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Influenza D viruses (IDV) belong to a new genus in the family Orthomyxoviridae. IDV is the aetiologic agent of acute, mild respiratory disease in ungulate species with agricultural importance (cattle, pigs, sheep, goats, camels, etc.). Despite the initial isolate being of porcine origin, serological data suggest cattle to be the primary host of IDV. The study aims were twofold: elucidating species‐specific replication kinetics of IDV in bovine and porcine hosts and defining the interspecies potential with two different IDV strains. Three calves and three pigs were intranasally inoculated with the prototypic strain D/swine/Oklahoma/1334/2017 or a genetically distinct cattle isolate, D/bovine/Texas/72/2017. Two days following infection, three naïve pigs and three naïve calves were co‐housed with inoculated calves and pigs, respectively. The species of IDV origin had no effect on virus replication kinetics in the upper respiratory tract of inoculated calves and pigs; similar shedding profiles were observed for each species and virus. However, interspecies transmission was found to be associated with virus origin species; D/bovine/Texas/72/2017 and D/swine/Oklahoma/1334/2017 were directly transmitted only to contact calves or pigs, respectively. Even so, transmission efficiency was higher for calves compared to pigs. Together, these data show that cattle and pigs are permissive for IDV replication, but IDV transmission may be species dependent. Host‐specific mutations likely influenced transmission efficiencies between agriculturally important mammalian species. [ABSTRACT FROM AUTHOR]

The newly identified influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range with a broad geographical distribution. Despite the first appearance in U.S. pig herds in 2011, subsequent studies demonstrated that IDV is widespread in global cattle populations, supporting a theory that IDV utilizes bovines as a primary reservoir. Our investigation of the two reference influenza D viruses, D/swine/Oklahoma/1334/2011 (OK/11), isolated from swine, and D/Bovine/Oklahoma/660/2013 (660/13), isolated from cattle, revealed that 660/13 replicated to titers approximately 100-fold higher than those for OK/11 in multiple cell lines. By using a recently developed IDV reversegenetics system derived from low-titer OK/11, we generated recombinant chimeric OK/11 viruses in which one of the seven genome segments was replaced with its counterpart from high-titer 660/13 virus. Further characterization demonstrated that the replication level of the chimeric OK/11 virus was significantly increased only when harboring the 660/13 nucleoprotein (NP) segment. Finally, through both gain-of-function and loss-of-function experiments, we identified that one amino acid residue at position 381, located in the body domain of NP protein, was a key determinant for the replication difference between the low-titer OK/11 virus and the high-titer 660/13 virus. Taken together, our findings provide important insight into IDV replication fitness mediated by the NP protein, which should facilitate future study of the infectious virus particle production mechanism of IDV. IMPORTANCE Little is known about the virus infection and production mechanism for newly discovered influenza D virus (IDV), which utilizes bovines as a primary reservoir, with frequent spillover to new hosts, including swine. In this study, we showed that of two well-characterized IDVs, 660/13 replicated more efficiently (approximately 100-fold higher) than OK/11. Using a recently developed IDV reverse-genetics system, we identified viral nucleoprotein (NP) as a primary determinant of the different replication capacities observed between these two nearly identical viruses. Mechanistic investigation further revealed that a mutation at NP position 381 evidently modulated virus fitness. Taken together, these observations indicate that IDV NP protein performs a critical role in infectious virus particle production. Our study thus illustrates an NP-based mechanism for efficient IDV infection and production in vitro. [ABSTRACT FROM AUTHOR]

In 2011, a new virus was isolated from swine with influenza-like symptoms in Oklahoma, USA. Later in 2013, it was also evidenced in cattle, considered as its natural reservoir, elsewhere in the USA. This new virus has certain resemblance with Influenza C virus (ICV), predominately a human pathogen. Due to its inability to re-assort with ICV, it is now classified as Influenza D virus (IDV) and is a new candidate in the Orthomyxoviridae family. It causes mild respiratory disease in several animal species and replicates in both upper and lower respiratory tract. To date, serological evidence was demonstrated in various animal species and humans in all continents except Australia. It is transmitted through direct contact or through aerosol routes. Not much is known about its potential impact to animal and human health but it poses a potential risk as an emerging threat to cattle-workers. Currently, limited data is available on its global occurrence and distribution. Therefore, in current review, we summarize the global available data regarding epidemiology, pathology, zoonotic potential and future perspectives of this virus. In conclusion, continuous surveillance and risk assessment of this emerging virus is required. [ABSTRACT FROM AUTHOR]

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-plasmidbased 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 replicationincompetent 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 crossrecognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reversegenetics system that can be used to further study replication, tropism, and pathogenesis of IDV. [ABSTRACT FROM AUTHOR]

• Influenza D has a wide host tropism in both domestic and wild animals despite a very specific receptor preference. • Differences in tissue tropism exist between the two major influenza D clades in water buffalo, Asian elephant, and hedgehog. • Despite positive serosurveillance data, wild Suidae and Cervidea do not express influenza D receptors. • Influenza D has potential to spread from domesticated animals to wildlife and vice versa. The influenza D virus (IDV) uses a trimeric hemagglutinin-esterase fusion protein (HEF) for attachment to 9-O-acetylated sialic acid receptors on the cell surface of host species. So far research has revealed that farm animals such as cattle, domestic pigs, goats, sheep and horses contain the necessary receptors on the epithelial surface of the respiratory tract to accommodate binding of the IDV HEF protein of both worldwide clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660). More recently, seroprevalence studies have identified IDV-seropositive wildlife such as wild boar, deer, dromedaries, and small ruminants. However, no research has thus far been conducted in wildlife to reveal the distribution of acetylated sialic acid receptors that accommodate binding of IDV. Using our previously developed tissue microarray (TMA) system, we developed TMAs containing respiratory tissues of various wild and domestic species including wild boar, deer, dromedary, springbok, water buffalo, tiger, hedgehog, and Asian elephant. Protein histochemical staining of these TMAs with HEF proteins showed no receptor binding for wild Suidae, Cervidae and tiger. However, receptors were present in dromedary, springbok, water buffalo, Asian elephant, and hedgehog. In contrast to previously tested farm animals, a difference in host tropism was observed between the D/OK and D/660 clade HEF proteins in Asian elephant, and water buffalo. These results show that IDV can attach to the respiratory tract of wildlife which might facilitate transmission of IDV between wildlife and domestic animals. [ABSTRACT FROM AUTHOR]

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids. [ABSTRACT FROM AUTHOR]