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Establishment of a Reverse Genetics System for Influenza D Virus.

ABSTRACT: Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil (U), contrary to previous reports, and we were then able to successfully generate recombinant IDV by cotransfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and nucleoprotein into human rectal tumor 18G (HRT-18G) cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that the recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles compared to that of the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain a recombinant virus with the same replicative capacity as the wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity, as well as in those contributing to the development of BRDC countermeasures.IMPORTANCE The bovine respiratory disease complex (BRDC) causes high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of the BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity and will also lead to the development of new countermeasures against the BRDC.

SUBMITTER: Ishida H

PROVIDER: S-EPMC7199395 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Establishment of a Reverse Genetics System for Influenza D Virus.

Ishida Hiroho H Murakami Shin S Kamiki Haruhiko H Matsugo Hiromichi H Takenaka-Uema Akiko A Horimoto Taisuke T

Journal of virology 20200504 10

Influenza D virus (IDV) was initially isolated in the United States in 2011. IDV is distributed worldwide and is one of the causative agents of the bovine respiratory disease complex (BRDC), which causes high morbidity and mortality in feedlot cattle. The molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establ ...[more]

PMID: 32102883

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Project description:Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmid-based system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replication-incompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3' conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reverse-genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.IMPORTANCE Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse-genetics system that can generate infectious viruses with replication kinetics similar to those of wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor-binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse-genetics system and minireplicon reporter assay described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.

Project description:Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1-2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. IMPORTANCE Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo. Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

Dataset Information

Establishment of a Reverse Genetics System for Influenza D Virus.

Publications

Establishment of a Reverse Genetics System for Influenza D Virus.

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