Abstract:Objective The aim of the present study was to further confirm the viral concentration of SHIV-KB9 virus for infection of Chinese rhesus monkeys，to test the animals' adaptability to the virus，to clarify the repeatability of the animal model，and try to establish a Chinese-origin rhesus macaque model of SHIV virus infection. Methods Four Chinese-origin rhesus macaques were used in this study. All the monkeys were screened by serological test to ensure that they were free from SIV，SRV and STLV infection. Then they were infected by i. v. injection of 10 × diluted viral series separately. Finally to determine if the Chinese-origin rhesus macaques were infected by flow cytometry，blood routine examination，virus isolation and PCR. Results The results of all tests proved that only the virus concentration higher than of 4. 8 × 105 copies/mL can infect the Chinese-origin rhesus macaques. Conclusion This results of this study further clarified the effective concentration of SHIV-KB9 virus for infection of Chinese rhesus monkeys，identified virological and immunological indicator of SHIV-KB9 infection of Chinese rhesus monkeys，and we have successfully established a Chinese rhesus monkeys model of SHIV-KB9 virus infection.

Abstract:Objective To engineer recombinant strains of simian-human immunodeficiency virus ( SHIV) stably expressing green fluorescent protein ( EGFP) . A replication-competent SHIV construct containing the green fluorescent gene with the ability to infect rhesus monkeys would serve as an important tool in AIDS research. Methods A SHIV strain was constructed by inserting the EGFP genes into the nef gene of SHIV KB9. The infection activity and bright fluorescence expression of the SHIV clone was determined in vitro in TZM-bl cells and macaque PBMCs. Results Replicationcompetent virus and bright fluorescence of infected cells were obtained with one construct，in which EGFP was inserted into the SHIV nef locus. This strain was infectious to rhesus PBMC and TZM-bl cells. Green fluorescing cells were detected by direct microscopic visualization. Conclusions A recombinant and replication-competent SHIV strain expressing EGFP is engineered. It is suggested that the SHIV-KB9nefGFP could be used as a tool to directly detect infected cells and aid in the immunophenotypic characterization of these cells.

Abstract:Objective To propagate SHIV1157ipd3N4 virus stock via passages in Chinese-origin monkey peripheral blood mononuclear cells ( PMBCs ). Methods The PBMCs from Chinese-origin rhesus macaque were cocultured with SHIV1157ipd3N4，a CCR5-tropic chimeric simian-human immunodeficiency virus (SHIV) strain. The level of P24 antigen in the supernatant was tested continuously. The virus stock was collected when the viral duplication reached a peak. The env gene of SHIV1157ipd3N4 was analyzed，and the viral load，P24 antigen level and TCID50 were determined. The rhesus monkey G1004V was infected with this batch of SHIV1157ipd3N4 intravenously. The viral load in plasma and the changes of CD4 + / CD8 + ratio were analyzed by real-time RT-PCR and flow cytometry. Results Totally 243 mL virus stock was propagated in monkeys PBMCs. The viral load was 1. 586 × 108 copies/mL，the level of P24 antigen was 1. 16 × 103 pg /mL and had 3. 16 ×103 50% tissue culture infective dose (TCID50 ) in 1 mL of cell-free SHIV1157ipd3N4. There was no variation in the gp120 sequence of env gene and this SHIV1157ipd3N4 stock was still exclusively CCR5-tropic. The monkey G1004V was infected and had a high level of viral loads in plasma. Conclusion This panel of pathogenic R5-tropic SHIV1-157ipd3N4 was adapted in Chinese-origin rhesus monkeys PBMCs，and is stable and suitable to serve as an animal model.

Abstract:Objective To establish a guinea pig model of influenza A /California /7 /2009(CA7) virus infection and evaluate the intervention with hormone methylprednisolone for viral pneumonia due to this infection. Methods Female Hartley strain guinea pigs ( 200-300 g ) were divided into four groups: control，model，methylprednisolone group 1 and methylprednisolone group 2. The animals were anesthetized by inhalation of ether and inoculated intranasally inoculated with 0. 3 mL of virus CA7. Methylprednisolone was administered beginning at postinfection day 3 or 5，recommended dose lasting for 3 days and then quarter of recommended dose for another 3 days. The animals were observed daily for 14 days for clinical signs of infection and survival. Results The guinea pig model for A /California /7 /2009(CA7)virus infection was established successfully. The lung inflammation of methylprednisolone group 1 was alleviated compared with that of both model group and methylprednisolone group 2，but the survival rate was lower compared with that of both two groups. The lung inflammation of methylprednisolone group 2 was alleviated in comparison with that in the model group but aggravated compared with that of methylprednisolone group 1，while the survival rate was not affected. Conclusion Guinea pigs can be infected with influenza A virus A /California /7 /2009，and it is better to administer methylprednisolone on dpi 5 than dpi 3.

Abstract:Objective To study the mechanism of the influenza virus infection in BALB / c mice with A / California /7 /2009Influenza H1N1 virus. Methods Sixty BALB / c mice were randomly divided into 2 groups:experimental group and control group，each group consisted of 30 mice. The CA7 influenza virus was used to establish influenza models.The expressions of lung IL-6，TNF-α，and IL-2 in experimental group of 5th day control group were detected by enzyme linked immunosorbent assay(ELISA) . Results The lung tissue IL-6，TNF-α levels were significantly higher，IL-2 levels were significantly lower than that in the control group，the differences were significant ( P < 0. 05 ) . Conclusions The cytokines IL-2，IL-6 and TNF-α may participate in the whole process of influenza A H1N1 virus infection and their lung levels are positively correlated with the severity of the disease.

Abstract:Objective To evaluate the antiviral activity of oseltamivir phosphate，ribavirin and rimantadine hydrochloride on influenza A (H1N1) in vitro. Methods Drugs diluted in FCS-free DMEM media supplemented with 2 μg /mL TPCK-treated trypsin was applied to confluent MDCK cell monolayers for 1 h prior to addition of virus，at a challenge dose of one hundred times of the TCID50 . The IC50 of drugs were calculated using neutral red assay after 3 days treatment at 34℃ .Results The therapeutic index (TI) of oseltamivir phosphate against influenza A ( H1N1) virus was 61. 90，ribavirin was 42. 86，and rimantadine hydrochloride was 6. 22. Ubenimex was lower than 1. 00. Conclusions Influenza virus is sensitive to oseltamivir phosphate and ribavirin. The antiviral effect of rimantadine hydrochloride is significantly lower than that of oseltamivir phosphate and ribavirin.

Abstract:Objective The ELISA kit for HIV-1 p24 is sometimes used for SIV p27 detection. The aim of this study was to eclarify whether there is any dependence and correlation between the results of detecting SIV p27 antigen by HIV-1 p24 ELISA kit and SIV p27 ELISA kit. Methods SIV p27 antigens were detected qualitatively and quantitatively by HIV-1 p24 ELISA kit and SIV p27 ELISA kit，respectively，and the results were compared by regression and correlation analysis. Results The sensitivity of detecting SIV p27 antigen was 150 pg /mL by HIV-1 p24 ELISA kit while 62. 5 pg /mL by SIV p27 ELISA kit. The qualitative results obtained by HIV-1 p24 kit consisted with those by SIV p27 kit on detecting SIV p27 antigen in virus stock and plasma. The statistical analysis of quantitative results indicated linear regression R2 = 0. 857，linear correlation r = 0. 926，P < 0. 01 in virus stock，while R2 = 0. 512，r = 0. 716，P < 0. 05 in plasma. The quantitative results turned out to be positive linear regression，which was higher in virus stock and lower in plasma. Conclusions HIV-1 p24 ELISA kit can be used for detecting SIV p27 antigen in virus stock and plasma qualitatively replacing SIV p27 ELISA kit，but quantitatively was appropriate in virus stock only.

Abstract:Objective To propagate and titrate the adapted RT-SHIV from Chinese rhesus monkey using TZM-bl，CEMx174 and PBMCs cells，and to compare the possible difference of these viruses which propagated in PBMC and CEMx174，respectively. Methods Rhesus macaques free-living in China were infected with RT-SHIV intravenously.Blood samples were collected regularly and viral loads were monitored. Peripheral blood mononuclear cells ( PBMCs) were examined when the viral load reached the peak，and were co-cultured with PBMCs from uninfected rhesus or CEMx174 cells. The P24 antigen level from supernatant was monitored regularly. The co-cultured virus was collected，packed and frozen when the viral replication reaches the peak. The collected viruses as a stock were charactarized with the RNA viral loads，P24 antigen level and titration of TCID50. Results In this study，in tatol of 78 mL of RT-SHIV in monkeys’PBMCs and 85 mL of RT-SHIV was propagated in CEMx174 cells，respectively. The similarity between RT gene sequence and original sequence was 99% ，only two mutation of amino acid at 254 and 265. RT-SHIV ( PBMC) and RT-SHIV (CEMx174) viral loads were titrated with TZM-bl，CEMx174 and PBMCs，and their results were 1. 641 × 108 copies/mL and 8. 375 × 108 copies/mL. respectively，P24 antigenlevel were 20. 745 ng /mL and 4. 28 ng /mL，respectively，titrations of TCID50 in TZM-bl，CEMx174，PBMC were 3. 16 × 105TCID50 /mL and 1 × 104 TCID50 /mL;5 × 102 TCID50 /mL and 5 × 105 TCID50 /mL;5 × 102 TCID50 /mL and 5 × 103 TCID50 /mL，respectively. Conclusions RT-SHIV propagated from PBMCs has higher infective ablity than that from the other cells in vitro.

Abstract:Objective To explore the characteristic changes of SHIV-XJ02170 during serial passages in Chinese rhesus macaques in late infection stage and to identify the sequence variation of env gene. Methods Viral bloods from macaque G0401V infected with SHIV-XJ02170 in late infection stage of 5 years were used for passaging 2 monkeys (G0401V→G0402V→G0403V)without CD8 + T deletion and 1 monkey ( G0401V→G0404V) with CD8 + T deletion. Flow cytometry，viral load detection and sequence alignment were used to analyze the change features during serial passages.Results SHIV-XJ02170 incubated in G0401V showed stable infectivity during late infection for 5 years and its virulence was enhanced after passaging in vivo，especially after CD8 + T cell deletion by injection of CM-T807. G0402V showed a sharp decline of weight and CD4 + T cell counts in peripheral blood，and died suddenly on day 41 after infection.Monkeys G0403 and G0404 showed a viral load peak on day 14，then maintained low viral load level (104 － 105 copies/mL) up to now after passaging (141 d) . Sequence alignment analysis showed significant variation in env region and changes of Nglycosites in gp120 region. Conclusions The infectivity of SHIV-XJ02170 is significantly enhanced during serial passages in vivo and this study provides a firm basis for SHIV-XJ02170 infectious molecular clone construction.

Abstract:Objective Nonhuman primate models are increasingly used in the screening of candidate AIDS vaccines and immunization strategies for advancement to large-scale human trials. The predictive value of such macaque studies is largely dependent upon the fidelity of the model system in mimicking human immunodeficiency virustype 1 ( HIV-1) infection in terms of viral transmission，replication，and pathogenesis. The aim of this study was to establish a Chineseorigin rhesus monkey model of CCR5-specific chimeric simian / human immunodeficiency virus ( SHIVSF162p3 ) infection，induced by repeated low-dose intravaginal infection，and explore whether this new chimeric model could be more efficient in AIDS vaccine research. Methods Totally 6 female Chinese-origin rhesus monkeys were enrolled in this study. All animals were intra-vaginally challenged 13 times with 1 mL of a phosphate buffered virus solution containing the heterologous SHIVSF162p3，with 20 TCID50 for the first 7 challenges and 30 TCID50 for the remaining 6 challenges. Challenges were done every 4-7 days. Whole blood was collected and plasma virus was quantified by real-time SYBR green RT-PCR and T cell subsets were determined by flow cytometry analysis. Results Systemtic infection was successfully established in 6 Chinese-origin rhesus macaques after 13 challenges. The peak of viral loads was between 106 copies/mL to 108 copies/mL，wherease their CD4 + /CD8 + ratio was decreased. Conclusions The results of this study provide a firm basis for establishing a Chinese rhesus macaques model of SHIV infection by repeated low dose of SHIVSF162p3，a route more close to natural way of AIDS infection. This model would be very useful for HIV-1 subtype B vaccine and pathogenesis studies.

Abstract:Objective Shellfish toxins are not only threatening to quality of marine aquatic products but also become a potential threat to human health. As the structure diversity and function mechanism complexity，the monitoring programs of shellfish toxins using animal models are costly，difficult to quantitate and not meet 3R principles. There is a pressing need to develop new methods to reduce quantity of used animals and to relieve animal distress. Recently，many new assays based on action mechanisms have been developed including functional，immunological，chemical analysis and biosensor methods. These methods promote the efficacy of shellfish toxin monitoring，and will be accepted by administration for regulation programs provided these approaches be scientifically validated and standardized.

Abstract:Cholangiocarcinoma is a strongly aggressive malignancy of bile ducts. The worldwide incidence and mortality of cholangiocarcinoma ( CC) is steadily rising. Establishing suitable animal models of cholangiocarcinoma is beneficial for its early diagnosis and therapy. We review the current status of establishment methods of animal models of cholangiocarcinoma.

Abstract:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis infection，which is one of the major infectious diseases that are harmful to human health. The vaccine currently used，Bacille Calmette and Guerin (BCG) ，is effective in preventing the most severe disseminated forms of disease in children and newborns，but its efficacy against active TB in adults has been challenged by several clinical studies. In the last few years，the search for a new vaccine has obtained a new momentum. These recent advances in the clinical testing of new TB vaccines are very exciting and promising. However，there is a need to continue the search for additional vaccine candidates.

Abstract:Bronchial asthma ( asthma for short) is a common chronic disease. With the increase of allergic patients，it is increasingly important to emphasize the need of mouse allergic asthma models in research of this disease. This review summarizes recent domestic and overseas literatures on mouse allergic asthma experiments through comprehensive analysis on mouse selection，models making and model evaluation.

Abstract:Small model organisms such as yeast，nematodes，fruit fly and zebrafish have been widely used in biological research. They are used in research of viral mechanism and led to new understandings of virus-host interaction and antiviral immune mechanisms. The present review summarizes the versatility of these animal models and their main contributions in the elucidation of virus mechanisms.

Abstract:Currently，studies on canine reproductive regulation technology are far less developed than that in other mammals. Compared with other mammals，canine reproductive physiology has unique characteristics which make canine reproductive biotechnology faced with some difficulties. This review summaries the following aspects，including induction of estrus in dogs，preservation of semen and artificial insemination ( AI) ，embryo transfer ( ET) and clone，and discusses its current progress and prospect.