Abstract:The outbreak of human infections caused by novel avian-origin influenza A (H7N9) in China has brought serious and huge challenge for our health, agriculture, and economy. Although the epidemic situation has been controlled, the succeeding sporadic cases warn us that the epidemic may not have faded away. There are still too many uncertain issues about the H7N9 virus. Based on this situation, this review will focus on the research progress and perspectives for future critical H7N9 influenza virus topics, and serve as a modest spur to induce other researchers to come forward with their valuable contributions.

Abstract:Objective To analyze the pathological damages of the lung and restoration of BALB/c mice infected by H7N9 virus, H5N1 virus or H1N1 virus. Methods SPF BALB/c mice were challenged with H7N9 virus, H5N1virus or H1N1 virus, respectively. The survival rate and clinical symptoms of mice were evaluated, while the pathological lesions of the lung were analyzed after H&E staining. Furthermore, the proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry to evaluate the restoration after virus infection. Results The BALB/c mice were successfully infected by the three strains of influenza viruses. The survival rate was presented H7N9 >H1N1 >H5N1, but a opposite tendency was found in the pathological changes, and the highest expression level of PCNA after H7N9 infection was detected among the three strains of viruses. Conclusions Lighter lesion with an increased restoration is found in the lung of mice infected after the novel H7N9 virus. There is an excessive immunologic reaction and a high mortality in mice infected with the H5N1 virus.

Abstract:Objective To better understand the dynamic pathological changes in the lungs of mouse and ferret models infected with novel avian-origin H7N9 subtype influenza virus. Methods A/Anhui/1/2013 (H7N9) virus was inoculated by intranasal instillation to mice and ferrets. Autopsies of 2-3 mice each time were performed on days 1, 2, 3, 5, 7, 14 and 28 post inoculation (d.p.i.) and 1 ferret on 3, 7, 14 and 28 d.p.i. Clinical signs and gross examination were conducted and histopathological analysis was performed. Results Animals developed typical clinical signs including body weight loss (mice and ferrets), ruffled fur (mice), sneezing (ferrets), diarrhea(ferrets)and death (mice). Focal infection observed by gross examination and bronchitis and pneumonia determined by histology were seen in the lung in the mouse and ferret models. Inflammatory reaction was started from 2 d.p.i., most severe on 7-9 d.p.i. and absorbed from 14 to 28 d.p.i.. Lymphocytes and macrophages, especially CD8⁺ lymphocytes were increased in the lungs of infected mouse and ferret models. Conclusions Bronchitis and pneumonia can be induced in mice and ferrets inoculated with H7N9 virus. Inflammatory reactions are most severe on 7-9 d.p.i. and absorbed from 14 d.p.i., and infiltration of CD8⁺ lymphocytes is evident. Observation of pathological changes of the lungs in mouse and ferrets models enables detailed studies of the pathogenesis, diagnosis, treatment and prevention of this illness, and lays foundation for related drug or vaccine evaluation.

Abstract:Objective The aim of this study was to establish a mouse model of H7N9 avian influenza A virus infection. Methods Seventy SPF healthy female BALB/c mice were used in this study. A/Anhui/1/2013 (H7N9) avian influenza virus was administered by intranasal instillation to BALB/c mice, inoculated in a dose of 50 μL 1×10⁸, 1×10⁷ or 1×10⁶ TCID₅₀, respectively, 10 mice in each group. Other 30 mice were used for virus titration and pathological examination. Ten mice were given saline as control group. The changes of body weight, clinical signs and death of the mice were observed every 24 h from 1 to 14 d. Blood and several organ samples were taken for pathological examination, and avian influenza A (H7N9) virus was detected with virus titration and immunohistochemistery (IHC). Results The mice developed typical clinical signs including body weight loss, ruffled fur and humped back. The peak of virus shedding from respiratory tract was observed on 2 days post inoculation (d.p.i.), and histopathological examination observed interstitial pneumonia. The virus was also detected in the brain, liver, spleen, kidney and intestine from inoculated mice. The inoculation of H7N9 virus elicited seroconversion titers up to 160. There was reduction of lymphocytes and increase of neutrophils in the blood. Conclusions The mouse model of H7N9 avian influenza virus infection established in this study show similar signs of human avian influenza. Therefore, it provides a useful working basis for research of the pathogenesis, drug development, and vaccine evaluation of this disease.

Abstract:Objective To establish adapted strain of seasonal H1N1 influenza virus in mice and infection model. Methods The mice model was induced by nose dropping method with influenza virus, by continuous passage in the lung tissue of BALB/c, to get the adapted strain of seasonal H1N1 in mice and seasonal influenza virus infection model, and the lung index and death rate was observed. Results After 8 times consecutive passages in the lung tissue of BALB/c, virulence of wild type seasonal H1N1 virus was increased to a high level. Genome sequencing and alignment indicated that the HA gene was mutated. Conclusion The virulent mouse-adapted variant A/Brisbane/59/2007-MA can be acquired from the avirulent A/Brisbane/59/2007 strain by continuous passage in the lung tissues of mice. Mutation of Thr to Ile at residue 89 of HA protein is the key virulence determinant.

Abstract:Objective Increasing number of human infection caused by the novel avian-origin H7N9 subtype influenza virus underscores the need to better understand the pathogenesis and transmissibility of this virus in mammals. Methods Mice inoculated with H7N9 influenza virus and put into the cage with naive mice, which were then specifically measured their clinical signs, virus shedding, tissue dissemination and pathology. The secretion from the cohabitant mice was inoculated to other normal mice to analyze the possible transmission routes. Results H7N9 influenza virus replicated in the lung, intestine and brain of the experimental mice. It was worth noting that H7N9 virus could transmit between mice in close contact by mucosa and fecal-oral routes, with the highest concentration of virus found in pharyngeal secretions. Conclusions Our results demonstrate that the avian-origin H7N9 subtype influenza virus can infect mice without adaptation and spreads by mouse-to-mouse transmission. The secretions of infected mice are source of infection for mice in direct contact, and it is similar to the influenza virus transmission between humans.

Abstract:Objective the novel avian-origin H7N9 subtype influenza virus caused outbreak of human infections in China since March 2013. The aim of this study was to better understand the pathogenicity and transmissibility of the H7N9 influenza virus in ferrets to verify whether this new emerging infectious H7N9 virus can infect humans, and to compare the transmission ability of this virus with that of other influenza virus subtypes in ferrets. Methods A/Anhui/1/2013 (H7N9) virus was administered by intranasal instillation to groups of ferrets. Clinical signs, virus shedding from respiratory tract and pathological changes were recorded. Meanwhile, the replication ability, virulence and pathogenicity were investigated in ferret models and compared with those of 2009 pandemic H1N1 and H5N1 influenza viruses. Results In the ferret models, based on the clinical signs, mortality, virus dissemination, and pathological analysis, the H7N9 influenza virus was found to be less pathogenic than the H5N1 virus but was comparable with the 2009 pandemic H1N1 virus. The H7N9 virus could replicate in the upper and lower respiratory tract, heart, liver, and olfactory bulb. It is worth noting that the H7N9 virus exhibited low level of transmission between ferrets via respiratory droplets. There were four mutations in the virus isolated from the contact ferrets. Conclusions These data indicate that H7N9 subtype influenza virus is transmissible among ferrets, and the pathogenicity of avian-origin H7N9 subtype influenza virus is lower than that of H5N1 subtype influenza virus between ferrets, and is comparable to that of H1N1 influenza virus.

Abstract:Objective To Study the effect of ginsenoside Rb1 on antioxidant enzyme in mouse acute lung injury induced by A/swine/HeBei/012/2008/swine influenza virus(H9N2 SIV).Methods One hundred and ninety-five 6- to 8-week-old BALB/c mice were randomly divided into three groups, 65 in each.The mice in the control group were inoculated intranasally with an equivalent dilution of noninfectious allantoic fluid, and that of both the acute lung injury group(ALI group)and ginsenoside Rb1 group(G-Rb1 group)were inoculated intranasally with H9N2 SIV diluted in sterile saline, and in addition, the mice of the G-Rb1 group were treated with ginsenoside Rb1 10 mg/(kg·bw) by intraperitoneal injection continuously for seven days.Clinical signs and body weight loss were observed in eight infected mice of each group.At the same time, at the indicated time points after infection, histopathology of the lung was observed and the activities of T-SOD, MPO, CAT and GSH-PX were detected in the mouse lungs.Results After the first 2 days of infection, the mice of the ALI group showed depression, ruffled fur, reduced feed intake and weight loss. Furthermore, pulmonary edema, hemorrhage, and a number of inflammatory cells exuding from the pulmonary alveoli were observed in the lungs of infected mice. Lung coefficient and lung wet/dry weight ratio was increased gradually. The changes began to decline on the 8th day and tend to be normal on the 14th day. The organs of mice of the control group showed no abnormality.For mice in the G-Rb1 group, clinical symptoms were significantly improved, survival time was prolonged, and mortality was decreased. On the 4th, 6th and 8th day after infection：the activity of both T-SOD and CAT was significantly reduced(P<0.01)in the mice of ALI and G-Rb1 groups compared with that of the control group, but the index of G-Rb1 group was significantly higher than that of the ALI group(P<0.05).At each time point after infection, the GSH-PX activity was significantly lower(P<0.01)in the ALI group compared with that of the control group, but the GSH-PX activity of G-Rb1 group was significantly higher than that of the ALI group(P<0.01), with a significant difference between the two groups(P<0.01).Conclusions G-Rb1 can improve the activity of antioxidase in mouse acute lung injury induced by H9N2 SIV, and to some extent, G-Rb1 can ameliorate the acute lung injury induced by H9N2 SIV infection.

Abstract:Objective To explore whether seasonal influenza split vaccine has immune protective effect on H7N9 influenza virus infection in mice. Methods Seasonal influenza split vaccine and PBS were used to immunize mice by intraperitoneal injection. 14 days later, the mice were booster immunized with A/Anhui/(H7N9) virus. Then the mice were challenged with H7N9 influenza virus in a dose of 5 LD₅₀. H7N9 influenza virus was preincubated with mouse serum immunized with vaccine and titered by micro-neutralization and hemagglutination inhibition (HI) assay. The protective effect was judged by survival rate, body weight loss and residue virus titer in the lungs and turbinates 2 and 4 days post inoculation. Results After infection, the body weight of mice in the vaccine and model groups were declined, the survival rate of the vaccine group was 10% and all mice of the model group died. The virus titers of the turbinates in the vaccine group were significantly higher than that in model group. The neutralizing antibodies raised in the sera of all mice vaccinated with seasonal influenza split vaccine did not neutralize H7N9. Conclusion Seasonal influenza split vaccine cannot effectively protect mice against H7N9 influenza virus infection.

Abstract:Objective To get the Banna minipig inbred line (BMI) GHR gene sequence, predict its function by bioinformatics analysis and obtain the GHR mRNA multi-tissues transcription profile. Methods BMI GHR cDNA sequence was cloned from liver RNA by RT-PCR. Then the products were inserted into pMD18-T vector for cloning, sequencing and bioinformatics analysis. The GHR mRNA expression in different tissues was determined by semi-quantitative RT-PCR. Results The GHR cDNA sequence was cloned and the GenBank accession No. was KC999114. The length of encoding sequence was 1917 bp and encoded a protein of 638 amino acids. The results of bioinformatics analysis showed that four amino acid substitutions were found at the intracellular domain of GHR between BMI and Landrace pigs. The substitutions were p. E381D, p. A409S, p. L556V and p. A580G. Analysis of the GHR mRNA expression in various tissues revealed that it was expressed in almost all tissues. It was most highly expressed in the muscle, moderate in small intestine, heart, liver, nerve fiber, spleen and ovary while weakly expressed in the lung, stomach, cerebrum, pancreas and kidney. Conclusions We have cloned complete GHR coding sequence, performed bioinformatics analysis and tissue expression profile analysis. It provides the primary foundation for further insight into the dwarf mechanism of BMI.

Abstract:Objective To investigate the effects of growth arrest-specific gene 6 (GAS6) inactivation on maintaining mouse energy metabolic homeostasis. Methods Axl gene encodes the major receptor of Gas6. Wild type gene (Axl^+/+) and Axl gene-deficient (Axl^-/-) mice were used as research models. For each genotype mice, the fasting glucose, blood lipids, aliphatic index in the blood, and the mRNA expression and protein phosphorylation levels of PI3K, AKT and glucose transporter 4 (GLUT4) in skeletal muscle were determined. After high fat high cholesterol diet-induced type 2 diabetes mellitus (T2DM) models from the two genotype mice were generated, the plasma Gas6 concentrations of the mice were detected respectively so as to compare the effect of Axl deficiency on the success ratio of induced T2DM models. Results Abnormal blood lipids in Axl^-/- mice were observed, and the fat deposition rate was significantly higher than that of the wild-type mice as well (P<0.01). The stability of blood glucose in Axl^-/- mice was impaired, in which the fasting glucose level in Axl^-/- mice was significantly higher than that in the wild-type mice, and the mRNA level of Glut4 in skeletal muscle was increased significantly, while the phosphorylation levels of PI3K, AKT and GLUT4 proteins were slightly decreased. The GAS6 plasma concentrations in the T2DM mouse models were significantly lower than that in the respective control groups, which was irrelevant to genotypes. Surprisingly, the success rate of induction of T2DM in Axl^-/- mice was twice as high as that of the wild type mice (P<0.01). Conclusion Activation of GAS6/AXL signaling pathway contributes to lower blood glucose and inhibit fatty deposits to a certain extent.

Abstract:Objective To observe the effects of different modeling methods on the wound morphology and healing time of chronic skin ulcers in diabetic rats. Methods SD rats (n=50) were randomly divided into five groups, 10 rats in each group: skin defect group (group QS: dermal deficiency), diabetes group (group DM: STZ injection+skin excision), diabetes plus Staphylococcus aureus group (group DMJJ: STZ injection+skin excision+bacterial infection), diabetes plus hydrocortisone group (group DMJS: STZ injection+skin excision+hydrocortisone intervention) and diabetes plus hydrocortisone and implantation of foreign body group (DMYW group: STZ injection+skin excision+hydrocortisone intervention + foreign body embedded). The rats were measured for body weight and wound healing every day, and blood glucose after stable diabetes once a week. The rats were sacrificed 12 days later and the skin lesions were examined by histopathology.Results The healing rate of the DMYW group was significantly slower than that in the other groups (P<0.01). At 12 days after modeling, the healing rate of the DMYW group was significantly lower than that of the remaining groups (P<0.01), while the healing rates were not significantly different among the remaining groups.Conclusions The modeling method of DMYW group can show skin wounds similar to the clinical characteristics of "Yin syndrome", and the addition of foreign body implantation significantly prolongs the rat skin healing time.

Abstract:Objectives To investigate the estrus cycle in Mongolian Gerbils, to explore the mechanisms of their regularity, and to optimize the judgment criteria of estrus cycle phases. Methods Vaginal smears were consecutively taken from the gerbils for 18 d, and keratinocyte count was used to observe the vaginal exfoliative cytology under light microscope in 50 female gerbils. The advantages and disadvantages of Wright's staining, HE staining and direct examination under light microscope in distinguishing the four phases of estrus cycle were compared. Results There were three types of estrus cycles is Mongolian Gerbils as stable form, unstable form and pseudopregnancy form. Among these, the stable form accounted for 68.6%. The estrus cycles lasted for (106.3±35.0) hours. Moreover, it could be distinguished into four phases. The proportion of cornified cells in the four phases were: proestrus (13.5±7.8)%, estrus (86.7±9.9)%, metoestrus (27.9±12.8)%, and intermediate stage (3.3±2.8)%. Conclusions Cornified cell count can be used to assess the estrus cycle and each phase correctly. Direct examination under light microscope is ideal for revealing the morphology of vaginal exfoliative cells rapidly. The study of estrus cycle in Mongolian Gerbils may provide a theoretical basis for superovulation, fertilization in vitro, embryo freezing, and biological purification and so on.

Abstract:Objective To monitor the growth and reproductive performance of a rare closed colony of Gobiocypris rarus (Ihb: IHB) and to provide basic information for its management and application. Methods Egg production, spawning interval, diameter of egg membrane, fertilization rate and hatching rate were monitored for 50 pairs of parents fish of wild gathered fish (P0), F1, F2, F3 and F4 generations. Moreover, body size of newly hatched offspring (F1 to F4), and at 7, 30, 60 and 90 days were measured to examine their growth profile. Results There were certain differences in growth between generations. F1 had a larger total length of the newly hatched larva and 7-day old larva than those of F2 to F4. But while in the fish at 30, 60 and 90 days, those of F2, F3, and F4 had a larger body size than that of F1, and exhibited higher growth indexes. Good performance on fertilization rate and hatching rate was shown, though there was certain fluctuation between generations. No difference of spawning interval of the closed colony were found between generations. The wild gathered fish (P0) seemed to have larger egg diameter and egg production per batch than those of F1 to F4. Conclusions It is suggested that differences in growth of the fish may be related with culture conditions, and the higher egg production may be related with the larger body size of P0. The results indicate that the closed colony preserves good performance of growth and reproduction. No increasing or decreasing tendency is found during the passage process. This Ihb: IHB colony meets the requirement of quality control of laboratory animal closed colony.

Abstract:Objective To establish a rat model simulating high-altitude pulmonary edema by using hypobaric chamber. Methods Sixty healthy Wistar rats (male:female=1:1) were randomly divided into five groups: control group, hypoxia 24 h group, hypoxia 48 h group, hypoxia 72 h group, and hypoxia 7 d group. Except for the control group, hypoxia group rats were put in hypobaric chamber for the given time. The lung water content, contents of IL-6 and TNF-α in the lung homogenates were assessed and histological examination was conducted on all animals. Results Compared with the control group, the lung water content, content of IL-6 and TNF-α in the lung homogenates were significantly increased in the hypoxia 24 h group, hypoxia 48 h group and hypoxia 72 h group, while lung water content, content of IL-6 and TNF-α in the lung homogenates were lower than that in the hypoxia 72 h group. Interstitial pulmonary edema and inflammatory cell infiltration, alveolar septal widening and interstitial hyperemia were found in the hypoxia 72 h group. Conclusion A rat model of high altitude pulmonary edema is successful set up by putting the rats in hypobaric chamber for 72 h.

Abstract:Objective To study the differentially expressed proteins in the skin of Chinese hamster, and explore the mechanism of albinism. Method Two-dimensional gel electrophoresis was performed to separate the differentially expressed proteins, and subsequently they were identified by mass spectrometry. Results Totally 2700-3000 spots were detected in the samples. Sixty-four differentially expressed spots were detected. Among them 33 significant differential protein spots were observed. There were 14 spots matched with real proteins in the 33 spots, and only 11 differential proteins were found. These identified proteins could be divided into 4 categories according to their functions: (1) glycometabolism proteins; (2) transport proteins; (3) cytoskeletal proteins; (4) other proteins. Conclusions There are significant differences between the Chinese hamster and albino ones. Some proteins are involved in the pathogenesis mechanism of albinism, which may be helpful in the study of albinism and may become therapeutic target sites.

Abstract:Objective To establish a BALB/c mouse model of endometriosis dysmenorrhea. Methods Auto-transplantation of uterine tissue into the peritoneum was applied to generate endometriosis model in 60 healthy, 6-8-week old female BALB/c mice. The mice were divided into 4 groups: operation+estrogen+oxytocin group, operation+estrogen group, operation+ oxytocin group, and operation group. Besides, an estrogen+oxytocin group and sham operation group were also set up. Different regimens were applied to treat the mice from 1st to 12th day after operation, observed the writhing response, and collected tissue samples from the ectopic foci to examine the histopathological characteristics in order to screen the best regimen. Results Except for the estrogen+oxytocin group and sham operation group, the ectopic foci of all the other groups developed well. The lesion volumes of the operation+estrogen+oxytocin group and operation+ estrogen group were significantly larger than that in the other groups(P<0.01). The incidence of writhing response of the operation+estrogen+oxytocin group was 100%, and there were statistically significant differences among different groups (P<0.01). The writhing latency and writhing frequency of the operation+estrogen+oxytocin group were significantly different from that in the other groups (P<0.01 or P<0.05). Conclusions Operation+estrogen+ oxytocin is the best method to establish a mouse model of endometriosis dysmenorrhea, and is simple to perform. This animal model can be used to investigate the mechanisms and treatment of endometriosis dysmenorrhea.

Abstract:Objective To determine the blood biochemical parameters of wild and cage reared rhesus macaques in Anhui Province, and compare the differences between the two kinds of macaques and B virus (BV) positive and negative infection. Methods Fourteen blood biochemical indexes of Anhui rhesus macaques were measured with an automatic blood biochemical analyzer. The differences of biochemical parameters between wild and breeding, and BV positive and negative infection were analyzed. Results The blood biochemical indexes of males were higher than those of females. ALP, TG and GGT showed significant difference between males and females of wild macaques. ALP, ALB, Ca, TG, Cr and GGT showed significant difference between males and females of breeding macaques. The biochemical indexes of BV positive infected monkeys were higher than that of BV negative infected monkeys. Conclusion The blood biochemical parameters are different between wild and breeding macaques, males and females, and BV positive and negative infected monkeys.

Abstract:Objective To investigate the prevalence of avian influenza in birds in the flower & bird markets in Beijing, and to provide basic evidence for prevention and control of influenza. Methods An influenza surveillance was carried out in two bigger flower & bird markets in Beijing, and 161 anal samples were obtained from 100 sparrows, 50 swallows, 10 pigeons, and one parrot. Their virus isolation was performed in SPF embryonated chicken eggs. Result No avian influenza virus was isolated from these bird samples. However, Newcastle disease virus was detected in one pigeon, and avian paramyxovirus type 2 was isolated in tree sparrows. Conclusions The basic data of epidemiological evidence of avian influenza is obtained from flower & bird markets in Beijing. H7N9 influenza virus has not been detected in birds in two flower & bird markets in Beijing. The data are helpful for prevention and control of influenza.

Abstract:As one of the major domestic minipig strains, Guangxi Bama minipigs have the following characteristics: genetic stability, fecund species, lighter weight, large area of body surface covered with white hairs, many tissues and organs and biochemical indicators similar to those of humans and so on. All these characteristics make them being widely applied in medical research. Because of the similar anatomy and physiology in cardiovascular system, Bama minipigs have been used in the research of cardiovascular system. In our country, Bama minipigs are used for establishing models of cardiovascular diseases such as myocardial ischemia, patent foramen ovale and so on. Pigs are omnivore-animals and similar to human beings in lipid metabolism, so they can be used in the study of endocrine diseases. Bama minipigs have been used in modeling, genetic susceptibility and complication prevention of diabetes. Bama mini-pigs' digestive system is similar to that of humans, so they can be used as a model of digestive system diseases including chronic pancreatitis, rupture of colon and bilioenteric anastomosis. The aspect that large area of their body surface is covered with white hairs except their head and tail makes them ideal model of skin wound and healing of burns. Bama minipigs can be used for stomatological research for their similarity to human beings in the structure of the teeth and large crack. Bama minipigs have been applied for the research of pulp necrosis and the way of maxillary expansion. They are similar to human beings in anatomy, physiology and pathology, which makes them an appropriate provider for zenotransplantation. In the research of traditional Chinese medicine, Bama minipigs has been used as liver, spleen and femoral arteriovenous fistula hemorrhagic model to study curative effect and mechanism of traditional Chinese preparation.