The tree shrew is a promising model for the study of influenza B virus infection

Abstract

Background: Influenza B virus is a main causative pathogen of annual influenza epidemics, however, research on influenza B virus in general lags behind that on influenza A viruses, one of the important reasons is studies on influenza B viruses in animal models are limited. Here we investigated the tree shrew as a potential model for influenza B virus studies.

Methods: Tree shrews and ferrets were inoculated with either a Yamagata or Victoria lineage influenza B virus. Symptoms including nasal discharge and weight loss were observed. Nasal wash and respiratory tissues were collected at 2, 4 and 6 days post inoculation (DPI). Viral titers were measured in nasal washes and tissues were used for pathological examination and extraction of mRNA for measurement of cytokine expression.

Results: Clinical signs and pathological changes were also evident in the respiratory tracts of tree shrews and ferrets. Although nasal symptoms including sneezing and rhinorrhea were evident in ferrets infected with influenza B virus, tree shrews showed no significant respiratory symptoms, only milder nasal secretions appeared. Weight loss was observed in tree shrews but not ferrets. V0215 and Y12 replicated in all three animal (ferrets, tree shrews and mice) models with peak titers evident on 2DPI. There were no significant differences in peak viral titers in ferrets and tree shrews inoculated with Y12 at 2 and 4DPI, but viral titers were detected at 6DPI in tree shrews. Tree shrews infected with influenza B virus showed similar seroconversion and respiratory tract pathology to ferrets. Elevated levels of cytokines were detected in the tissues isolated from the respiratory tract after infection with either V0215 or Y12 compared to the levels in the uninfected control in both animals. Overall, the tree shrew was sensitive to infection and disease by influenza B virus.

Conclusion: The tree shrew to be a promising model for influenza B virus research.

Keywords: Animal model; Ferret; Influenza B virus; List of Abbreviations.; Mouse; Tree shrew.

Fig. 1

Influenza B viruses can replicate in tree shrews, ferrets and mice. Tree shrews (a), ferrets (b) and mice (c) were inoculated with 1.0 × 10⁶TCID₅₀ of virus intranasally. At 2, 4 and 6 days post inoculation (DPI), nasal washes were collected and virus titers were determined by tissue culture infectious dose 50% (TCID₅₀) assay in Madin Darby canine kidney (MDCK) cells●Yamagata strain B/Guangzhou/12/2016 (Y12), ○Victoria strain B/Guangzhou/0215/2012 (V0215). Lines indicate means. **p < 0.01, *p < 0.05

Fig. 2

Tree shrews show weight loss post inoculation with influenza B viruses but ferrets do not. Tree shrews did not show an increase in body temperature greater than 0.5 °C above pre-inoculation (a) and up to 10% body weight loss (c) post inoculation with influenza B viruses. Ferrets inoculated with B/Guangzhou/12/2016 showed a mean body temperature increase of over 1 °C at one day post inoculation and at other timepoints temperatures were approximately 0.5 °C above pre-inoculation (b). Ferrets did not lose weight post inoculation with either virus (d). Body temperature and weight were measured daily after inoculation with 1.0 × 10⁶TCID₅₀ of virus intranasally. The body temperature at 0 day was the average of five days before infection. ●Yamagata strain B/Guangzhou/12/2016 (Y12), ○Victoria strain B/Guangzhou/0215/2012 (V0215)

Fig. 3

Both Yamagata and Victoria lineage influenza B viruses caused 100% mortality in BALB/c mice. Mice inoculated with either Yamagata strain B/Guangzhou/12/2016 (Y12) or Victoria strain B/Guangzhou/0215/2012 (V0215) began losing weight at two days post inoculation (a). All mice were killed by eight days post inoculation (b). ●Yamagata strain B/Guangzhou/12/2016 (Y12), ○Victoria strain B/Guangzhou/0215/2012 (V0215)

Fig. 4

Inoculation of tree shrews with influenza B viruses causes inflammation in tissues of the respiratory tract. mRNA expression of IL-6 (a), IL-8 (b), IL-10 (c), IP-10 (d), TNF-α (e) and TGF-β (f) mRNA were increased at 2, 4 and 6 days post inoculation (DPI) in tissues of the respiratory tracts of tree shrews inoculated with influenza B virus. Statistically significant differences in fold expression levels of IL-6 (a) and IP-10 (d) were observed between tree shrews inoculated with the Yamagata or Victoria lineage virus. mRNA expression was detected via real-time PCR (SYBR Green) using GAPDH expression as a control. Fold changes are in comparison to uninoculated animals. Tree shrews were inoculated with either Yamagata strain B/Guangzhou/12/2016 (Y12) or Victoria strain B/Guangzhou/0215/2012 (V0215). **p < 0.01, *p < 0.05

Fig. 5

Inoculation of ferrets with influenza B viruses causes inflammation in tissues of the respiratory tract. mRNA expression of IL-6 (a), IL-8 (b), IL-10 (c), IP-10 (d), TNF-α (e) and TGF-β (f) mRNA were increased at 2, 4 and 6 days post inoculation (DPI) in tissues of the respiratory tracts of ferrets inoculated with influenza B virus. mRNA expression was detected via real-time PCR (SYBR Green) using GAPDH expression as a control. Fold changes are in comparison to uninoculated animals. Ferrets were inoculated with either Yamagata strain B/Guangzhou/12/2016 (Y12) or Victoria strain B/Guangzhou/0215/2012 (V0215)

Fig. 6

Inoculation of BALB/c mice with influenza B viruses causes inflammation in tissues of the respiratory tract. mRNA expression of IL-6 (a), IL-8 (b), IL-10 (c), IP-10 (d), TNF-α (e) and TGF-β (f) mRNA were increased at 2, 4 and 6 days post inoculation (DPI) in tissues of the respiratory tracts of mice inoculated with influenza B virus. The Yamagata strain B/Guangzhou/12/2016 elicited greater increases in the expression of IL-6 at two days post inoculation (DPI) (a), IL-10 at two and six DPI (c), IP-10 at all time points (d) and TNF-α at 6DPI (e). mRNA expression was detected via real-time PCR (SYBR Green) using GAPDH expression as a control. Fold changes are in comparison to uninoculated animals. Mice were inoculated with either Yamagata strain B/Guangzhou/12/2016 (Y12) or Victoria strain B/Guangzhou/0215/2012 (V0215)

Fig. 7

Influenza B virus inoculation caused pathology in the nasal turbinates, trachea and lungs of trees shrews. Nasal turbinates (a, d, g, j, m, p), trachea (b, e, h, k, n, q), and lungs (c, f, i, l, o, r) were collected at 2, 4 and 6 days post inoculation (DPI) from animals inoculated with either Yamagata strain B/Guangzhou/12/2016 or Victoria strain B/Guangzhou/0215/2012 and stained with hematoxylin and eosin. Images were examined at magnification of × 200

Fig. 8

Influenza B virus inoculation caused pathology in the nasal turbinates, trachea and lungs of ferrets. Nasal turbinates (a, d, g, j, m, p), trachea (b, e, h, k, n, q), and lungs (c, f, i, l, o, r) were collected at 2, 4 and 6 days post inoculation (DPI) from animals inoculated with either Yamagata strain B/Guangzhou/12/2016 or Victoria strain B/Guangzhou/0215/2012 and stained with hematoxylin and eosin. Images were examined at magnification of × 200

Fig. 9

Influenza B virus inoculation caused pathology in the lungs of mice. Lungs were collected at 2 (a, d), 4 (b, e) and 6 (c, f) days post inoculation (DPI) from mice inoculated with either Yamagata strain B/Guangzhou/12/2016 or Victoria strain B/Guangzhou/0215/2012 and stained with hematoxylin and eosin. Images were examined at magnification of × 200