Detection of influenza D virus among swine and cattle, Italy

Recent studies have identified a new genus of the Orthomyxoviridae family. The virus, distantly related to human influenza C virus, has been provisionally designated as influenza D virus. This novel virus was identified for the first time in pigs with influenza-like illness, but subsequent serologic and virologic surveys have suggested cattle as a possible reservoir. Moreover, the virus was shown to infect ferrets used in laboratories as surrogates for humans when investigating influenza infection. In a serologic study conducted on 316 human samples, low antibody titers and a low level of positive samples were detected, suggesting that humans are a possible host to be studied in depth. To investigate the circulation of influenza D viruses among pigs and cattle in Italy, we performed biomolecular and virological tests on clinical samples collected from respiratory outbreaks in Po Valley, the area in Italy with the highest density of swine and cattle farms.

Clinical specimens were screened from swine (n = 150) and cattle (n = 150) for influenza D virus by reverse transcription quantitative PCR. Three nasal swab samples were found positive: 1 from a sow and 2 from cattle, collected from 3 farms located at linear distances ranging from 47 to 80 km. All positive samples were confirmed by partial polymerase basic 1 gene sequencing and submitted to viral isolation in cell cultures as previously described. The virus was isolated on CACO-2 and HRT18 cell cultures only from the sow sample. Cell cultures were tested by using reverse transcription quantitative PCR. Viral RNA was isolated from clinical samples or cell culture by using One-For-All Vet Kit (QIAGEN, Milan, Italy). Full-genome amplification from influenza D virus–positive samples was achieved as previously described. A sequencing library of the purified amplicons was prepared by using NEXTERA-XT kit and sequenced by using a Miseq Reagent Kit v2 in a 250-cycle paired-end run (both from Illumina Inc., San Diego, CA, USA). Sequencing reads were assembled de novo or by using D/swine/Oklahoma/1334/2011 (GenBank accession nos. JQ922305–JQ922311) as a template by Seqman NGen DNASTAR version 11.2.1 (DNASTAR, Madison, WI, USA). Gene sequences from the 3 influenza D viruses isolated in Italy and all the available influenza D virus sequences retrieved from GenBank were aligned with ClustalW by using MEGA5. We analyzed the predicted amino acid sequences for each gene.  Emerging Infectious Diseases  Original web page at Emerging Infectious Diseases