Experimental infection of pigs with the human 1918 pandemic influenza virus

Abstract

Swine influenza was first recognized as a disease entity during the 1918 "Spanish flu" pandemic. The aim of this work was to determine the virulence of a plasmid-derived human 1918 pandemic H1N1 influenza virus (reconstructed 1918, or 1918/rec, virus) in swine using a plasmid-derived A/swine/Iowa/15/1930 H1N1 virus (1930/rec virus), representing the first isolated influenza virus, as a reference. Four-week-old piglets were inoculated intratracheally with either the 1930/rec or the 1918/rec virus or intranasally with the 1918/rec virus. A transient increase in temperature and mild respiratory signs developed postinoculation in all virus-inoculated groups. In contrast to other mammalian hosts (mice, ferrets, and macaques) where infection with the 1918/rec virus was lethal, the pigs did not develop severe respiratory distress or become moribund. Virus titers in the lower respiratory tract as well as macro- and microscopic lesions at 3 and 5 days postinfection (dpi) were comparable between the 1930/rec and 1918/rec virus-inoculated animals. In contrast to the 1930/rec virus-infected animals, at 7 dpi prominent lung lesions were present in only the 1918/rec virus-infected animals, and all the piglets developed antibodies at 7 dpi. Presented data support the hypothesis that the 1918 pandemic influenza virus was able to infect and replicate in swine, causing a respiratory disease, and that the virus was likely introduced into the pig population during the 1918 pandemic, resulting in the current lineage of the classical H1N1 swine influenza viruses.

FIG. 1.

Lethality of the rescued viruses in mice, indicated by the number of surviving animals. Mice were inoculated intranasally with 10⁴ TCID₅₀ per animal of the 1930/rec or the 1918/rec virus. Controls were inoculated intranasally with PBS.

FIG. 2.

Extent of gross lung lesions in the infected piglets. Lung lesions at 5 and 7 dpi in the 1930/rec and 1918/rec virus-infected animals and in the 1918/rec virus-inoculated pigs also at 12 (pig 106) and 17 (pigs 107 and 108) dpi. Total extent of the lung lesions (white columns) and the extent of the lesions in the diaphragmatic lobes (black columns) are given as the total lung surface percentage for individual animals. While the macroscopic lesions in the lungs of the 1930/rec virus-infected animals did not appear to be spreading, the lung lesions in the 1918/rec virus-infected animals were more extensive later times postinfection (after 7 dpi).

FIG. 3.

Gross lung lesions in the infected piglets. A few plum-colored, depressed lobules can be observed in the apical, cardiac, and anterior diaphragmatic lobes of piglets inoculated with the 1930/rec influenza virus. The extent of the lesions appeared to be the same at 3 and 5 dpi (Fig. A and B, respectively). The 1918/rec virus-inoculated piglets had more extensive lesions, and the plum-colored depressed lobules could be observed in the diaphragmatic and cardiac lobes at 5 dpi (C) and 7 dpi (D), while the lesions in 1930/rec virus-inoculated piglets remained localized in the apical lobes. At 17 dpi, plum-colored, depressed lobules were observed throughout the entire lungs of both 1918/rec virus-infected piglets although both lungs also had uniform purple discoloration of the dorsal portion of the diaphragmatic lobe, suggesting hypostatic congestion, likely developed during euthanasia. Panel E illustrates the spider-like/petechial hemorrhages present both on the ventral and dorsal side of lungs at 17 dpi.

FIG. 4.

Microscopic lesions and antigen presence in lungs of infected animals. Microscopic lesions in lungs of pigs inoculated with the 1918/rec or the 1930/rec influenza virus at 5 dpi, using serial lung sections with H&E and IHC staining. H&E staining reveals mononuclear cells infiltrating the small bronchioles in the 1930/rec virus-infected pig (A) compared to the severely affected lungs of the 1918/rec virus-infected piglet with bronchiolitis and pneumonitis and hyperplasia of the bronchiolar epithelium (C). Panel D shows positive IHC staining for influenza virus antigen in bronchiolar epithelial cells in the 1918/rec virus-infected piglet; lower-intensity staining in only few cells of the same type in the 1930/rec virus-infected animal is shown in panel B. Note intense staining both in nuclei and cytoplasm in the 1918/rec virus-infected animal. Bar, 20 μm.

FIG. 5.

Detection of viral RNA in BALFs of infected pigs using real-time RT-PCR. Copy numbers in log₁₀ per 1 ml of BALFs obtained by real-time RT-PCR targeting the matrix protein gene are given for individual animals. The white columns represent piglets inoculated with the 1930/rec influenza virus at 3 dpi (pigs 66, 67, and 83) 5 dpi (pigs 69, 71, 84, and 85), and at 7 dpi (pigs 86 and 87). The gray columns represent piglets inoculated with the 1918/rec virus at 3 dpi (pigs 88, 89, 97, 98, and 103) and at 5 dpi (pigs 90, 91, 99, 100, and 104). Black diamonds indicate samples where no viral RNA was detected: at 7 dpi, pigs 92, 93, 101, 102, and 105 inoculated with the 1918/rec virus and piglets 70 and 72 inoculated with the 1930/rec virus; at 3 dpi, pig 82 inoculated with the 1930/rec virus. Viral RNA was not detected in the 1918/rec virus-inoculated piglets after 7 dpi.