Background
Influenza B viruses (IBVs) are one of the main causes of seasonal influenza cases, accounting for up to 40% of total cases [
1]. However, during the 2011/2012 and 2015/2016 influenza seasons in China, 53.7 and 52.2% of influenza notifications were due to IBV infections [
2]. There are two lineages of IBVs currently in circulation; B/Victoria/2/1987-like (Victoria) and B/Yamagata/16/1988-like (Yamagata). Generally, IBVs cause a less severe respiratory disease compared to influenza A viruses (IAVs) [
3]. However, IBVs cause more complications in children, including neurological, muscular [
4] and cardiac-associated events [
5]. Recent studies also showed that the antiviral drug Oseltamivir, the drug most commonly used to treat influenza, was less effective against IBV compared to IAV [
6‐
8].
The tree shrew (
Tupaia belangeri), is a small non-primate mammal that has been used as an animal model to study viral infection [
9]. Tree shrews are a potentially promising model for the study of IBVs as they are more closely related to primates compared to ferrets, which are commonly used in influenza virus research [
10,
11]. Tree shrews are also smaller than ferrets, easier to handle and more cost-effective. Unlike mice, tree shrews display symptoms including fever, rhinitis and mild pneumonia post inoculation with IAVs, and show a distribution of α2,3-linked and α2,6-linked sialic acids in their respiratory tract similar to humans [
10,
12]. As such, here we conducted a study to determine if the tree shrew is a useful animal model for the study of IBV infection. We compared the tree shrew to two animal models commonly used in influenza research; ferrets and mice, using two strains of IBVs from Yamagata and Victoria lineages that were isolated from clinical specimens. Overall, we found the tree shrew to be sensitive to IBV inoculation, displaying a clear symptomology, viral shedding and seroconversion, thus demonstrating their utility as a model for future studies of IBVs.
Materials and methods
Cells and viruses
IBVs B/Guangzhou/0215/2012 (Victoria-like, V0215) and B/Guangzhou/12/2016 (Yamagata-like, Y12) were obtained from the State Key Laboratory of Respiratory Disease of Guangzhou Medical University. These viruses were isolated during our previous study of IBVs isolated from human cases (partial data was published in Chinese) [
13]. Briefly, our study of 8 viruses in the mouse model revealed that only the V0215 and Y12 viruses were capable of replication in the mouse model without prior adaptation. Therefore, these viruses were selected for this present study. Madin–Darby canine kidney (MDCK) cells (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum. All the viruses were cultured in MDCK cells cultures in DMEM without bovine serum and containing 1 μg/mL of L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) treated trypsin (Sigma). Virus titers were determined via the 50% tissue culture infective dose (TCID
50) method using MDCK cells and the Reed–Muench method [
14].
Animals
Male tree shrews weighing 100 to 155 g and special-pathogen-free female six-week-old BALB/c mice were obtained from the Animal Experimental Centre of Kunming Medical University. Female ferrets weighing 600 to 700 g were obtained from Wuxi Coral Reef Biotechnology Co., Ltd. (Wuxi city, Jiangsu province, China). Ferrets and tree shrews were confirmed to be seronegative by virus neutralization assay to V0215 and Y12 viruses prior to commencement of experiments. All the animals were fed in IVC cages in a BSL-2 animal house at the Animal Experimental Centre of Kunming Medical University. Animals were acclimatized to their housing for 2 days prior to use in these studies.
Animals were divided into four groups; Group One comprised of ten tree shrews and ten ferrets. These animals were implanted with transponder microchips (IPTT-300, Bio Medic Data Systems Inc., Seaford, Delaware) to allow daily monitoring of body temperature for 5 days prior to intranasal inoculation with 1.0 × 10
6 TCID
50 of V0215 (five ferrets and five tree shrews) or Y12 (five ferrets and five tree shrews) in 250 μL of phosphate buffered saline, pH 7.4 (PBS). After inoculation, body weight and temperature were measured daily and nasal washes were collected using 1 mL of PBS at 2, 4 and 6 days post inoculation (DPI) to measure viral titers. Serum were collected 21 DPI to measure influenza-specific antibody titers. Seroconversion of the animals was tested by HI assays [
15].
Group Two comprised of seven ferrets and 21 tree shrews. Three ferrets and nine tree shrews were inoculated with 1.0 × 106 TCID50 of V0215 or Y12 and one ferret and three tree shrews were intranasally inoculated with equal PBS as negative controls. At 2, 4 and 6 DPI one ferret and three tree shrews inoculated with each virus were killed to collect respiratory tissues for pathology studies and real time PCR. The animals inoculated with PBS were killed at 6 DPI.
Group Three consisted of 15 mice. Five mice were intranasally inoculated with either 1.0 × 106TCID50 of V0215 or Y12 or equal PBS. These mice were observed for 15 days for mortality and morbidity.
Group four consisted of 35 mice. 15 mice were intranasally inoculated with 1.0 × 106TCID50 of either virus in 50 μL of PBS and five were inoculated with equal PBS as a negative control. At 2, 4 and 6DPI five mice inoculated with either V0215 or Y12 virus were killed and lungs were collected for pathology studies and real time PCR. The five mice inoculated with PBS were killed at 6DPI and lungs were collected for pathology studies.
Quantification of mRNA
Tissues were homogenized and total RNA was extracted using Trizol reagent (Invitrogen) according to manufacturer’s instructions. Reverse transcription was performed using PrimeScript RT Master Mix kit (TAKARA) according to manufacturer’s instructions. Real-time PCR was conducted using TB Green
premix Taq II (TAKARA) to measure cytokine and chemokine mRNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. The primers were designed with Primer Primier 5.0 (Additional file
1: Table S1).
Histology
The tissues isolated from the respiratory tract were first fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 μm and stained with hematoxylin and eosin (H&E) after dehydration, the pathological sections were observed and photographed under a microscope (Nikon, ECLIPSE Ni).
Statistical analysis
Data were analyzed using SPSS (version 13.0). The data were expressed as mean ± standard deviation of the mean (SD). Significance was determined by single-tailed student’s t-test when comparing two groups, Multiple group comparisons were performed via one-way-ANOVA test base on variance homogeneity. p < 0.05 was considered significant.
Discussion
As one of the major causes of seasonal influenza, IBV seriously threatens human health. As vaccines and antiviral compounds are the main interventions against IBV, it is essential to have animal models that replicate human disease to use in their development. We previously inoculated mice with IBVs isolated from clinical samples and identified two IBVs capable of causing lethal infections in mice without adaptation; the Victoria-like virus B/Guangzhou/0215/2012 (V0215) and the Yamagata-like virus B/Guangzhou/12/2016 (Y12). In this current study, we used these viruses to compare IBV infection in tree shrews to ferrets and mice, to determine if tree shrews could be a model for future studies of IBVs.
Previously, we demonstrated that tree shrews present a useful alternative model for the study of subtype H1N1 IAV infection. Evidence of viral replication in the upper respiratory tract was observed, as were fever, nasopharyngeal secretions and inflammation. Additionally, the distribution of α2,3-linked and α2,6-linked sialic acids were similar to that found in the human respiratory tract. However, the effects of IBV in tree shrews were unknown. Our results showed that Yamagata and Victoria-lineage IBVs were capable of replication in the respiratory tract of tree shrews, ferrets and mice. Clinical signs and pathological changes were also evident in the respiratory tracts of these animals. Ferrets infected with influenza B virus may have mild respiratory and systemic symptoms, including fever, runny nose, sneezing, however, these nasal and systemic symptoms of tree shrews are not as obvious as ferrets, But in other aspects, tree shrews appeared to be more sensitive to IBV inoculation compared to ferrets, as weight loss was observed in tree shrews but not in ferrets. However, the overall severity of infection in these two animal models remained mild, which is comparable to what is observed in humans. In the course of this study, we observed that the main symptoms in mice infected with influenza virus include ruffled fur, arched back, shortness of breath and malaise. From the analogue comparison of symptoms after virus infection, tree shrews and ferrets were superior to mice as a model for human IBV infection. Effective replication of viruses in host is the main pathogenic mechanism. Previously, it was considered that unadapted influenza B virus could not be infected or replicated effectively in laboratory animals such as mice [
16,
17], because influenza B virus exhibit a limited host range and lacked natural animal hosts [
18,
19]. In this study, however, tree shrews, ferrets and mice were susceptible to two lineages of unadapted human influenza B (V0215 and Y12). The results showed that the respiratory tract of these animals infected with influenza B virus could replicate the virus effectively, which the peak of virus replication was 2 days after infection. The peak and period of respiratory viral shedding of tree shrews infected with influenza B virus were similar to those of ferrets, and similar to the respiratory viral shedding symptoms of human influenza diseases,such as viral shedding time of duration and peak time of viral shedding. [
20,
21], whereas the viral shedding cycle of mice increased significantly, and there were still high viral titers in the lung tissues until 6DPI. We also found differences in susceptibility of three animals to above two influenza B viruses. There were no significant differences in titers between V0215 and Y12 inoculated ferrets, however, viral titers were significantly higher in tree shrews and mice inoculated with Y12 compared to V0215 at both 2 and 4DPI. This difference may be related to the different virulence and tissue tropism of V0215 and Y12, as well as host differences [
22,
23]. Although there was no difference in viral titers between tree shrews and ferrets infected with Y12, viral titers were detected at 6DPI in tree shrews but not in ferrets. Histopathology of respiratory tissues obtained from tree shrews inoculated by IBVs revealed rhinitis and pneumonia in all animals. Because Y12 has longer viral shedding time and higher viral titer in tree shrews, it appeared that Y12 caused more severe disease in tree shrews compared to V0215 by pathological examination.
The antibody response of hosts infected with influenza virus is an important immune and epidemic prevention mechanism. Highly virulent influenza virus strains produce higher antibody titers, which are related to the severity of influenza infection [
24,
25]. A survey of serum antibodies against influenza viruses in Pennsylvania showed that the titers of antibodies in most people infected with A/California/7/2009 (H1N1) and B/Brisbane/60/2008 (B/Victoria) ranged from 0 to 1:640 and 0–1:640, respectively. A few patients infected with H1N1 virus can reach 1:1280 in some months, some even greater than 1:2560 [
26], Tree shrews and ferrets infected with IBV 0215 (B/Victoria) and 12 (B/Yamagata) can seroconvert, indicating that tree shrews have certain similarities in adaptive immunity with humans and ferrets, and could therefore be used in the development and research of new vaccines.
The immune response formed by cytokines and chemokines is the first line of defense against influenza virus infection and the key factor of the immune pathogenesis of influenza virus infection in human and animal models [
27]. Moderate inflammation can inhibit the replication and transmission of influenza virus and prevent the development of disease, while excessive inflammation is closely related to clinical severe pathological injury [
28]. Especially in some highly pathogenic avian influenza patients, excessive inflammation can promote the imbalance of immune function and the excessive release of a large number of inflammatory cytokines, termed a “cytokine storm”, leading to multiple organ dysfunction or acute respiratory distress syndrome [
29]. In this study, we detected six cytokines in respiratory tract tissues of tree shrews, ferrets and mice. Elevated levels of cytokines were detected in the tissues isolated from the respiratory tract of tree shrews, ferrets and mice after infection with either V0215 or Y12 compared to the levels in the uninfected control, suggesting that infection with IBV can trigger innate immune responses in experimental animals including tree shrews. The expression of these cytokines correlated well with the pathological changes of respiratory tract tissues, as observed in similar reports using the ferret model [
30,
31]. Similarly, many pro-inflammatory cytokines and chemokines such as IL-6, IL-8, IP-10, MIG, TNF-α and so on were significantly up-regulated in Influenza virus infection patients [
32,
33]. In addition, IL-10 is an anti-inflammatory cytokine, which can strongly inhibit the synthesis of TNF-α, IL-1, IL-6, IL-8 and other inflammatory mediators. In this study, IL-10 increased significantly in tree shrews and mice tissues, suggesting that similar to human infection, tree shrews infected with influenza can also form a feedback regulation mechanism between pro-inflammatory and anti-inflammatory responses, maintaining the balance between inflammatory and anti-inflammatory mediators in the body [
34].
Therefore, considering their small size, ease of handling and reduced cost of the tree shrews in comparison to ferrets, they present as a useful model for the study of IBV infection.