Detection of Influenza D Virus among Swine and Cattle, Italy

To the Editor: Recent studies have identified a new genus of the Orthomyxoviridae family (1–5). 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 (1), but subsequent serologic and virologic surveys have suggested cattle as a possible reservoir (2–4). Moreover, the virus was shown to infect ferrets used in laboratories as surrogates for humans when investigating influenza infection (1). In a serologic study conducted on 316 human samples, low antibody titers and a low level of positive samples (1.3%) were detected (1), 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.


To the Editor:
Recent studies have identified a new genus of the Orthomyxoviridae family (1)(2)(3)(4)(5). 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 (1), but subsequent serologic and virologic surveys have suggested cattle as a possible reservoir (2)(3)(4). Moreover, the virus was shown to infect ferrets used in laboratories as surrogates for humans when investigating influenza infection (1). In a serologic study conducted on 316 human samples, low antibody titers and a low level of positive samples (1.3%) were detected (1), 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.
We screened clinical specimens from swine (n = 150) and cattle (n = 150) for influenza D virus by reverse transcription quantitative PCR (1). 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 (5,6). The virus was isolated on CACO-2 and HRT18 cell cultures only from the sow sample (D/swine/Italy/199723-3/2015). 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 (3). 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 (7). We analyzed the predicted amino acid sequences for each gene.
Phylogenetic trees of the individual segments were inferred by using the maximum-likelihood method implemented in the IQ-TREE package 0.9.6 (8). The robustness of the maximum-likelihood trees was evaluated by bootstrap analysis by comparison to 1,000 bootstrap samples. The swine isolate D/swine/Italy/199723-3/2015 was fully sequenced (GenBank accession nos. KT592530-KT592536). To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV) (Nairovirus, Bunyaviridae), the causative agent of Crimean-Congo hemorrhagic fever, has been detected in sub-Saharan Africa, southeastern Europe, the Middle East, and central Asia. The virus has been detected in >31 species of ticks and is transmitted to humans by bite of infected ticks (mainly of the genus Hyalomma) or by contact with body fluids or tissue of viremic patients or livestock. The disease is characterized by fever, myalgia, headache, vomiting, and sometimes hemorrhage; reported mortality rate is 10%-50% (1).
CCHFV strains currently constitute 7 evolutionary lineages, 1 of which (Europe 2) contains the prototype strain AP92, which was isolated in 1975 from Rhipicephalus bursa ticks collected from goats in Greece (2). This strain seems to have low or no pathogenicity for humans; only a few mild cases have been reported (3). This observation is supported by the relatively high (14.4%) seroprevalence but no clinical cases in humans in northwestern Greece (4). The documented tick carriers of this strain are R. bursa and Hyalomma marginatum (5).
Hyalomma aegyptium ticks are highly host specific; adults feed almost entirely on tortoises of the genus Testudo (6) and occassionally on hedgehogs and hares. Unlike adult ticks, the larvae and nymphs are less host specific and feed on a wide spectrum of hosts (e.g., other reptiles, birds, and mammals [including humans]) (7). This trait elevates the epidemiologic role of the tick as a possible bridge vector connecting wildlife, domestic animals, and humans.
To determine the biological and epidemiological role of H. aegyptium ticks, during 2009-2010, we collected 56 adult ticks from 12 Testudo graeca tortoises at a locality near the city of Aflou in Laghouat Province, Algeria. We tested the ticks for probable CCHFV infection by using nested reverse transcription PCR (8), which amplifies a partial fragment of the CCHFV small RNA segment. We slightly modified the assay: reverse transcription time was 60 minutes and annealing temperature was 52°C (9).
Our findings demonstrate the presence of CCHFV in Algeria, either recently introduced or overlooked. The nearest location where CCHFV has been reported is the Zouala region in Morocco, where the virus was detected in H. marginatum tick larvae and nymphs collected from