Objective To establish a simple and reliable experimental rodent model sensitive to coxsackievirus A16 ( CVA16) .Methods Mongolian gerbils with different age were selected and inoculated intraperitoneally with live CVA16, and the gerbils were observed daily until 14 days postinoculation to screen for the most optimal ages sensitive to the virus.The dose-dependent symptoms were evaluated and the 50% lethal dose (LD50) was determined.The virus titers were measured in blood and various tissues of CVA 16-infected Mongolian gerbils 3 days post-infecton.Finally, the gerbils were immunized twice with inactivated CVA 16 vaccine at day 1 and day 11, respectively, followed by challenge with the virus with a dose of LD50 at day 14.The gerbils were then observed for another 2 weeks to record their body weight , symptom and mortality rate .Their blood samples were collected from the eyes , and CVA16-specific neutralizing antibodytiters and total antibody titers was checked by microneutralization test and ELISA , respectively .Results Various clinical symptoms, such as inactivity, hind limb weakness, paralysis and even death occurred in gerbils following CAV 16 infection. 7-day-old and 14-day-old gerbils are susceptible to CVA 16 infection whereas 28-day-old gerbils are resistant .The most sensitive and appropriate age is 14-day-old.The 50%lethal dose was determined to be 1×104.5 CCID50.High titers of the virus were confirmed in blood and various tissues of Mongolian gerbils contracted CAV 163 days post-infecton.The survival rate is 87.5%for 14-day-old gerbils preimmunized with two doses of inactivated CVA 16 vaccine and challenged with the virus.The geometric mean titers ( GMTs) of neutralizing antibody was 28.14, and the seroprevalence was 87.5%.Conclusions Mongolian gerbils is sensitive to CVA16 and the virus reproduces actively in Vivo.Thus, it can be used as a reliable small animal model for studies of CVA 16 pathogenesis , vaccine development and drug evaluation .

Objective To develop a novel anti-Enterovirus 71 (EV71) vaccine as recombinant virus-like particles.Methods By utilizing the foreign antigen presentation and virus-like particles forming features of Norovirus casipid VP1 P domain (NoVP), two pET-28a (+)-based recombinant expression plasmids containing either NoVP alone or NoVP with three specific epitopes SP55, SP70 and VP2-28 of EV71 capsid proteins tandemly inserted at the surface loop site were constructed and transferred to Escherichia coli.The recombinant fusion proteins of NoVP + EV71-SP55-SP70-VP2-28 and NoVP were induced expression and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and transmission electron microscopy (TEM) observation.BALB/C mice were randomly divided into three groups:group A immunized with the recombinant fusion protein, group B immunized with NoVP and group C injected with 10 mmol/L Tris plus 20 mmol/L NaCl (pH 9.0).Enzymelinked immunosorbent assay (ELISA) was used to test specific antibodies in the serum of the mice, besides, the serums were mixed with the EV71 H3-TY strain and Vero cells, then specific antibody titer was examined by microneutralization test.One way ANOVA and Bonferroni test were used to analyze data.Results Both recombinant fusion protein and NoVP were expressed in Escherichia coli in inclusion bodies form.SDS-PAGE demonstrated that the relative molecular weights of recombinant fusion protein and NoVP protein were approximately 43 × 103 and 36 × 103, respectively;positive protein band of about 43 × 103 (relative molecular mass) was detected in recombinant fusion protein by Western Blot.Virus-like particles derived from the recombinant fusion proteins were observed under TEM.ELISA showed that absorbance 490 (A490) of mice serum added in SP70 peptide was significantly higher than those of group B and C (F =13.860,P ＜0.05).And microneutralization test demonstrated that the serum from group A was able to neutralize EV71 at a geometric mean titer above 1:38.Conclusion A novel virus-like particles vaccine against EV71 with good antigenicity and specificity has been prepared, which is able to induce high titer of neutralizing antibody against EV71 in mice.

Objective To identify immunodominant B linear cell epitopes in capsid proteins VP1 to VP3 of Coxsackievirus A16 (CVA16) strain YY157.Methods The protean algorithms of bioinformatic software Lasergene were used to analyze antigenicity, hydrophilicity and surface probability of amino acid sequence of CVA16 capsid proteins VP1 to VP3.Multiple regions containing potential lineal B cell epitopes were predicted and their corresponding average indexes were calculated by BepiPred 1.0 Server.Corresponding peptides were synthesized and examined in peptide-enzyme-linked immunosorbent assay (ELISA) individually to check whether it reacted positively or negatively toward sera from children with confirmed CVA16 infection.Results Totally 21 possible B cell linear epitopes were predicted according to their relatively strong antigenicity, hydrophilicity and surface probability.The corresponding synthetic peptides reacted positively with sera of CVA16-infected children in a varying extent.Conclusion Immunodominant B cell linear epitopes of capsid proteins VP1 to VP3 of CVA16 strain YY157 are successfully predicted and confirmed.

Objective To predict and identify liner B-cell epitopes in the hemagglutinin ( HA) of human-infected avian-origin H7N9 influenza virus and analyze the specificity of H7 subtype.Methods Three serum samples collected at different times from the same patient who was confirmed to be infected with H7N9 influenza virus were provided by Shaoxing People’s Hospital, and one serum sample from healthy person was collected as the control.The extracellular region of HA protein was predicted by TMHMM Sever v.2.0.The potential B-cell epitopes were predicted by DNAStar Lasergene’ s Protean, BcePred and ABCpred tools, and the immunogenicity of the predicted B cell antigen epitopes was assessed by indirect enzyme-linked immunosordent assay ( ELISA ) .H7 subtype specificity was analyzed by comparing HA protein amino acid sequence with H7N9 and H1-H16 subtype influenza virus from Genbank using Clustal X 2.1 software, and Cn3D 4.3.1 software was used to detect the distribution and 3D structure of predicted epitopes on the HA protein of H7N9.Results The potential B-cell epitopes may be located in 172-183, 363-380, 452-472 and 491-506 of extracellular N-terminus of HA protein.ELISA showed that four predicted eptiopes specifically reacted with positive serums from patient.Multi-sequence alignment demonstrated that peptide 172-183 and 363-380 had higher H7 subtype specificity compared with amino acid sequences of other subtypes.Moreover, the predicted linear B-cell epitopes all located on the surface of HA protein according to the 3D structure analysis.Conclusion Four potential B-cell epitopes were identified, in which peptide 172-183 and 363-380 have higher H7 subtype specificity, and may be used in the design of epitope-based vaccines and diagnostics tests.

Objective To investigate the effect of Andrographis paniculata Nees(APN)extract on Coxsackievirus A16(CVA16)in vitro.Methods African green monkey kidney-derived Vero cells(Vero cells)were treated with APN extract at the concentration of 500.0,250.0,125.0,60.0,30.0,15.0,7.5 and 3.8 μg/mL,the cytotoxicity was determined with cell counting Kit-8 and the IC50was calculated by Probit unit regression method.Direct inactivating activity on CVA16,blocking of CVA16 adsorbing Vero cells and inhibition of CVA16 replication in Vero cells were determined and compared between Ribavirin(RBV) and APN extract with CVA16-infected Vero cells.SPSS 24.0 software was used to analyze the data.Results The selected concentrations of APN extract and RBV for experiment were 15.0, 7.5 and 3.8 μg/mL according to cytotoxicity test.Both of APN extract and RBV had neither direct inactivation on CVA 16 nor blocking of CVA16 adsorbing at the concentration of 15.0,7.5 and 3.8 μg/mL(F=1.54,1.52 and 0.67, 1.68,all P>0.05).However,both drugs had the capability of inhibiting CVA 16 replication in Vero cells at the concentration of 15.0 and 7.5 μg/mL(t=6.87,11.76 and 7.71,12.84,all P<0.05).Conclusion Experimental result shows that APN extract can effectively inhibit CVA 16 replication in Vero cells in vitro.

Objective: To study infection of coxsackievirus A6 (CV-A6) in Mongolian gerbils. Methods: To screen the optimal ages of Mongolian gerbils, five groups with different ages were infected with 1×10⁵ TCID₅₀ dose of CV-A6 XS45 strain by intraperitoneal, and symptom scores of Mongolian gerbils were collected. Then to estimate the dose-effect, three doses of virus were injected to the Mongolian gerbils. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry(IHC) were used to determine virus load and tissues infection in muscle, brain and intestinal tract. Results: Mongolian gerbils infected with 1×10⁵ TCID₅₀ dose CV-A6 consistently exhibited clinical signs, and the morbidity (death) rates of five age groups were up to 100%. There was a positive correlation between the trend of symptom scores changes and ages. The morbidity (death) rates of three doses (1×10³ TCID₅₀, 1×10⁴ TCID₅₀, 1×10⁵ TCID₅₀) also were up to 100% in 28 days Mongolian gerbils. The correlation between the trend of symptom scores changes and doses were negative. Virus loads were detected in muscle, brain and intestinal tract of pathogenesis animal. The virus loads of muscle were higher than others. IHC results showed virus infection and cytopathic effects in three tissues. Conclusions Mongolian gerbils had high susceptibility to CV-A6, and were best for animal model of CV-A6 infection.

Objective: To identify immunodominant linear B cell epitopes in neuraminidase of avian influenza virus H7N9. Methods: By using protean algorithms of bioinformatic software DNAStar, antigenicity, hydrophilicity and surface probability of the H7N9 neuraminidase sequence was analyzed and their corresponding average indexes were calculated. Multiple regions containing potential linear B cell epitopes were predicted. Corresponding peptides were synthesized artificially and used in peptide-ELISA individually to check their reactivity to confirmed H7N9 positive human sera, and H7N9 negative human sera was used as control. Results: Seven potential linear B cell epitopes, namely A to G, were predicted according to their relatively strong antigenicity, hydrophilicity and surface probability. All corresponding synthetic peptides reacted strongly with H7N9 positive sera. Conclusions Immunodominant linear B cell epitopes in neuraminidase of H7N9 were successfully predicted and confirmed. It will facilitate to clarify molecular basis of the antigen specificity and to make respective antibodies.

Esophageal cancer is a common malignant tumor of digestive tract. Remarkable regional difference is a prominent feature of the clinical epidemiology of esophageal cancer. They are mainly manifested in incidence rate, incidence type, onset age, and gene mutation. These differences may be related to dietary habits, lifestyle, and environmental factors. In recent years, research on the regional differences in esophageal cancer has gradually deepened. This article summarizes the differences in incidence rate, incidence type, gene mutations, epigenetics, risk factors, and prognosis of esophageal cancer in different regions, including Asia (China, India, Japan, and other countries), Europe, America (the United States), Africa, and other regions. Understanding these differences can help doctors and public health experts understand the risk factors and causes of esophageal cancer and further develop highly effective prevention and treatment strategies to reduce the occurrence and mortality rate of this malignancy.