Characterization of swine-origin H1N1 canine influenza viruses

Abstract

Host switch events of influenza A viruses (IAVs) continuously pose a zoonotic threat to humans. In 2013, swine-origin H1N1 IAVs emerged in dogs soon after they were detected in swine in the Guangxi province of China. This host switch was followed by multiple reassortment events between these H1N1 and previously circulating H3N2 canine IAVs (IAVs-C) in dogs. To evaluate the phenotype of these newly identified viruses, we characterized three swine-origin H1N1 IAVs-C and one reassortant H1N1 IAV-C. We found that H1N1 IAVs-C predominantly bound to human-type receptors, efficiently transmitted via direct contact in guinea pigs and replicated in human lung cells. Moreover, the swine-origin H1N1 IAVs-C were lethal in mice and were transmissible by respiratory droplets in guinea pigs. Importantly, sporadic human infections with these viruses have been detected, and preexisting immunity in humans might not be sufficient to prevent infections with these new viruses. Our results show the potential of H1N1 IAVs-C to infect and transmit in humans, suggesting that these viruses should be closely monitored in the future.

Keywords: H1N1; Host switch; canine; influenza A viruses; reassortant.

Figure 1.

Receptor-binding specificity of H1N1 IAVs-C using a flow cytometry-based binding assay. The binding of virus to five different Neu5Ac-linked glycans was tested: (A) WZ11, (B) LZ56, (C) WZ2, (D) LZ36, (E) Cal09, (F) Illi15, (G) Beth15, and (H) Viet05. E, F, G, and H were included in the analysis for comparison and as controls. α-2,3 glycans are in blue and α-2,6 glycans are in red. Structure of glycans is provided in Figure S1A.

Figure 2.

Virulence and replication of H1N1 IAVs-C in mice. To test the viral replication, mice in each group were infected intranasally (i.n.) with 10⁶ PFU: (A) WZ11, (B) LZ56, (C) WZ2, (D) LZ36, (E) Cal05, or (F) Illi15. Virus titers (upper panels) of lung and nasal turbinate on 3 and 5 days post-infection (d.p.i.) were shown as the mean titer of three mice. Five mice per group were i.n. inoculated with 10², 10³, 10⁴, 10⁵, or 10⁶ PFU of infected virus. Body weight (middle panels) and survival (lower panels) and were monitored daily. PFU, plaque-forming units; MLD₅₀, 50% mouse-lethal dose.

Figure 3.

Direct contact transmission and aerosol droplet transmission of H1N1 IAVs-C in guinea pigs. Groups of three guinea pigs were inoculated i.n. with 10⁶ PFU of tested viruses: (A) WZ11, (B) LZ56, (C) WZ2, (D) LZ36, (E) Cal09, or (F) Illi15. Three naïve guinea pigs were each placed in the same cage for direct contact transmission or in the adjacent cage for aerosol droplet transmission at 24 h post-inoculation (h.p.i.). Nasal washes were collected every 2 days from all animals beginning on 2 d.p.i. (one day post-exposure) for the detection of virus shedding. Each colour bar represents the virus titer from an individual animal. ND, not done.

Figure 4.

Phylogenetic trees of the HA (A) gene of the H1Nx and NA (B) gene of HxN1 IAVs. Representative H1 and N1 sequences were downloaded from the Influenza Virus Resource at NCBI’s GenBank. Alignments and phylogenetic trees were generated using MAFFT. The trees were based on amino acid sequence (open reading frame) of HA and NA. Avian isolates were in blue; swine isolates were in pink; human isolates were in green; canine isolates were in red. Red star indicates virus used in this study.

Figure 5.

HI profiles and NI profiles for adult human against H1N1 IAVs-C. (A) HI titers of adult human plasma samples (n = 36) measured against H1N1 IAVs-C (B) NI titers of plasma against the NA of H1N1 IAVs-C. One-way analysis of variance (ANOVA) was used to determine whether there were any statistical significance between the HI titers and NI titers of the canine IAVs to Cal09 data set (*p ≤ 0.05, ***p ≤ 0.001). The same point indicated by its colour and shape represents the same donor. Bars represent the geometric mean. The dashed line indicates the starting serum dilutions used in both HI and NI assays. HI, hemagglutination inhibition; NI, neuraminidase inhibition.

Figure 6.

Growth properties of H1N1 IAVs-C in differentiated HTBE cells (A & B) and MDCK cells (C & D). Cells were infected with viruses at MOI of 0.01 and incubated at 37°C (A & C) and 33°C (B & D). Supernatants were harvested at the indicated time points and the virus titers were determined by means of plaque assays in MDCK cells. Error bars indicated SDs from three independent experiments. HTBE, human tracheobronchial epithelial; MDCK, main Darby canine kidney; MOI, multiplicity of infection.