase complex that stabilize the Venus gene. We found that some of these amino acids are located near the template exit or the product exit of the viral polymerase, suggesting that these amino acids contribute to the stability of the Venus gene by affecting the binding affinity between the polymerase complex and the RNA template and product. IMPORTANCE The reverse genetics method of influenza virus generation has enabled us to generate recombinant viruses bearing modified viral proteins. Recombinant influenza viruses expressing foreign genes have become useful tools in basic research, and such viruses can be utilized as efficient virus vectors or multivalent vaccines. However, the insertion of a foreign gene into the influenza virus genome often impairs virus replication, and the inserted genes are unstable. Elucidation of the mechanisms of foreign gene stabilization will help us to establish useful recombinant influenza viruses.