Research Article: A Multiplex RT-PCR Assay for Detection and Differentiation of Avian-Origin Canine H3N2, Equine-Origin H3N8, Human-Origin H3N2, and H1N1/2009 Canine Influenza Viruses

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Chenxi Wang, Qian Wang, Junyi Hu, Honglei Sun, Juan Pu, Jinhua Liu, Yipeng Sun, Yue Wang.


Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.

Partial Text

Canine influenza virus (CIV) is an emerging pathogen that causes acute respiratory infection in dogs [1,2]. Influenza viruses that originated from avian, equine, and human hosts have transmitted to dogs. Among these viruses, H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 have resulted in widespread outbreaks [3–6]. H3N8 CIV was first isolated from racing greyhounds that showed respiratory signs in Florida in January 2004 [4]. Phylogenetic analysis suggested that H3N8 CIV was directly transmitted from horses [4], especially considering that H3N8 CIV had spread among several greyhound racetracks in different states and became the dominant CIV subtype circulated in pet dogs in North America [7,8]. The equine H3N8 influenza virus also caused infection in English foxhounds from the United Kingdom in 2002, which emerged independently of the cases from the United States [9]. Since 2007, an avian-origin H3N2 CIV has been detected in domestic dogs in South Korea and China. Experimental infection of cH3N2 CIVs can lead to direct transmission between dogs [2,5,10]. Serological and virological data have also documented the sporadic transmission and subclinical infection of dogs with human influenza H3N2 viruses [3, 11–13]. During the H1N1/2009 pandemic in human, evidence of H1N1/2009 influenza virus infection was found in dogs in Italy and China [6,14]. All eight genes of the two H1N1/2009 viruses isolated from dogs in China were found to be closely related to H1N1/2009 influenza virus circulated in humans, with nucleotide identities of 98.9%–100% to a human representative H1N1/2009 strain, A/California/04/2009 [6]. Results of a recent serological survey showed seropositivity rates of cH3N2, H1N1/2009, and hH3N2 to be 3.5%, 1.5%, and 1.2%, respectively, which emphasized the co-circulation of different CIVs in China [15].

Previous studies have showed that multiple viruses of different subtypes and host origins have transmitted to dogs. Particularly, virological and serological surveys have documented that H1N1/2009, cH3N2, hH3N2, and equine-origin H3N8 influenza viruses are consistently circulating in dogs [3–6]. A few methods for detecting equine-origin H3N8 and cH3N2 CIV subtypes have been developed [30–32]. However, no study has described a method for distinguishing different influenza virus subtypes that circulate in dogs. Here, we developed an mRT-PCR assay to simultaneously identify and differentiate H1N1/2009, cH3N2, hH3N2, and equine-origin H3N8 influenza viruses in a single sample that contained almost all of the influenza viruses that circulate in dogs. This method permits one-step detection of the CIVs and enables subtyping of those CIVs when dogs are co-infected with several subtypes.