Comparative medical characteristics of ZDF-T2DM rats during the course of development to late stage disease

Background: There are few reports on the comparative medical characteristics of type 2 diabetes models in late stage. An analysis of comparative medical characteristics of Zucker diabetic fatty type 2 diabetes mellitus(ZDF-T2DM) rats during the course of development to late stage disease was performed.Methods: In this study, ZDF rats were fed with high-sugar and high-fat diets to raise the fasting blood glucose, and develop of type 2 diabetes. At the late stage of T2DM, the preliminary comparative medical characteristics of the T2DM model were analyzed through the detection of clinical indicators, histopathology, related cytokine levels, and insulin-related signaling molecule expression levels.Results: In the T2DM group, the fasting blood glucose was higher than 6.8 mmol/L,the serum insulin, leptin, and adiponectin levels were significantly decreased, and glucose intolerance and insulin resistance were measured as clinical indicators. Regarding pathological indicators, a large number of pancreatic islet cells showed the reduction of insulin secretion, resulting in damaged glycogen synthesis and liver steatosis. At the molecular level, the insulin signal transduction pathway was inhibited by decreasing the insulin receptor substrate 1(IRS1), insulin receptor substrate 2(IRS2), phosphatidylinositol 3 kinase(PI3K), and glycogen synthesis kinase 3β(GSK-3β) expression levels.Conclusion: The results show that the ZDF/T2DM rats have typical clinical,histopathological, and molecular characteristics of human T2DM and thus can be used as an effective model for T2DM drug development and treatment of advanced T2DM.

Molecular Characterization of Coxsackievirus B1 Strains Isolated from Patients with Hand Foot and Mouth Disease in Yunnan,Southwest China

Coxsackievirus(CV)B belongs to the species Enterovirus B,genus Enterovirus of the family Picornaviridae.Enterovirus B(EV-B)includes 63 serotypes:CVB1-6;CVA9;echoviruses E1-7,9,11-21,24-27,and 29-33;EV-B69,EV-B 73-75,EV-B77-88,EV-B 93,EV-B 97-101,EV-B 106-107.

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

A Comparison of the Biological Characteristics of EV71 C4 Subtypes from Different Epidemic Strains

The comparative analysis of the biological characterization and the genetic background study of EV71 circulating strains is commonly recognized as basic work necessary for development of an effective EV71 vaccine. In this study, we sequenced five EV71 circulating strains, isolated from Fuyang, Hefei, Kunming and Shenzhen city of China and named them FY-23, FY-22, H44, K9 and S1 respectively. The sequence alignment demonstrated their genotypes be C4. The genetic distance of the VP1 gene from these isolates suggested that they were highly co-related with genetic identity similar to other previously reported EV71 strains in China. Additionally, these strains were identified to display some obvious proliferation dynamics and plaque morphology when propagated in Vero cells. However, a distinctive difference in pathogenic ability in neonatal mice was found. Some differences in cross neutralization test & immunogenic analysis were also found. All these results are related to the biological characterization of circulating EV71 strains in China and aid in the development of an EV71 vaccine in the future.

Genetic Analysis of the VP1 Region of Human Enterovirus 71 Strains Isolated in Fuyang,China,During 2008

Enterovirus 71 （EV71） is a common cause of Hand, foot, and mouth disease （HFMD） and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences （891 nueleotides） from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous tO members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine（H） at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.

Experimental infection of tree shrews(Tupaia belangeri) with Coxsackie virus A16

Coxsackie virus A16(CA16) is commonly recognized as one of the main human pathogens of hand-foot-mouth disease(HFMD). The clinical manifestations of HFMD include vesicles of hand, foot and mouth in young children and severe inflammatory CNS lesions. In this study, experimentally CA16 infected tree shrews(Tupaia belangeri) were used to investigate CA16 pathogenesis. The results showed that both the body temperature and the percentages of blood neutrophilic granulocytes / monocytes of CA16 infected tree shrews increased at 4-7 days post infection. Dynamic distributions of CA16 in different tissues and stools were found at different infection stages. Moreover, the pathological changes in CNS and other organs were also observed. These findings indicate that tree shrews can be used as a viable animal model to study CA16 infection.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Sequence Variation in the Gp120 region of SHIV-CN97001 during in vivo Passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.

Isolation and Complete Genomic Sequence Analysis of a New Sindbis-like Virus

The Sindbis-like virus was first discovered in China in 1986. Its complete genomic sequence consists of more than 11 000 bp encoding more than 3 700 amino acids. It contains a 5＇ non-transcriptional region （5＇-NTR） in a non-structural region, four non-structural proteins （nsP1, nsP2, nsP3, nsP4） regions, capsids in conserved and non-conserved regions and structural El, E2, E3, 6K regions and a 3＇ non-transcriptional region （3＇-NTR）. The Sindbis-IMB was isolated from the blood of a patient suspected to have encephalitis, and was followed by identification and passage. The virus RNA was extracted from virus supernatant in infected cells and the whole genome was divided into 12 fragments; RT-PCR was then performed to amplify the 12 fragments for complete sequencing. The results showed that the whole genomic sequence of Sindbis-IMB consists of 11 717 bp encoding 3 773 amino acids. Homology comparison with other Sindbis-like isolates demonstrated the highest similarity was the YN87448 with a variation of 1% strain isolated in Yunnan Province and the second highest to the SAAR86 strain with a variation of -1.2%. The nucleotide sequence variations were present in non-structural regions, resulting in amino acids K, E, N, R, H, and L in protein sequences in positions 230, 231,443,781, 1 582, and 1746 in the new isolation respectively. Furthermore, three additional amino acids-glutamic acid, serine and alanine-were noted in nsp4 terminus as compared to the YN87448 isolate

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization （M-FISH） for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model

Understanding the pathogenesis of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and clarifying antiviral immunity in hosts are critical aspects for the development of vaccines and antivirals.Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation.However,normal adult mice are not susceptible to SARS-CoV-2.Here,we developed a viral receptor(human angiotensin-converting enzyme 2,hACE2)pulmonary transfection mouse model to establish SARS-CoV-2 infection rapidly in the mouse lung.Based on the model,the virus successfully infected the mouse lung 2 days after transfection.Viral RNA/protein,innate immune cell infiltration,inflammatory cytokine expression,and pathological changes in the infected lungs were observed after infection.Further studies indicated that neutrophils were the first and most abundant leukocytes to infiltrate the infected lungs after viral infection.In addition,using infected CXCL5-knockout mice,chemokine CXCL5 was responsible for neutrophil recruitment.CXCL5 knockout decreased lung inflammation without diminishing viral clearance,suggesting a potential target for controlling pneumonia.

The miRNAs of Herpes Simplex Virus(HSV)

Herpes simplex virus (HSV) is a group of common human pathogens with two serotypes HSV-1 and HSV-2.The prevalence of HSV is worldwide.It primarily infects humans through epithelial cells,when it introduces a latent infection into the nervous system.During viral latency,only a region known as the latency-associated transcript (LAT) is expressed.The discovery of HSV miRNAs helps to draw a larger picture of the infection and pathogenesis of the virus.This review summarizes miRNAs found in HSV-1 and HSV-2 so far.The functional studies of miRNAs in HSV to date indicate that they play a stage-specific role coordinated with viral proteins to maintain the virus life cycle.

Differentiation of rhesus adipose stem cells into dopaminergic neurons

LIM homeobox transcription factor la （Lmxla） has the capacity to initiate the development program of neuronal cells and promote the differentiation of embryonic stem cells into dopaminergic neurons. In this study, rhesus adipose stem cells were infected with recombinant adenovirus carrying the Lmxla gene and co-cultured with embryonic rat neural stem cells. Cell differentiation was induced using sonic hedgehog and fibroblast growth factor-8. Immunofluorescence staining showed that cells were positive for neuron-specific enolase and ^-tubulin II1. Reverse transcription-PCR results demonstrated that rhesus adipose stem cells were not only positive for neuron-specific enolase and I^-tubulin III, but also positive for the dopaminergic neuron marker, tyrosine hydroxylase, neurofilament, glial cell line-derived neurotrophic factor family receptor a2 and nuclear receptor related factor 1. The number of Lmxla gene-infected cells expressing the dopaminergic neuron marker was substantially greater than the number of cells not infected with Lmxla gene. These results suggest that Lmxla-mediated regulation combined with the strategy of co-culture with neural stem cells can robustly promote the differentiation of rhesus adipose stem cells into dopaminergic neurons.

Screening of a High Growth Influenza B Virus Strain in Vero Cells

Due to the insufficient supply of embryonated chicken eggs,the preparation of large quantities of inactivated influenza vaccines will require an alternative virus culture system after the emergence or reemergence of a pandemic influenza virus.The Vero cell is one of the ideal options since it was used for producing many kinds of human vaccines.However,most of the influenza viruses can not grow well in Vero cells.To develop a new influenza vaccine with Vero cells as a substrate,the virus needs to adapt to this cell substrate to maintain high growth characteristics.By serial passages in Vero cells,the B/Yunnan/2/2005va(B)strain was successfully adapted to Vero cells,with the hemagglutination titer(HAT)of the virus reaching 1:512.The high growth characteristic of this strain is stable up to 21 passages.The strain was identified by hemagglutination inhibition (HAI)test and sequencing respectively;the HA1 gene sequence of the virus was cloned and analyzed.The screening and establishment of high growth B virus provides an important tool for influenza vaccine production in Vero cells.

Dysbiosis of gut microbiome affecting small intestine morphology and immune balance:a rhesus macaque model

There is a growing appreciation for the specific health benefits conferred by commensal microbiota on their hosts.Clinical microbiota analysis and animal studies in germ-free or antibiotic-treated mice have been crucial for improving our understanding of the role of the microbiome on the host mucosal surface;however,studies on the mechanisms involved in microbiome-host interactions remain limited to small animal models.Here,we demonstrated that rhesus monkeys under short-term broad-spectrum antibiotic treatment could be used as a model to study the gut mucosal host-microbiome niche and immune balance with steady health status.Results showed that the diversity and community structure of the gut commensal bacteria in rhesus monkeys were both disrupted after antibiotic treatment.Furthermore,the 16S rDNA amplicon sequencing results indicated that Escherichia-Shigella were predominant in stool samples 9 d of treatment,and the abundances of bacterial functional genes and predicted KEGG pathways were significantly changed.In addition to inducing aberrant morphology of small intestinal villi,the depletion of gut commensal bacteria led to increased proportions of CD3+T,CD4+T,and CD16+NK cells in peripheral blood mononuclear cells(PBMCs),but decreased numbers of Treg and CD20+B cells.The transcriptome of PBMCs from antibiotic-treated monkeys showed that the immune balance was affected by modulation of the expression of many functional genes,including IL-13,VCAM1,and LGR4.

Expression and Immunoreactivity of a Human Group A Rotavirus Vp4

Rotavirus capsid protein Vp4 plays an important role in the virus adhering and entering the cells. In this study, a Vp4 gene cloned from a rotavirus strain TB-Chen was highly expressed in E.coli BL21 (DE3). The results of the Western blot showed that the protein possesses specific immuno-reactivities and can be specifically recognized by guinea pig antibodies against rotavirus strain SA11 or Wa. Some Vp4 dimers were formed during renaturation. These data obtained from this study provide a strong basis for further study on the structure and function of the Vp4.

Expression and immunoreactivity of HCV/HBV epitopes

AIM： To develop the epitope-based vaccines to prevent Hepatitis C virus （HCV）/Hepatitis B virus （HBV） infections. METHODS： The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD （residuals aa2-21） and C2 VKFPGGGQIVGGVYLLPRR （residuals aa22-40）, envelope epitope E GHRMAWDMMMNWSP （residuals aa315-328） and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC （residuals aa124-147） were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E. coli cells （pET-3 system）. Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined. RESULTS： The expressed chimedc protein carrying the HCV epitopes C1, C2, E （two times）, L3C1-I2E-L1C2- L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with anti- HCV and anti-HBs antibodies. CONCLUSION： These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.

Diagnostic value of bone marrow cell morphology in visceral leishmaniasis-associated hemophagocytic syndrome:Two case reports

BACKGROUND Visceral leishmaniasis related-hemophagocytic lymphohistiocytosis(VL-HLH)is a hemophagocytic syndrome caused by Leishmania infection.VL-HLH is rare,especially in nonendemic areas where the disease is severe,and mortality rates are high.The key to diagnosing VL-HLH is to find the pathogen;therefore,the Leishmania must be accurately identified for timely clinical treatment.CASE SUMMARY We retrospectively analyzed the clinical data,laboratory examination results,and bone marrow cell morphology of two children with VL-HLH diagnosed via bone marrow cell morphology at Kunming Children’s Hospital of Yunnan,China.Both cases suspected of having malignant tumors at other hospitals and who were unresponsive to treatment were transferred to Kunming Children’s Hospital.They are Han Chinese girls,one was 2 years old and the other one is 9 mo old.They had repeated fevers,pancytopenia,hepatosplenomegaly,hypertriglyceridemia,and hypofibrinogenemia over a long period and met the HLH-2004 criteria.Their HLH genetic test results were negative.Both children underwent chemotherapy as per the HLH-2004 chemotherapy regimen,but it was ineffective and accompanied by serious infections.We found Leishmania amastigotes in their bone marrow via morphological examination of their bone marrow cells,which showed hemophagocytic cells;thus,the children were diagnosed with VL-HLH.After being transferred to a specialty hospital for treatment,the condition was well-controlled.CONCLUSION Morphological examination of bone marrow cells plays an important role in diagnosing VL-HLH.When clinically diagnosing secondary HLH,VL-HLH should be considered in addition to common pathogens,especially in patients for whom HLH-2004 chemotherapy regimens are ineffective.For infants and young children,bone marrow cytology examinations should be performed several times and as early as possible to find the pathogens to reduce potential misdiagnoses.

Isolation and characterization of a new candidate human inactivated rotavirus vaccine strain from hospitalized children in Yunnan,China:2010-2013

AIM To determine the distribution of rotavirus VP7 gene in hospitalized children in Yunnan, China. METHODS A total of 366 stool specimens were collected from hospitalized children in hospitals in Yunnan Province from September 2010 to December 2013. The genomic RNA electropherotypes and the G genotypes of the rotaviruses were determined. A phylogenetic analysis of the VP7 gene was performed. Rotavirus isolation was performed, and characterized by plaque, minimum essential medium, and all genes sequence analysis. Quantification of antibodies for inactivated vaccine prepared with ZTR-68 was examined by enzyme-linked immunosorbent assay and microneutralization assay.RESULTS Group A human rotavirus was detected in 177 of 366(48.4%) stool samples using a colloidal gold device assay. The temporal distribution of rotavirus cases showed significant correlation with the mean air temperature. Rotaviruses were isolated from 13% of the rotavirus-positive samples. The predominant genotype was G1(43.5%), followed by G3(21.7%), G9(17.4%), G2(4.3%), G4(8.7%), and mixed(4.3%) among a total of 23 rotavirus isolates. A rotavirus strain was isolated from a rotavirus-positive stool sample of a 4-month-old child in The First People's Hospital of Zhaotong(2010) for use as a candidate human inactivated rotavirus vaccine strain and for further research, and was designated ZTR-68. The genotype of 11 gene segments of strain ZTR-68(RVA/Human-wt/CHN/ZTR-68/2010/G1P[8]) was characterized. The genotype constellation of strain ZTR-68 was identified as G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. The VP7 and VP4 genotypes of strain ZTR-68 were similar to Wa-like strains.CONCLUSIONS A high prevalence of the G1, G2, and G3 genotypes was detected from 2010 to 2012. However, a dominant prevalence of the G9 genotype was identified as the cause of gastroenteritis in children in Yunnan, China, in 2013. A candidate human inactivated rotavirus vaccine strain, designated ZTR-68 was isolated, characterized, and showed immunogenicity. Our data will be useful for the future formulation and development of a vaccine in China.

Complete Nucleotide Sequence of a Mumps Virus SP Strain Isolated in China

The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4% –6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.