(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >10⁵ genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

The Respirovirus genus, family Paramamixoviridae, includes respiratory viral pathogens. Here we report the identification and genetic characterization of a respirovirus in an Alpine chamois showing interstitial pneumonia associated with catarrhal bronchopneumonia. The full-genome characterization of this respirovirus, named ChamoisRV/IT2014, revealed low similarities to caprine respirovirus (77.1%), bovine respirovirus (74.5%) and human respirovirus (72.0%). The phylogenetic analyses based on the full-length genome sequence of the novel isolate and reference respirovirus strains showed that ChamoisRV/IT2014 clustered with caprine respirovirus but formed a separate branch. The phylogenetic tree topology of complete large protein amino acid sequences, representing the current species demarcation criterion for Respirovirus genus, showed a 0.05 branch length of ChamoisRV/IT2014 sequence between the nearest node and the tip of the branch, suggesting that this virus belongs to a novel species. This new isolate in a new host species raises several questions to be addressed on the epidemiological role of chamois and the risks of cross-transmission between wild ruminants and livestock.