Padykula, I. | Search bioRxiv

bioRxiv2024

Pandemic Risk Assessment for a Swine Influenza A Virus in Comparative Human Substrates

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify what animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019, using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest selection of models is important for meaningful risk assessment. Article Summary LineA novel swine influenza A virus was rigorously assessed for pandemic potential in animal models and human surrogate culture models, illustrating a wide range in potential public health risk dependent on the model utilized.

microbiology↗

bioRxiv2019

Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. Viral culture coupled with third-generation sequencing were tested for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus, epizootic hemorrhagic disease virus, infectious bronchitis virus, two influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20x coverage depth for all seven viruses, including a sample containing two viruses. Each lineage-typing region had at least 26x coverage depth for all viruses. Furthermore, analyzing the canine distemper virus sample through a pipeline devoid of canine distemper virus reference sequences modeled the ability of this protocol to detect unknown viruses. These results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, nonsegmented, and segmented RNA viruses.

pathology↗

bioRxiv2018

Plasmodium male gametocyte development and transmission are critically regulated by general and transmission-specific members of the CAF1/CCR4/NOT complex

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites also regulate the stability and turnover of transcripts to provide more comprehensive gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized PyCCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase, which prevents PyCCR4-1 association with the complex, leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that PyCCR4-1 does not have gross changes in UTR or poly(A) tail length. We conclude that general and transmission-specialized members of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.\n\nAUTHOR SUMMARYMalaria is a disease caused by Plasmodium parasites, which are transmitted during an infectious blood meal by anopheline mosquitoes. Transmission of the sexual stages of the parasite to mosquitoes requires the proper regulation of specific mRNAs. While much work has been done to characterize regulation of mRNAs in female gametocytes, little has been done to assess this regulation in male gametocytes. Here, we demonstrate that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in both male and female parasites, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are less able to productively infect mosquitoes. We find that PyCCR4-1 requires its association with PyCAF1 and by proxy, the rest of the complex, in order to perform its functions based upon experiments in both Plasmodium yoelii and Plasmodium falciparum. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly and indirectly to modulate transcript abundance. These findings demonstrate that the combined effects of the CAF1/CCR4/NOT complex upon specific mRNAs are important for both male and female gametocytes, and that this regulation is required for efficient transmission to the mosquito vector.

microbiology↗