Establishment of a Reverse Genetics System for Influenza D Virus

Abstract

250 words 15 Text 4,170 words 16 2 ABSTRACT Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is 17distributed worldwide and is one of the causative agents of bovine respiratory disease 18 complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. 19 Molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics 20 systems are vital tools not only for studying the biology of viruses, but also for use in 21 applications such as recombinant vaccine viruses. Here, we report the establishment of a 22 plasmid-based reverse genetics system for IDV. We first verified that the 3′-terminal 23 nucleotide of each 7-segmented genomic RNA contained uracil in contrary to the 24 previous report, and were then able to successfully generate recombinant IDV by 25 co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids 26 expressing polymerase proteins and NP into HRT-18G cells. The recombinant virus had 27 a growth deficit compared to the wild-type virus, and we determined the reason for this 28 growth difference by examining the genomic RNA content of the viral particles. We 29 found that recombinant virus incorporated an unbalanced ratio of viral RNA segments 30 into particles as compared to the wild-type virus, and thus we adjusted the amount of 31 each plasmid used in transfection to obtain recombinant virus with the same replicative 32 capacity as wild-type virus. Our work here in establishing a reverse genetics system for 33 IDV will have a broad range of applications, including uses in studies focused on better 34 understanding IDV replication and pathogenicity as well as those contributing to the 35 development of BRDC countermeasures. 36 37 IMPORTANCE Bovine respiratory disease complex (BRDC) exhibits high mortality 38 and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is 39 considered to be a causative agent of BRDC. Here, we developed a reverse genetics 40 3 system that allows for the generation of IDV from cloned cDNAs, and the introduction 41 of mutations into the IDV genome. This reverse genetics system will become a 42 powerful tool for use in studies related to understanding the molecular mechanisms of 43 viral replication and pathogenicity, and will also lead to the development of new 44 countermeasures against BRDC. 45 46 KEYWORD bovine respiratory disease complex, influenza D virus, mutant, 47 recombinant virus, reverse genetics, transfection 48 49 50 Influenza D virus (IDV), a member of the family Orthomyxoviridae, was first isolated 52 from pigs with respiratory illness in Oklahoma, USA, in 2011 (1, 2). Epidemiological 53 analyses revealed that cattle are the main host of the virus, due to their high 54 seroprevalence for IDV (2, 3). Further epidemiological studies revealed that IDVs 55 circulate in cattle in many countries including the USA (2-4), Mexico (5), China (6), 56 Japan (7, 8), France (9), Italy (10), Ireland (11), Luxembourg (12), and African 57 countries (13). Furthermore, serolo...

Establishment of a reverse genetic system from a bovine derived Influenza D virus isolate

“…During this study we did not determine the 5′ and 3′ NCRs of each genomic segment. However, because of the importance of the NCR in viral replication and the previous reported sequence discrepancy among different IDV isolates in public sequence repositories, we decided to determine the 5′ and 3′ NCR sequences of the genomic segments from one of our bovine IDV isolates [38]. Based on the Ct-value and available amount of nasopharyngeal material, this was chosen to be the D/Switzerland/bovine/CN286 isolate (Glaus et al, Manuscript in preparation).…”

The first decade of research advances in influenza D virus

“…The underlying mechanisms that contribute to the unique biology and cross-species transmission of IDV are still not fully understood. The recent development of robust reversegenetics systems for IDV [69,73] will facilitate future studies aiming to advance our understanding of the biology of IDV at the molecular level and illuminate the viral determinants driving cross-species transmission.…”

“…It is worth mentioning that two independent groups have recently developed a plasmid-based IDV reverse-genetics system. One involves a 7-plasmid system [69], while the other relies on an 11-plasmid method [73]. Viruses rescued Fig.…”

The host cellular protein Ndufaf4 interacts with the vesicular stomatitis virus M protein and affects viral propagation

“…This domain is also involved in the viral budding process by recruiting cellular partners. The globular domain of M can inhibit the host antiviral response to promote viral replication by inhibiting host gene expression or impairing the type I IFN system [3]. To determine which domain of M participates in the M-Ndufaf4 interaction, each domain of M was cloned into the PGBKT7 plasmid, and its ability to bind Ndufaf4 was determined in the Y2H system.…”

“…The M protein of VSV is a multifunctional protein that plays an pivotal role in modulation of the host antiviral immune response [3,4], suppression of host transcription/translation [5,6], virion assembly/budding [7][8][9][10], and the induction of apoptosis [11,12]. To perform numerous functions, viral proteins may need to interact with speci c host proteins.…”