Background
On 19 February 2013, the first patient infected with the novel influenza A H7N9 virus from an avian source showed signs of sickness. More than 347 laboratory-confirmed cases have been reported in mainland China, with 109 cases resulting in death. Most cases of H7N9 infection have occurred in elderly men who had recently been exposed to live poultry. Patients with laboratory-confirmed H7N9 infections had an exposure history that included direct contact with respiratory secretions or fecal material. Exposure to live poultry is a case-fatality risk associated with influenza A H7N9 virus infection [
1‐
6]. Live bird markets are suspected of being the source of human infections; shutting down live poultry markets resulted in an immediate reduction in cases [
7]. There is a precedent for the human transmission of H7 influenza, with limited person-to-person transmission observed in an H7N7 outbreak in the Netherlands in 2003 [
8]. Three suspected family clusters of cases were reported [
9], indicating the possibility of limited human-to-human transmission of the H7N9 virus [
10].
Previous studies have reported aerosol transmission of the H7N9 virus among ferrets [
11]. Other researchers have used a mouse model to study contact-dependent transmission of influenza A virus [
12]. We evaluated the transmission of the H7N9 virus in mice to provide insights into potential human transmissibility.
Discussion
A second wave of H7N9 viral infections in humans occurred in 2014 across China, with over 50 deaths recorded in 1 month. The Chinese government enforced a temporary closure of live poultry markets in affected provinces, resulting in an immediate reduction in the number of human cases. This provided further evidence of the role live poultry markets play in the spread of the virus, and clearly showed that closure of the markets was an effective control strategy [
7]. However, the transmission routes of the H7N9 virus remained unknown, therefore it was still considered a serious threat to human health worldwide [
13].
The mouse is a typical model used to evaluate the virulence of influenza viruses, and in its quantitation. It has been reported that a murine model can be used to study contact-dependent transmission of the influenza A virus [
12]. In the current study, H7N9 virus-infected mice displayed symptoms of lethality, with pathological changes seen in mice that came in contact with infected mice. We conducted a study on the aerosol transmission of H7N9 in ferrets [
11], and used a murine model to study other potential routes of transmission.
Our results indicated that the novel H7N9 virus could efficiently replicate in mice without prior host adaptation. We inoculated BALB/c mice intranasally with A/Anhui/1/2013 to determine H7N9 virus replication, morbidity (as measured by weight loss), and the LD
50 in mice. The LD
50 titers for the H7N9 virus were lower than those for H5N1 and H1N1 viruses. Mice inoculated with H7N9 virus showed severe morbidity (30% weight loss), a value comparable to that seen for HPAI H5N1 and H1N1 virus infections, which have a high pathogenicity phenotype in this model. Our findings were similar to those reported in previous studies [
14]. The H7N9 virus was detected in extra-respiratory intestines, liver, spleen, kidneys, and brains of infected mice, with viral antigen detected in the same tissues. Weight loss was used as a measurable outcome and a marker of virulence following virus infection in these mice [
15‐
20]. In our study, the duration of H7N9 virus infection in mice was about 12 days, a similar duration to that seen with human H7N9 infections (4–11 days) [
15,
21‐
23]. Virus was detected at higher titers in the lungs than in nasal samples (Table
1). From our transmission experiments we found that virus titers in the lungs of co-housed mice at 5 dpi were higher than those at 7 dpi, with the proportion of infected mice higher than at 7 dpi (Table
2). Results with the H7N9-infected mice were similar to those for H1N1-infected mice. It was previously reported that an avian H7N9 virus effectively replicated in mice with minimal symptoms and that the virus could be transmitted in mice without any pathogenic effects observed [
24]. Our results indicate that the H7N9 virus could be transmitted in mice, however weight loss and ruffled coats were observed. The virus was detected at different time points and in several tissues, although virus titer was low.
The transmission of H7N9 influenza virus in ferrets has been shown to occur
via aerosols and respiratory droplets [
11,
14]. Our results show that the H7N9 influenza virus can also be transmitted between mice
via eye secretions (Table
4). Virus in the lung tissues was detected at 4 dpi following ocular administration of ES inocula in mice.
Eye and pharynx secretions, and feces from infected mice proved to be infectious
via mucosal, intravenous, and oral routes. Virus was present in lung and intestinal samples (Table
3); at 3 and 7 dpi, mild dilatation of the interstitial pulmonary vasculature was observed in the lungs (Figure
3b). Pathological changes in the lungs were milder in naïve mice than in the infected mice; this difference positively correlated with the amount of virus detected in naïve mice.
Our results indicate that influenza virus can be detected in secretions from the eyes, throat and feces. Virus titer was higher in throat secretions than from eye secretions or fecal samples. Secretions from H7N9-infected mice are able to transmit virus by the ocular, oral, and blood-borne routes. An understanding of viral pathogenesis and of the several transmission routes of the virus would allow for various interventions in animals to prevent a future human pandemic [
25].
Materials and methods
Viruses
Influenza virus A/Anhui/1/2013 was isolated from a patient with a laboratory-confirmed human A (H7N9) virus infection. The patient was a 35-year-old woman who lived in the Anhui Province of China. On day 6 after the onset of illness, she developed acute respiratory distress syndrome, septic shock, and acute renal damage; the patient died 13 days later [
1]. A throat swab (TS) was collected from the patient and propagated in the allantoic sac and amniotic cavity of 9–11-day-old embryonic chicken eggs. The propagated virus was then passaged once in Madin-Darby canine kidney (MDCK) cells. A Q226L (H3 numbering) substitution at the 210-loop of the HA gene was found in this virus. This site has been shown to change receptor binding from avians to humans, possibly increasing the ability of the virus to be transmitted by airborne routes [
26]. In addition, the virus encoded PB2 627 K, which is essential for efficient replication of avian influenza viruses in mammals. The highly pathogenic avian influenza A (H5N1) virus A/Shenzhen/406H/06 and 2009 pandemic A (H1N1) virus A/California/07/2009 were also isolated from two patients and propagated in embryonic chicken eggs and MDCK cells. The A (A/Shenzhen/406H/06; H5N1) and 2009 pandemic A (A/California/07/2009; H1N1) viruses were obtained from the University of Hong Kong. MDCK cells were maintained in Eagle’s minimal essential medium (EMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin, and were incubated at 37°C/5% CO
2.
H7N9 influenza virus in mice
Murine studies were performed in an animal biosafety level 3 (ABSL3) facility using HEPA-filtered isolators. All procedures in this study involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (ILAS-PC-2013-006).
The 50% mouse lethal dose (LD
50) of the virus was determined by intranasally inoculating five groups of mice (
n =10 mice per group) with 10-fold serial dilutions of the virus in a volume of 50 μl. The LD
50 was calculated by the method of Reed and Muench [
27]. The H5N1 A/Shenzhen/406H/06 and H1N1 A/CA/07/09 strains were used as controls. Mice were infected with 10
6 TCID
50 of H7N9, H1N1, or H5N1 virus to compare the virulence of strains.
We anesthetized female 4-week-old mice using isoflurane, and then inoculated them intranasally with the appropriate virus. Heart, liver, spleen, lungs, kidney, intestines, brain tissue specimens and turbinate samples were collected from mice and placed into 10% (w/v) phosphate-buffered saline (PBS) on 1, 2, 3, 5, and 7 dpi (Additional file
2: Table S1). Viruses were titrated in MDCK cells and virus titer was expressed as TCID
50, as per the Reed-Muench method. We euthanized six mice for every virus at each time point. Additionally, three mice were sacrificed for every virus at each time point, with lungs, intestines, and brain tissues collected for immunohistochemical and pathological analysis. The remaining 10 mice in each group were observed daily for weight loss and mortality until 14 dpi.
Murine model of H7N9 virus transmission
Three groups of mice were used in this study. For each group, three 4-week-old BALB/c mice were infected, and seven naïve mice were placed in direct contact with infected mice (Additional file
2: Table S1). The three infected mice were lightly anesthetized and inoculated intranasally with 50 μl of 10
7 TCID
50 virus in PBS as previously described. At 24 hpi, all materials in the cage were replaced and sterilized, and the seven naïve mice (co-housed mice) were placed in the same cage. The seven co-housed mice were observed daily, with detailed recording of clinical signs and weight loss continuing until 14 dpi. To monitor viral shedding, nasal washes were collected from seven co-housed mice at 3, 5, and 7 dpi. One group of mice was used for each time point. We dripped 50 μl of PBS into the nose of each mouse; the liquid was then allowed to fall back into a collection tube. This was repeated three times. The seven co-housed mice were then euthanized at 3, 5, and 7 dpi. The lungs, kidney, brain, and intestines were collected, and immersed into 1 ml of PBS. Virus titers in the tissues were determined using MDCK cells as previously described [
27]. The number of co-housed mice that became infected was calculated by determining the virus titer in the lungs, brain, and nose at 5 and 7 days after co-housing. Pathological examination of the lungs and intestines occurred on days 3 and 7. Sera were obtained from co-housed mice on 14 dpi to confirm seroconversion using hemagglutination inhibition (HI) assays with 0.5% chicken erythrocytes.
Secretions from H7N9-infected mice
To collect eye secretions (ESs), a swab pre-wetted with PBS was used to wipe the eyes of six mice (5–7 wipes) at 1, 2, and 3 dpi, and then placed into 1 ml of PBS. Throat swabs (TSs) were collected from inoculated mice at 1, 2, and 3 dpi, and transferred to 1 ml of PBS. The ESs and TSs were vortexed and centrifuged (300 g, 10 min), and the supernatants filtered. Five pellets of fecal (F) samples from inoculated animals were collected at 5 dpi and placed into 10% (w/v) PBS, vortexed, and centrifuged (300 g, 10 min). Supernatants were then filtered. The liquid from the ES, TS, and F samples were used to intranasally inoculate mice on days 1, 2, and 3, respectively. Six inoculated mice were then euthanized at 4 dpi (Additional file
2: Table S1). Tissues from the lungs, nasal cavity, and intestines were collected in 1 ml of PBS. Viral titers in tissues were determined using MDCK cells as previously described.
Transmission route of H7N9 in mice
Conjunctival secretions from H7N9-infected mice were used to wipe the eyes of twelve naïve mice (Eye). Pharynx secretions were injected into the tail veins (150 μl per mouse) of naïve mice (IV). The secretion was concentrated using a hyperfiltration tube (Millipore Amicon). The virus titer of conjunctival secretions was 10
3.8 TCID
50, while that for pharynx secretions was 10
3.8 TCID
50. Similar virus titers were seen for the fecal suspensions. Six naïve mice were given the fecal suspensions from H7N9-infected by gavage administration (Or; 200 μl per mouse). Six mice infected through Or, Eye and IV routes were autopsied at 3 and 5 dpi. Three mice were used for the isolation of lung, tracheal, and intestinal tissues for pathological and histochemical examinations at 5 dpi. Three mice were used to obtain nasal washes and lung tissues to determine virus titers at 3 and 5 dpi (Additional file
2: Table S1).
Virus titrations
Virus titrations were performed by end-point titration in MDCK cells. MDCK cells were inoculated with 10
0.5-fold serial dilutions of homogenized tissues, nasal washes, and TSs. At 1 hpi, cells were washed once with PBS and incubated in 200 μl of infection medium (EMEM, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml TPCK-trypsin). At 3 dpi, supernatants of infected cell cultures were tested for agglutinating activity using turkey erythrocytes as an indicator of cellular infection. Infectious titers were calculated from five replicates using the Reed-Muench method [
27].
Histopathology and immunohistochemistry (IHC)
Animal necropsies were performed according to standard protocols. Samples for histological examination, comprising formalin-inflated lungs, were stored in 10% neutral-buffered formalin, embedded in paraffin, and sectioned (4-μm thickness). The sections were stained with hematoxylin and eosin (HE) for examination by light microscopy, or via an immunohistochemical method using a monoclonal antibody against the nucleoprotein of influenza A virus. All slides were examined by a pathologist with 10 years’ experience, and results were confirmed by a second pathologist.
HI assays
Standard HI assays were performed on post-exposure mouse sera using 0.5% turkey erythrocytes against homologous virus. Sera were collected from inoculated or naïve mice at 14 dpi or 14 days after coming into contact with infected mice, respectively, and tested for the presence of H7N9-specific antibodies.
Statistical analysis
Differences in body weight, viral copy numbers, and virus titers among groups were analyzed by one-way ANOVA and post-hoc Bonferroni correction. Differences between two groups were analyzed by Student’s t-test using SPSS 11.5. A P- value less than 0.05 was considered statistically significant.
Acknowledgements
This work was supported by grants from the National Science and Technology Major Projects of Infectious Disease (2012ZX10004501-004, 2012ZX10004404, and 2012ZX10004301-8), the special project on human H7N9 viruses from the Ministry of Science and Technology of China (KJYJ-2013-01-04), the National Natural Science Foundation of China (31370203), the Fundamental Research Funds for the Central Universities (2012Y02 and 2012D15), and the Open Fund of the Key Laboratory of Human Disease Comparative Medicine, Ministry of Health (DWS201214).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CQ was the principal investigator, designed and supervised the study, and wrote the grant application. LB designed the animal studies. LB, HZ, TC, FL, YY, YS, and PY performed the animal studies. QL and HJ performed the cell culture experiments. WD, YX, LH, and YL performed the histopathological and immunohistochemical analyses. XL, WH, XZ, YL, and JG performed genome sequencing and analyses. LB, LX, and QC drafted the manuscript. All authors contributed to the review and revision of the manuscript and have read and approved its final version.