Establishment of a Reverse Genetics System for Influenza D Virus.

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.

[L-Lysine-α-Oxidase in vitro Activity in Experiments on Models of Viruses Sindbis, Forest-Spring Encephalitis, Western Nile, Tyaginya and Dhori].

The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses.

Mx1 sensitivity: Batken virus is an orthomyxovirus closely related to Dhori virus.

Batken virus, isolated from mosquitoes and ticks, was tentatively classified as a member of the family Bunyaviridae. Here we show that Batken virus is inhibited by the interferon-induced Mx1 protein of mice which selectively blocks the growth of orthomyxoviruses, including Thogoto and Dhori viruses. Furthermore, we show that Batken virus multiplication is characterized by accumulation of viral proteins in the nucleus and by budding of viral particles from the cell surface. Serological cross-reactions between Batken and Dhori viruses revealed a phylogenetic relationship of these viruses, as previously also proposed by D. K. Lvov. Fragments of the Batken virus glycoprotein and nucleoprotein genes were amplified by RT-PCR. The deduced amino acid sequences were similar to the corresponding Dhori virus sequences. Therefore, Batken virus should be classified into the newly established genus Thogotovirus of the family Orthomyxoviridae. Finally, our results demonstrate that Mx1 susceptibility of orthomyxoviruses is a reliable marker in the hunt for new family members.