The serological evidence in humans supports a negligible risk of zoonotic infection from porcine circovirus type 2

There are two porcine circovirus (PCV) genotypes, PCV-1 and PCV-2. In pigs, PCV-1 infection is asymptomatic but PCV-2 infection can cause severe respiratory disease and other pathology. Although humans ingest PCV-contaminated foods and are exposed to PCV through other sources, the potential of PCV-2 as a zoonotic agent in humans and other species has not been fully explored. Here, four recombinant proteins derived from the PCV-2 capsid gene were examined as antigens using the Luciferase Immunoprecipitation System (LIPS) assay for serological analysis of PCV-2 infection. PCV-2-CAP-Δ1 was the optimum recombinant protein in the LIPS assay with a sensitivity of 93% and specificity of 100% using porcine samples. Testing of healthy human blood donors, equine and bovine serum samples failed to demonstrate the presence of anti-PCV-2 antibodies. Additionally, analysis of two high-risk human groups, cystic fibrosis patients taking porcine derived oral supplements and type I diabetes patients who had undergone porcine islet cell transplantation, showed no evidence of anti-PCV-2 antibodies. These results extend the extensively demonstrated use of LIPS as a robust approach for identifying humoral responses and provide evidence that PCV-2 is likely not infectious in humans.     

A reference map and identification of porcine testis proteins using 2-DE and MS

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.     

猪圆环病毒2型(PCV2)核衣壳蛋白免疫原性的鉴定和应用及其多抗在PCV2感染诊断中的应用

猪圆环病毒2型ORF2编码与病毒毒力相关的结构蛋白--核衣壳蛋白(Cap),该蛋白可以用于PCV2感染的血清学调查,但不同区域的PCV2分离株的ORF2特别是其抗原表位序列存在一定的突变.本研究将PCV2浙江分离株ORF2的主要抗原表位以及PCV1 ORF2进行了原核表达,将分别纯化的融合蛋白Cap2s和Cap1s免疫SPF兔后制备多抗,并进一步分析了纯化蛋白的免疫原性和多抗的特性.Western blot结果表明无论Cap2s和Cap1s均能与两个多抗发生交叉反应,而PCV2或PCV1阳性猪血清只能分别特异性地识别Cap2s和Cap1s.IFA结果则证明两个多抗对于天然Cap蛋白无交叉反应性.利用Cap2s作为包被抗原对13个猪场的259份血清样品的PCV2抗体进行ELISA检测,平均阳性率为80.69%(209/259),而各猪场的阳性率差异较大(48.28%～100%).以上结果表明Cap2s可作为一个型特异性抗原用于浙江省本地猪场猪群血清中PCV2抗体的监控,而其多抗也可用于免疫组化对PCV2感染进行有效诊断.     

Proteomic analysis of primary porcine endothelial cells after infection by classical swine fever virus

Endothelial cells are the main target of classical swine fever virus during infection, and extensive hemorrhage is the most typical clinical sign of classical swine fever. To investigate the molecular mechanism of hemorrhagic pathogenesis, two-dimensional difference gel electrophoresis with fluorescent dyes (2D-DIGE) was used to analyze the proteomic profile of primary porcine umbilical vein endothelial cells (PUVECs) following CSFV infection. Of 15 protein spots with differential expression, 8 were characterized by MALDI-TOF-MS/MS in infected PUVECs at 48 h p.i.: moesin, peroxiredoxin 6, stathmin-1, a protein similar to nascent polypeptide-associated complex alpha subunit isoform 2, phosphoglycerate kinase 1, glucosidase II, transketolase and α-tubulin. These could be sorted into 5 functional groups: glycometabolism, cell proliferation, anti-oxidative stress, inflammatory response and cytoskeleton. Western blot and real-time RT-PCR analysis confirmed the down-regulation of phosphoglycerate kinase 1 (PGK1) and up-regulation of moesin identified by 2D-DIGE. Pathway analysis of these 15 differentially expressed proteins showed that CSFV infection altered the metabolism, cytoskeleton and cell proliferation of PUVECs, and that consequently an inflammatory response was induced.     

猪圆环病毒2型的PCR检测方法应用

本研究登录Genbank对猪圆环病毒2型基因的序列进行分析,用Primer 5.0设计了ORF2基因的扩增引物,试图选择一种比较合理的PCR方法检查PCV2感染的病原,以期这种PCR方法可以有效分析猪综合征障碍病毒(PRRSV)、猪细小病毒(PPV)、猪瘟病毒的扩增(HCV)、猪伪狂犬病毒(PRV)等比较常见的病原。研究结果表明,在PCV2进行扩增阳性样品检测中,发现一条异常447 bp的DNA条带,对产物测序结果进行扩增分析,证明其是PCV ORF2基因序列。敏感性检验分析表明检测样本DNA浓度时达到了9.8×10^-4ng/μL。PCR实验具有良好的稳定性和重复性。根据对66例临床病猪的分析表明,在PCV2的检测中阳性感染率是27.26%,这可能受到HCV、PRRSV、PRV、PPV等多种病毒的感染,其中混合感染的比例达到了72.23%。     

Effect of natural or vaccine-induced porcine circovirus type 2 (PCV2) immunity on fetal infection after artificial insemination with PCV2 spiked semen

The objectives of this study were to determine if vaccination against porcine circovirus type 2 (PCV2) or previous PCV2 infection of the dam are sufficient to prevent fetal infection when dams are artificially inseminated with PCV2-spiked semen. Nine sows (Sus domestica) were allocated into three groups of three dams each: The PCV2 naïve negative control Group 1 was artificially inseminated with extended PCV2 DNA negative semen during estrus, whereas the extended semen used in the vaccinated Group 2 (PCV2 vaccine was given 8 wk before insemination) and PCV2-exposed Group 3 (infected with PCV2 12 wk before insemination) was spiked with 5 mL of PCV2 inoculum with a titer of 10^4.2 tissue culture infectious dose (TCID₅₀) per milliliter at each breeding. The dams in the vaccinated and PCV2-exposed groups were positive for PCV2 antibody but negative for PCV2 DNA in serum at the time of insemination. Three negative control dams, two vaccinated dams, and three dams with previous PCV2 exposure became pregnant and maintained pregnancy to term. After artificial insemination, viremia was detected in one of three vaccinated dams and in two of three dams with previous PCV2 exposure. At farrowing, PCV2 infection was not detected in any piglets or fetuses expelled from the negative control dams or from dams with previous PCV2 exposure. In litters of the vaccinated dams, 15 of 24 live-born piglets were PCV2 viremic at birth, with 6 of 26 fetuses having detectable PCV2 antigen in tissues. In conclusion, vaccine-induced immunity did not prevent fetal infection in this sow model using semen spiked with PCV2.     

MicroRNA-98促进猪圆环病毒2型在3D4/21细胞中的复制

【目的】通过高通量测序的方法获得PCV2感染3D4/21细胞的miRNAs表达谱,并探讨miRNA-98在PCV2复制中的作用。【方法】本研究以猪肺泡巨噬细胞系3D4/21细胞为细胞模型,对PCV2感染过程中的3D4/21细胞进行miRNAs差异表达分析,筛选与病毒复制相关的特异性miRNAs,并探讨其在PCV2复制中的作用。【结果】经高通量测序,获得PCV2感染3D4/21细胞的miRNAs表达谱,结合实验室前期研究筛选获得miRNA-98。实验表明,miRNA-98的表达量随PCV2感染时间的延长而持续升高,其变化趋势与Cap蛋白表达变化基本一致,由此推测miRNA-98与PCV2复制正相关。过表达miRNA-98可显著上调Cap蛋白的表达量和PCV2的复制。进一步的研究表明,miRNA-98参与调节宿主免疫相关细胞因子的表达和PCV2的复制。【结论】miRNA-98可通过调节免疫相关细胞因子的表达调控宿主免疫功能,帮助PCV2逃逸宿主免疫,促进PCV2在3D4/21细胞中的复制。这些发现不仅为深入了解PCV2与宿主之间的关系提供了新视角,还有望为猪圆环病毒相关疾病的防控提供新的抗病毒策略。     

重组表达猪圆环病毒2型衣壳蛋白的抗原特性分析

将猪圆环病毒2型(PCV2 )去核定位信号衣壳蛋白(Nuclearlocalizationsignal_defectedcapsidprotein ,dCap)与谷胱甘肽_S_转移酶(GST)融合,在大肠杆菌中表达,经纯化和凝血酶剪切分别获得纯化的GST_dCap融合蛋白和dCap蛋白,Westernblot结果表明二者都能与猪抗PCV2血清发生特异性反应。dCap蛋白免疫小鼠制备的单克隆抗体,不仅能特异地与GST_dCap融合蛋白、dCap蛋白和纯化的PCV2粒子发生反应,而且能特异地与PK_15细胞内的PCV2病毒颗粒发生反应,其中抗dCap蛋白的单克隆抗体4C4、3F6和2G7具有阻止病毒感染细胞的能力。表明原核表达的dCap蛋白完全或部分正确模拟了PCV2天然衣壳蛋白的构像,PCV2衣壳蛋白存在阻止PCV2病毒感染细胞的功能性表位。同时重组PCV2dCap蛋白的获得为进一步研究Cap蛋白晶体结构和将重组的dCap蛋白作为抗原建立血清学诊断试剂及疫苗研究提供了基础     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.     

安徽省猪圆环病毒2型毒株的分离与鉴定

目的从病原学角度了解安徽省猪群中PCV2的感染情况。方法PCR检测临床疑似PCV2感染的病料,阳性病料接种于PK15细胞中进行PCV2的分离和增殖,再经PCR检测、间接免疫荧光试验、电镜观察,以及ORF2基因序列分析等鉴定。结果9份临床病料中均扩增出630bp的PCV2特异性DNA片段,分离鉴定获得5株PCV2,分离毒株之间及其与27株国内外参考毒株之间的ORF2基因序列同源性在87.3%～99.8%。结论首次从病原学角度证实安徽省猪群中PCV2感染的普遍存在,分离毒株与国内外参考毒株的同源性均较高。     

Interaction of porcine circovirus type 2 replication with intracellular redox status in vitro

Abstract

Objectives

Redox status influences replication of some viruses but its effect on porcine circovirus type 2 (PCV2), the primary causative agent of the emerging swine disease post-weaning multisystemic wasting syndrome is not known. The interaction of PCV2 replication with intracellular redox status in PK15 cells was examined in this study.

Methods

Intracellular glutathione (GSH) was measured spectrophotometrically by reaction with 5, 5′-dithiobis (2-nitrobenzoic acid). Total superoxide dismutase activity (SOD) was assayed by inhibition of oxyamine oxidation by the xanthine oxidase system. Malondialdehyde (MDA) was assayed spectrophotometrically using the thiobarbituric acid reaction. Both quantification of PCV2 DNA by real-time polymerase chain reaction and indirect immunofluorescence of PCV2-infected cells were used to evaluate the replication of PCV2.

Results

Both GSH and SOD decreased significantly at 48 hours after PCV2 infection, whereas MDA concentration increased significantly after 48 hour post-infection. Furthermore, PCV2 replication in PK15 cells was significantly impaired after the elevation of intracellular GSH through treatment with the antioxidant N-acetyl-l-cysteine (NAC), a precursor in GSH synthesis. In contrast, PCV2 replication in PK15 cells was enhanced after reduction of GSH levels through H₂O₂-mediated oxidation. In addition, NAC treatment blocked the increase of virus replication induced by H₂O₂.

Conclusions

This study suggests that PCV2 infection induces oxidative stress and that intracellular redox status influences PCV2 replication in PK15 cells.     

Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus type 2 infected PK-15 cells

The infection of host cells by porcine circovirus type 2 (PCV2) leads to extensive modulation of the gene expression levels of target cells. To uncover the pathogenesis and virus-host interactions of PCV2, a quantitative proteomic study using the stable isotope labeling with amino acids in cell culture (SILAC), coupled with mass spectrometry, was performed on PCV2-infected PK-15 cells. The SILAC-based approach identified 1341 proteins, 163 of which showed significant change in level at 72 h after infection (79 up-regulated and 84 down-regulated). The modulated proteins included a number of proteins involved in substrate transport, cytoskeletal changes, and the stress response. Changes in the expression levels of selected proteins were verified by Western blot analysis. Ingenuity Pathway Analysis was used to reveal protein and interactive pathway regulation in response to PCV2 infection. Functional network and pathway analyses could provide insights into the complexity and dynamics of virus-host cell interactions and may accelerate our understanding of the mechanisms of PCV2 infection.     

家养野猪源猪圆环病毒2型安徽株的分离鉴定及其全基因组序列分析

目的对安徽省临诊疑似PMW S家养野猪病例进行猪圆环病毒2型(PCV2)分离鉴定,并对分离株的全基因组进行克隆与序列分析。方法应用PK-15细胞进行PCV2的分离与增殖,根据PCR、IFA、电镜技术进行PCV2分离株的鉴定,克隆分离株的全基因组,并对序列进行分析。结果获得1株来自安徽家养野猪源PCV2分离株,命名为YZ0901。该毒株全基因组长为1 767 bp,属于PCV2b基因型。与GenBank已发表的国内外参考毒株的同源性介于93.9%~99.2%,与安徽分离毒株彼此之间的同源性介于93.4%~99.5%。结论安徽省家养野猪中存在PCV2感染,分离毒株与家猪源病毒差异不大,PCV2核苷酸序列比较稳定,其进化不存在明显的地域相关性,家养野猪源PCV2的基因型与当地PCV2流行株的基因型密切相关。     

嵌合猪圆环病毒PCV1-2的构建及其感染性初步鉴定

猪Ⅱ型圆环病毒(PCV2)是当前严重危害养猪业的重要病原之一。目前,世界上还没有有效疫苗用于该病毒的免疫预防。该研究利用PCR方法,将PCV2的ORF2基因替换猪Ⅰ型圆环病毒(PCV1)的ORF2基因,构建了以PCV1基因组为骨架的嵌合病毒(PCV1-2)分子克隆(pSK2PCV1-2)。将该分子克隆转染PK-15细胞并连续盲传5代,用RT-PCR方法可以在转染后盲传的细胞中检测到PCV1的ORF1 mRNA和PCV2的ORF2 mRNA,但检测不到PCV1的ORF2 mRNA和PCV2的ORF1 mRNA。间接免疫荧光检测显示在盲传第5代的细胞中有PCV2 ORF2蛋白的表达,表达蛋白主要分布于细胞核。该研究初步证实构建的PCV1-2分子克隆转染细胞后可以形成具有感染性的嵌合病毒,从而为更深入研究嵌合病毒生物学特性奠定了基础。     

2010–2015年华东地区猪圆环病毒2型的分子流行病学分析

[目的] 猪圆环病毒2型（PCV2）是严重危害世界养猪业的重要传染病,即猪圆环病毒相关疾病（PCVAD）的重要病原,其致病机制及流行规律尚未阐明。本研究对2010-2015年间采集自中国华东地区的病料进行PCV2检测,以探讨中国华东地区PCV2的分子流行病学特征,分析PCV2的遗传演化规律。[方法] 本研究利用PCR方法对384份断奶仔猪多系统衰竭综合征疑似发病猪样本进行检测,并对随机选取的42份阳性样品进行PCV2全基因组的测定和分析。[结果] 华东地区普遍存在PCV2的感染,阳性率为41.15%。序列扩增获得42株PCV2全长基因组,同源性和系统进化树分析表明,基于PCV2 ORF2和全长核苷酸序列构建的系统进化树结构是基本一致的。从病料中测得的42株PCV2与11株参考毒株序列的ORF2基因的核苷酸和氨基酸序列同源性分别为87.0%-100.0%和82.5%-100.0%。遗传进化分析显示,53株PCV2毒株可分为4个基因型,即PCV2a、PCV2b、PCV2c和PCV2d。本文获得的42株序列ORF2基因的核苷酸和氨基酸序列同源性分别为89.6%-100.0%和86.8%-100%,可分为3个基因型,即PCV2a、PCV2b和PCV2d,其中69.05%（29/42）属于PCV2d基因型,为优势基因型;PCV2b未在安徽和浙江出现。Cap蛋白的氨基酸序列分析表明,42株PCV2毒株总体多样性较低,存在4个主要的高变区,且不同基因型具有代表性突变位点。本研究获得的42株Cap序列在抗体识别的关键位点存在一定程度变异,这是否导致PCV2抗原结构、致病性以及疫苗效果的改变,还有待深入分析。[结论] 2010-2015年间,PCV2在华东地区猪群中存在较高的感染率,遗传演化相对稳定,基因型无明显地域差异,且PCV2d为优势基因型。42株PCV2 Cap序列有明显差异,但同一基因型具有代表性突变位点。本研究为探讨华东地区PCV2的遗传进化和致病机理提供了参考依据。     

Ochratoxin A promotes porcine circovirus type 2 replication in vitro and in vivo

Ochratoxin A (OTA), a worldwide mycotoxin found in food and feeds, is a potent nephrotoxin in animals and humans. Porcine circovirus-associated disease (PCVAD), including porcine dermatitis and nephropathy syndrome, is a worldwide swine disease. To date, little is known concerning the relationship between OTA and porcine circovirus type 2 (PCV2), the primary causative agent of PCVAD. The effects of OTA on PCV2 replication and their mechanisms were investigated in vitro and in vivo. The results in vitro showed that low doses of OTA significantly increased PCV2 DNA copies and the number of infected cells. Maximum effects were observed at 0.05 μg/ml OTA. The results in vivo showed that PCV2 replication was significantly increased in serum and tissues of pigs fed 75 μg/kg OTA compared with the control group and pigs fed 150 μg/kg OTA. In addition, low doses of OTA significantly depleted reduced glutathione and mRNA expression of NF-E2-related factor 2 and γ-glutamylcysteine synthetase; increased reactive oxygen species, oxidants, and malondialdehyde; and induced p38 and ERK1/2 phosphorylation in PK15 cells. Adding N-acetyl-l-cysteine reversed the changes induced by OTA. Knockdown of p38 and ERK1/2 by their respective specific siRNAs or inhibition of p38 and ERK1/2 phosphorylation by their respective inhibitors (SB203580 and U0126) eliminated the increase in PCV2 replication induced by OTA. These data indicate that low doses of OTA promoted PCV2 replication in vitro and in vivo via the oxidative stress-mediated p38/ERK1/2 MAPK signaling pathway. This suggests that low doses of OTA are potentially harmful to animals, as they enhance virus replication, and partly explains why the morbidity and severity of PCVAD vary significantly in different pig farms.     

Differential expression of porcine testis proteins during postnatal development

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.     

Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH₄Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH₄Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% ± 110%, 1,212% ± 34%, and 1,100% ± 179%, respectively, in porcine kidney (PK-15) cells; by 128% ± 7%, 158% ± 3%, and 142% ± 11% in swine kidney cells; by 160% ± 28%, 446% ± 50%, and 162% ± 56% in swine testicle (ST) cells; and by 313% ± 25%, 611% ± 86%, and 352% ± 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.