Bovine respiratory disease complex (BRDC) is one of the most common causes of death in livestock in US feedlots and feedlots worldwide (10). During October 2016–January 2018, we collected 1,525 samples (mainly nasal swab and lung tissue specimens) from cattle in the Midwest of the United States and submitted them to Kansas State Veterinary Diagnostic Laboratory (Manhattan, Kansas, USA) for BRDC diagnostic testing. We screened samples for ICV by real-time reverse transcription PCR, as well as for 10 other BRDC-associated pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Bibersteinia trehalosi, Mycoplasma bovis, bovine viral diarrhea virus, bovine respiratory syncytial virus, bovine respiratory coronavirus, bovine herpesvirus 1, and influenza D virus; Technical Appendix,). We sequenced a 590-bp fragment of the matrix gene from 12 ICV-positive samples (GenBank accession nos. MH421865–73; Technical Appendix Table) to confirm the PCR results and perform a phylogenetic analysis. We selected 1 isolate (C/bovine/Montana/12/2016) for complete genome sequencing (GenBank accession nos. MH348113–9).

Of 1,525 samples, 64 (4.20%) were positive for ICV: 38 samples with a cycle threshold (C_t) <36 and 26 with a C_t 36–39. The most common pathogens were bovine respiratory coronavirus (34.98%), M. bovis (32.27%), and M. haemolytica (17.04%). The remaining BRDC pathogens were present but less prevalent: P. multocida (13.42%), H. somni (12.58%), influenza D virus (11.93%), bovine respiratory syncytial virus (9.19%), bovine viral diarrhea virus (7.05%), B. trehalosi (3.47%), and bovine herpesvirus 1 (2.95%).

Co-infections with >1 pathogen are common in BRDC cases. ICV-positive samples were also found to be positive for >1 bovine respiratory disease pathogen (n = 12, Table 1), the most common being M. bovis (9/12), followed by H. somni (7/12), and M. haemolytica (6/12). Among the ICV-positive samples, ICV12 was strongly positive (C_t 15.81); this sample was also positive for M. haemolytica and P. multocida, both bacterial pathogens commonly associated with secondary infections. Other BRDC pathogens associated with secondary infections (M. bovis, bovine viral diarrhea virus, and H. somni) were also detected in samples ICV4, ICV16, ICV18, and ICV20 (11–13). These results suggest that ICV is associated with bovine respiratory disease in cattle.

We further evaluated 12 strong positive (C_t<31) samples by sequencing a 590-bp fragment of their matrix gene. Alignment of the partial matrix gene sequences indicated that the isolates in 3 samples (ICV2, ICV3, and ICV4) obtained from different cattle on the same farm in Oklahoma were identical. Because these 3 influenza viruses were most likely the same strain, the virus in just 1 sample (ICV2) was used for phylogenetic analysis. The matrix gene sequence in sample ICV5 from Missouri (GenBank accession no. MH421866) was identical to that in ICV6 from Colorado (GenBank accession no. MH421867).

Figure

Figure. Phylogenetic tree of 10 bovine ICV isolates compared with 1 porcine and 195 human ICV isolates (not labeled), United States. Some subtrees containing only human isolates were collapsed to decrease the...

Phylogenetic analysis indicated that the bovine ICV isolates are closely related to the porcine and human ICV isolates, and the bovine ICV isolates are more closely related to each other (Table 2; Figure). The bovine ICV isolates’ partial matrix gene sequences shared high nucleotide identities (≈98%). For both partial matrix gene sequences and the whole genome sequence (7 segments), the nucleotide identity between bovine and human isolates was ≈95%. The full genome sequence of C/bovine/Montana/12/2016 from sample ICV12 had high nucleotide identity to C/Mississippi/80 (and several other human ICV strains), with an overall identity of 97.1%. Nucleotide identities between these 2 isolates were also high for each gene: 97.0% for polymerase basic 2, 97.7% for polymerase basic 1, 97.5% for polymerase 3, 96.2% for hemagglutininesterase, 96.8% for nucleoprotein, 96.8% for matrix, and 97.6% for nonstructural protein. The only porcine ICV isolate available was more closely related to human (≈98% identity) isolates than bovine (≈95% identity) isolates; the porcine ICV isolate had nearly the same identity that the human ICV isolates had among each other (Table 2).

The phylogenetic tree of the partial matrix gene sequences (Figure) further demonstrates the relationship between bovine and human ICV isolates. All bovine ICVs formed a separate clade on the phylogenetic tree, with a 99% bootstrap value. Of the 195 partial matrix gene sequences from human ICVs, the 10 corresponding sequences from bovine ICVs had the highest identities (average 96.70%) to those from C/Mississippi/80 (GenBank no. AB000720.1), C/Nara/82 (GenBank no. AB000723), and C/Kyoto/41/82 (GenBank no. AB000724) and the lowest identities (average 94.21%) to those from C/Yamagata/30/2014 (GenBank no. LC123874) and C/Yamagata/32/2014 (GenBank no. LC123875).

This study confirms the presence of ICV in US cattle with clinical signs of bovine respiratory disease. Although interspecies transmission of influenza viruses occurs between humans and other animals, we do not have data that indicates ICV is a zoonotic pathogen. However, the full genome sequence of C/bovine/Montana/12/2016 has 97.1% nucleotide identity with the human isolate C/Mississippi/80, which is within the range of average identities among human isolates. More detailed investigations are needed to confirm if ICV is involved in bovine respiratory disease, to characterize the relationship between bovine and human ICV strains, and to determine the zoonotic potential of bovine ICV isolates to cause human disease.