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1.

Background

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, activated during influenza A virus infection, can promote viral replication via multiple mechanisms. Direct binding of NS1 protein to p85β subunit of PI3K is required for activation of PI3K/Akt signaling. Binding and subsequent activation of PI3K is believed to be a conserved character of influenza A virus NS1 protein. Sequence variation of NS1 proteins in different influenza A viruses led us to investigate possible deviation from the conservativeness.

Results

In the present study, NS1 proteins from four different influenza A virus subtypes/strains were tested for their ability to bind p85β subunit of PI3K and to activate PI3K/Akt. All NS1 proteins efficiently bound to p85β and activated PI3K/Akt, with the exception of NS1 protein from an H5N1 virus (A/Chicken/Guangdong/1/05, abbreviated as GD05), which bound to p85β but failed to activate PI3K/Akt, implying that as-yet-unidentified domain(s) in NS1 may alternatively mediate the activation of PI3K. Moreover, PI3K inhibitor, LY294002, did not suppress but significantly increased the replication of GD05 virus.

Conclusions

Our study indicates that activation of PI3K/Akt by NS1 protein is not highly conserved among influenza A viruses and inhibition of the PI3K/Akt pathway as an anti-influenza strategy may not work for all influenza A viruses.  相似文献   

2.
A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.  相似文献   

3.
Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.  相似文献   

4.
BackgroundMalignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2).ObjectivesThis study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods.MethodsBlood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene.ResultsThe highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples.ConclusionsThis study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.  相似文献   

5.
Recently, a series of acute swine erysipelas outbreaks occurred in Eastern China. Eight strains isolated from cases of septicemia were determined as serotype 1a, and 4 of the isolates were resistant to acriflavine. One isolate strain named HX130709 was attenuated on agar media containing acriflavine dye. The 432-bp hypervariable region in spaA gene of the field and attenuated strains were amplified and sequenced. It was further compared with the vaccine strain G4T10, and thus, the eight field strains can be divided into four spaA-types. The partial spaA gene analysis also showed that no point mutations occurred among different archived passages of HX130709 during the attenuation. Results of pulsed-field gel electrophoresis showed that eight distinct patterns with 22 to 30 DNA fragment bands were produced from field strains, and twelve distinct patterns with 23 to 27 DNA fragment bands were produced from different passages of the attenuated strains. Mouse pathogenicity test showed that the mortality of the mice infected with 104 CFU field strains was 100% and the attenuation of strain HX130709 occurred between 46 and 50 passages. All the field and attenuated strains were highly sensitive to β-lactam antibiotics, tetracyclines and macrolides. So, we can make conclusions that the acute swine erysipelas outbreaks in Eastern China were caused by serotype 1a E. rhusiopathiae strains with different biochemical characteristics, and the virulence of serotype 1a E. rhusiopathiae strains is unrelated with some point mutations in 432-bp hypervariable region of the spaA gene.  相似文献   

6.
Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.  相似文献   

7.
Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. AF362776 and AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.  相似文献   

8.

Background

Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.

Methods

Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no M73220 and 16S variant 2 which is identical to GenBank no AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.

Results

Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.

Conclusion

Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.  相似文献   

9.

Background

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.

Methods

Five-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number M73220) and variant 2 (GenBank acc. no. AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).

Results

A. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.

Conclusion

The present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.  相似文献   

10.
The test was aimed to study the effect of insulin-like growth factor 1(IGF-1) on the proliferation of bovine mammary epithelial cells (BMECs),and provide a theoretical basis for the subsequent regulating mammary gland development using IGF-1. The optimal IGF-1 concentration for inhibiting BMECs apoptosis was obtained by measuring the apoptosis rate at 12, 24, 48 and 72 h after the addition of IGF-1 at four groups (0, 10, 50 and 100 μg/mL). Then divided into six groups:BMECs group, BMECs+IGF-1 group, BMECs+LY294002 group, BMECs+IGF-1+LY294002 group, BMECs+RAPA group and BMECs+IGF-1+RAPA group,and the apoptosis rates were detected by flow cytometry. The results showed that the heterologous IGF-1 had an inhibitory effect on the apoptosis of BMECs with an optimal concentration of 50 μg/mL. The apoptosis rates of BMECs+IGF-1+LY294002 and BMECs+IGF-1+RAPA groups were extremely significantly higher than BMECs+IGF-1 group (P<0.01). This study suggested that IGF-1 could activate the PI3K/Akt/mTOR signaling pathway and thereby inhibit the apoptosis of BMECs. Moreover, IGF-1 might also promote the "repair" mechanism for the inhibited PI3K/Akt/mTOR signaling pathway, and therefore make it reparticipate in BMECs life process.  相似文献   

11.
试验旨在研究胰岛素样生长因子-1（insulin-like growth factor 1,IGF-1）对奶牛乳腺上皮细胞（bovine mammary epithelial cells,BMECs）增殖的影响,为后期利用IGF-1调控乳腺发育奠定理论基础。以奶牛乳腺上皮细胞为材料,首先分4组分别外源添加0（对照组）、10、50、100 μg/mL IGF-1且分别培养12、24、48、72 h,测定抑制BMECs凋亡率的最佳浓度;然后分6组：单纯BMECs组、BMECs+IGF-1组、BMECs+LY294002组、BMECs+IGF-1+LY294002组、BMECs+RAPA组和BMECs+IGF-1+RAPA组,采用流式细胞术测定各组细胞凋亡率。结果表明,外源性添加IGF-1对BMECs凋亡率具有抑制作用,最佳浓度为50 μg/mL;BMECs+IGF-1+LY294002与BMECs+IGF-1+RAPA组细胞凋亡率均极显著高于BMECs+IGF-1组（P<0.01）。推断IGF-1能够活化PI3K/Akt/mTOR信号通路,参与BMECs凋亡的调控作用,进而抑制BMECs细胞凋亡;IGF-1可能会对被抑制PI3K/Akt/mTOR信号通路产生"修复"机制,使其能够重新参与BMECs的生命进程。  相似文献   

12.
The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.  相似文献   

13.
High-yield dairy cows are usually subject to high-intensive cell metabolism and produce excessive reactive oxygen species (ROS). Once ROS is beyond the threshold of scavenging ability, it can induce oxidative stress, imperilling the reproductive performance of cows. The study was to investigate the effects of vitamin E (VE) on H2O2-induced proliferation and apoptosis of bovine granulosa cells and the underlying molecular mechanism. Granulosa cells were pretreated with VE for 24 hr and then treated with H2O2 for 6 hr. The results showed that VE treatment decreased the intracellular ROS levels, increased the MDA content, and improved the antioxidant enzyme activity in a dose-dependent manner. Furthermore, VE treatment promoted the proliferation and inhibited apoptosis in granulosa cells by up-regulation of CCND1 and BCL2 levels and down-regulation of P21, BAX, and CASP3 levels. The cytoprotective effects of VE were attributed to the activation of the NRF2 signalling pathway. Knockdown of the NRF2 impaired the cytoprotective effects of VE on granulosa cells. Besides, the PI3K/AKT and ERK1/2, but not the p38 signalling pathway is involved in the regulation of VE-mediated cell proliferation and apoptosis. The PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited the VE-induced granulosa cell proliferation and promoted apoptosis, whereas the p38 inhibitor SB203580 had the opposite effects. These results were confirmed by proliferation and apoptosis-related gene expression at mRNA and protein levels. The results also showed that the PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited VE-induced NRF2, GCLC, GCLM, and HO-1 expression, whereas the p38 inhibitor SB203580 not. Overall, the results demonstrated that VE-regulated granulosa cell proliferation and apoptosis via NRF2-mediated defence system by activating the PI3K/AKT and ERK1/2 signalling pathway.  相似文献   

14.
Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.  相似文献   

15.
Insulin-like growth factor-I is involved in mammary gland development, promoting proliferation and inhibiting apoptosis of mammary epithelial cells (MECs). Mitogenic actions of IGF-I are mainly mediated by the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. We have found that in the presence of IGF-I bovine BME-UV1 MECs cultured on reconstituted basement membrane form large spheroids with disrupted polarity and no cavity in the center. These cells showed enhanced phosphorylation of Akt, decreased level of cleaved caspase-3, and sustained proliferative activity throughout the 16-d period of 3-dimensional culture. Inhibition of the PI3K/Akt pathway by a specific inhibitor of PI3K, LY294002, resulted in the restoration of the normal acinar phenotype. However, this effect was noted only when LY294002 was added in the second week of 3-dimensional culture, which corresponded with the time of cell cycle arrest and polarity formation under control conditions. Normal development of acini was also obtained when BME-UV1 cells were treated simultaneously with IGF-I and 17β-estradiol. The addition of 17β-estradiol regulated Akt activation, enabling the subsequent initiation of polarization processes. 17β-Estradiol also increased the level of IGFBP-3 protein in MECs cultured on Matrigel in the presence of IGF-I. The presented results indicate important interactions between signaling pathways activated by estrogen and IGF-I, which regulate alveologenesis in bovine mammary gland.  相似文献   

16.
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17.

Background

It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.

Methods

Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 106 infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.

Results

The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.

Conclusion

Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.  相似文献   

18.
以小鼠三叉神经节(TG)原代细胞为基础,应用实时荧光定量PCR(q-PCR)、Western blot等方法检测伪狂犬病病毒(pseudorabies virus,PRV)(MOI=1)感染TG细胞后不同时间点PI3K、Akt基因的转录水平和蛋白表达情况.PRV感染TG细胞后,利用PI3K特异性抑制剂LY294002处...  相似文献   

19.
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20.
The Akt/protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) pathways are involved in cell survival. This study examined the temporal profiles and localization of Akt/PKB and ERK1/2 activation in rat testis after ischemia/reperfusion (I/R). Testicular tissue was collected from normal control rats and rats exposed to reperfusion for 6, 24, and 48 hr after ischemic injury; the tissues were analyzed via Western blotting and immunohistochemistry. Western blot analysis showed that the levels of phosphorylated Akt/PKB (pAkt/PKB) and ERK1/2 (pERK1/2) increased significantly during the first 6-24 hr of reperfusion after ischemia. However, both of these activated proteins were decreased slightly at 48 hr after reperfusion. Immunohistochemically, low levels of pAkt/PKB expression were observed in Sertoli cells from the normal control. After I/R, pAkt/PKB expression increased mainly in the adluminal portion of the Sertoli cells, as well as in spermatogenic cells. In addition, pERK1/2 expression was observed in Sertoli and Leydig cells in the normal control. After I/R, pERK1/2 expression increased in some surviving spermatogenic cells (mainly spermatocytes), as well as in the adluminal portion of Sertoli cells. These results suggest that both Akt/PKB and ERK1/2 are involved in the survival of testicular cells during the early phase of testicular I/R. These pathways may represent important targets for increasing cell survival in testicular injury, including testicular torsion.  相似文献   

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