Infected Peripheral Blood Mononuclear Cells Research Articles

No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis)

Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes.

Hepatitis B virus replication is upregulated in proliferated peripheral blood lymphocytes.

Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin‑2 (IL‑2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT‑PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL‑2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.

Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients

Chlamydia pneumoniae is a cause of respiratory infection in adults and children. There is evidence for an association between atypical bacterial respiratory pathogens and the pathogenesis of asthma. We compared T helper (Th) responses in C. pneumoniae – infected peripheral blood mononuclear cells (PBMC) in patients with or without asthma. PBMC (1×106/mL) from asthmatic patients (N=11) and non-asthmatic controls (N=12) were infected or mock-infected for 1h +/− C. pneumoniae TW-183 at a multiplicity of infection (MOI)=1 and MOI=0.1, or cultured for 24h +/− Lactobacillus rhamnosus GG (LGG). Interleukin (IL)-4, IL-10, IL-12, Interferon (IFN)-gamma and total IgE levels were measured in supernatants (ELISA). C. pneumoniae infection led to an increase (>50%) of IgE levels in PBMC from asthmatics, compared with mock-infected on day 10; IgE wasn’t detected in non-asthmatics. C. pneumoniae – infected PBMC from asthmatics increased levels of IL-4 and IFN-gamma after 24h, compared with PBMC alone; levels of IL-10 and IL-12 were low. When uninfected-PBMC from asthmatics were LGG-stimulated, after 24h, IL-4 was undetectable, but IL-10, IL-12, and IFN-gamma increased, compared with PBMC alone. Thus, C. pneumoniae infection has the ability to induce allergic responses in PBMC of asthmatics, as evidenced by production of Th2 responses and IgE.

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells.

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

Occult Hepatitis C Virus Infection in Hemodialysis Patients; Single Center Study.

IntroductionA new form of hepatitis C virus infection, known as occult hepatitis C virus (HCV) infection, is characterized by the presence of HCV_RNA in the liver or peripheral blood mononuclear cells (PBMCs). However, no serological markers of infection occur and there is not as much damage to the liver damage as is produced by chronic hepatitis C. There is a high incidence of HCV infection among hemodialysis patients, there is significant concern about viral transmission. HCV infection is a major problem in hemodialysis (HD) units even though blood products are screened for anti-HCV antibodies and other precautions are taken. The aim of this study was to determine the prevalence of occult HCV infection in PBMC in chronic hemodialysis (CHD) patients in the dialysis unit at Theodor Bilharz Research Institute (TBRI) with HCV antibodies and HCV RNA negativity irrespective of their liver function tests.MethodsFifty-three patients who were repeatedly were anti-HCV negative and serum HCV-RNA negative and on regular hemodialysis for > six months were enrolled in the study, which was conducted in the dialysis unit of Nephrology Department at TBRI; there were 10 healthy matched controls. The patients were classified into two groups according to the result HCV RNA in their PBMCs. Serological markers of HCV infection, including anti-HCV antibody and serum HCV-RNA, were repeatedly negative for all patients included in the study. We collected serum and PBMC samples from the patients on the day they entered the study. The test of all serum samples for anti-HCV antibodies and HCV-RNA was repeated by RT-PCR to ensure that the patients did not have these HCV serologic markers, We also measured their ALT and GGT levels.ResultsOccult hepatitis C virus infection (OCI) was detected in 15.1% of our CHD patients without any evidence of chronic liver disease.ConclusionOccult HCV infection was present among the hemodialysis patients irrespective of whether they had persistent abnormal values of liver enzymes for which no cause had been identified. Further study is required to determine the clinical significance of occult HCV infections in these patients.

Leptin induces the phagocytosis and protective immune response in Leishmania donovani infected THP-1 cell line and human PBMCs

Visceral leishmaniasis (VL) is an infectious disease responsible for several deaths in malnourished children due to impaired cell-mediated immunity, which is accompanied by low circulating leptin levels. The cytokine function of leptin is implicated for several immune regulation activities such as hematopoiesis, angiogenesis, innate and adaptive immunity. Its deficiency associated with polarization of Th2 response, which coincides with VL pathogenesis. To determine the cytokine role of leptin in case of experimental VL, we tested the leptin associated Th1/Th2 type cytokine profile at mRNA level from Leishmania donovani infected human monocytic leukemia cell line (THP-1) and peripheral blood mononuclear cells (PBMCs). We also tested the effect of leptin on macrophages activation (viz. studying the phosphorylation of signaling moieties), phagocytic activity and intracellular reactive oxygen species (ROS) production during infection. We observed that leptin induced Th1 specific response by upregulation of IL-1α, IL-1β, IL-8 and TNF-α in THP-1 and IFN-γ, IL-12 and IL-2 in PBMCs. We also observed the downregulation of Th2 type cytokine i.e. IL-10 in THP-1 and unaltered expression of cytokines i.e. TGF-β, IL-10 and IL-4 in PBMCs. In addition, leptin stimulates the macrophages by inducing phosphorylation of Erk1/2 and Akt which are usually dephosphorylated in L. donovani infection. In concordance, leptin also induces the macrophage phagocytic activity by enhancing the intracellular ROS generation which helps in phagolysosome formation and oxidative killing of the parasite. In compilation, leptin is able to maintain the defensive environment against L. donovani infection through the classical macrophage activity.

In-vitro assessment of differential cytokine gene expression in response to infections with Egyptian classic and variant strains of highly pathogenic H5N1 avian influenza virus

In Egypt, two distinct genetic groups of HPAI H5N1 viruses are co-circulating: classic 2.2.1/C sub-clade and antigenic drift variant 2.2.1.1 clade isolated from vaccinated poultry flocks. The response of chicken innate immunity to both genotypes is not investigated, so far. In this study, expression of immune related genes (IL1b, IL4, IL6, IL8, IL10, IL18, IFNα and IFNγ) after infecting chicken macrophage cell line (HD11) and chicken peripheral blood Mononuclear cells (PBMC) with a classic and a variant strains was assayed using quantitative reverse-transcription real-time polymerase chain reaction assays (qRT-PCR). In HD11, the variant strain induced higher levels of IL1b and IL8 at 6hours post infection (hpi), IL4 at 24 / 48hpi and IFNα at 48hpi than the classic strain. Conversely, the classic strain induced about 10-fold increase of IFNγ at 24 and 48hpi and the virus replicated at higher level than the variant strain. The results of PBMC infection were similar to that reported from HD11 except for IFNγ gene expression that was higher at variant strain infected cells than that infected with the classic strain. After 24hpi skewing the innate immune response toward anti-inflammatory (humoral-associated) cytokines was different between HD11 (through IL4) and PBMC (through IL10). To sum up, the classic strain produced less cytokines which may indicate adaptation to evade the recognition by the innate immune system and explain its higher pathogenicity.

Expression of interleukin-1 beta and interleukin-6 in white-tailed deer infected with Epizootic Hemorrhagic Disease virus.

The pathogenesis of epizootic haemorrhagic disease (EHD) in white‑tailed deer (WTD) may be related to factors other than direct viral damage caused by replication in endothelium, such as the release of cytokines. This study focused on interleukin‑1 β (IL‑1) and interleukin‑6 (IL‑6), which have been shown to be variably upregulated in Bluetongue virus (BTV) infected cattle and sheep endothelial cultures possibly explaining species susceptibility to BTV. We evaluated circulating and tissue levels of IL‑1 and IL‑6 in WTD experimentally infected with EHD virus serotype 2 (EHDV‑2). Circulating levels of IL‑1 were assayed by ELISA. RT‑PCR and immunohistochemistry (IHC) were used to detect upregulation of IL‑1 and IL‑6 mRNA as well as protein expression, respectively. RT‑PCR was also used to determine whether IL‑1 and IL‑6 were upregulated in WTD peripheral blood mononuclear cells (PBMC) infected with EHDV‑2 in vitro. We found increased circulating levels of IL‑1 and upregulation of IL‑1 mRNA and protein expression and upregulation of IL‑6 mRNA in tissues of WTD infected with EHDV. Upregulation of mRNA levels of IL‑1 and IL‑6 in EHDV infected PBMCs was also observed. Findings suggest a role for IL‑1 and IL‑6 in the pathogenesis of EHD in WTD.

Identification of suitable reference gene in goat peripheral blood mononuclear cells (PBMCs) infected with peste des petits ruminants virus (PPRV)

Identification of an ideal, stably expressing reference gene regardless of treatment, such as virus infection, is an important prerequisite to normalize quantitative real-time PCR (qRT-PCR). Several studies have shown variable expression of reference genes in virus-infected cells. In the present study, we evaluated the expression stability of six commonly used housekeeping genes (ACTB, GAPDH, 18S rRNA, HSP 90, POLR2A, HPRT1) as reference genes in peste des petits ruminants virus (PPRV) infected and control (uninfected) goat PBMCs at different time points – 24h, 72h and 120h postinfection. Expression stability of six housekeeping genes in PPRV infected goat PBMCs was ranked using four different computational programs (NormFinder, Bestkeeper, geNorm, the comparative ΔCT method) based on their expression levels. Analysis revealed GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) as the most stable reference gene in PBMCs infected with PPRV. The other housekeeping genes evaluated in the study were found to vary in their expression due to virus infection and therefore are considered unreliable as reference genes for evaluating gene expression in PBMCs infected with PPRV.

Tax and Semaphorin 4D Released from Lymphocytes Infected with Human Lymphotropic Virus Type 1 and Their Effect on Neurite Growth.

Human lymphotropic virus type 1 (HTLV-1) is a retrovirus causing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurodegenerative central nervous system (CNS) axonopathy. This virus mainly infects CD4(+) T lymphocytes without evidence of neuronal infection. Viral Tax, secreted from infected lymphocytes infiltrated in the CNS, is proposed to alter intracellular pathways related to axonal cytoskeleton dynamics, producing neurological damage. Previous reports showed a higher proteolytic release of soluble Semaphorin 4D (sSEMA-4D) from CD4(+) T cells infected with HTLV-1. Soluble SEMA-4D binds to its receptor Plexin-B1, activating axonal growth collapse pathways in the CNS. In the current study, an increase was found in both SEMA-4D in CD4(+) T cells and sSEMA-4D released to the culture medium of peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients compared to asymptomatic carriers and healthy donors. After a 16-h culture, infected PBMCs showed significantly higher levels of CRMP-2 phosphorylated at Ser(522). The effect was blocked either with anti-Tax or anti-SEMA-4D antibodies. The interaction of Tax and sSEMA-4D was found in secreted medium of PBMCs in patients, which might be associated with a leading role of Tax with the SEMA-4D-Plexin-B1 signaling pathway. In infected PBMCs, the migratory response after transwell assay showed that sSEMA-4D responding cells were CD4(+)Tax(+) T cells with a high CRMP-2 pSer(522) content. In the present study, the participation of Tax-sSEMA-4D in the reduction in neurite growth in PC12 cells produced by MT2 (HTLV-1-infected cell line) culture medium was observed. These results lead to the participation of plexins in the reported effects of infected lymphocytes on neuronal cells.

In vitro antiretroviral activity and in vivo toxicity of the potential topical microbicide copper phthalocyanine sulfate.

BackgroundCopper has antimicrobial properties and has been studied for its activity against viruses, including HIV. Copper complexed within a phthalocyanine ring, forming copper (II) phthalocyanine sulfate (CuPcS), may have a role in microbicide development when used intravaginally.MethodsCuPcS toxicity was tested against cervical epithelial cells, TZM-BL cells, peripheral blood mononuclear cells (PBMC), and cervical explant tissues using cell viability assays. In vivo toxicity was assessed following intravaginal administration of CuPcS in female BALB/C mice and measured using a standardized histology grading system on reproductive tract tissues. Efficacy studies for preventing infection with HIV in the presence of various non-toxic concentrations of CuPcS were carried out in TZM-BL, PBMC, and cervical explant cultures using HIV-1BAL and various pseudovirus subtypes. Non-linear regression was applied to the data to determine the EC50/90 and CC50/90.ResultsCuPcS demonstrated inhibition of HIV infection in PBMCs at concentrations that were non-toxic in cervical epithelial cells and PBMCs with EC50 values of approximately 50 μg/mL. Reproductive tract tissue analysis revealed no toxicity at 100 mg/mL. Human cervical explant tissues challenged with HIV in the presence of CuPcS also revealed a dose–response effect at preventing HIV infection at non-toxic concentrations with an EC50 value of 65 μg/mL.ConclusionThese results suggest that CuPcS may be useful as a topical microbicide in concentrations that can be achieved in the female genital tract.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0358-5) contains supplementary material, which is available to authorized users.

Dissecting the contribution of EBNA3C domains important for EBV-induced B-cell growth and proliferation.

Epstein-Barr virus (EBV) is an oncogenic gammaherpes virus which is linked to pathogenesis of several human lymphatic malignancies. The EBV essential latent antigen EBNA3C is critical for efficient conversion of primary human B-lymphocytes to lymphoblastic cell lines and for continued LCL growth. EBNA3C, an EBV latent antigen with oncogenic potential can bind and regulate the functions of a wide range of cellular transcription factors. In our current reverse genetics study, we deleted the full length EBNA3C, and independently the RBP-Jκ and Nm23-H1 binding sites within EBNA3C using BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the EBV EBNA3C open reading frame (ORF) and more specifically the residues 621-675 which binds Nm23H1 and SUMO-1 showed a significant reduction in the ability of the cells to proliferate. Furthermore, they exhibited lower infectivity of human peripheral blood mononuclear cells (PBMCs). We also showed that recombinant EBV with deletions of the EBNA3C ORF, as well as a recombinant with residues 621-675 within EBNA3C ORF deleted had diminished abilities to activate CD40. Our study also revealed that the full length (1-992) and 621-675 aa deletions of EBNA3C when compared to wild type EBV infected PBMCs had differential expression patterns for the phosphorylation of MAP kinases specifically p38, JNK and ERK. Regulation of β-catenin also differed among wild type and EBNA3C deleted mutants. These temporal differences in signaling activities of these recombinant viruses in PBMCs is likely important in defining their functional importance in EBV-mediated B-cell transformation.

Thymosin α 1 potentiates the release by CD8+ cells of soluble factors able to inhibit HIV-1 and human T lymphotropic virus 1 infection in vitro

Background: Thymosin α-1 (Tα1) exploits a specific action on lymphoid cells and is able to induce in peripheral blood mononuclear cells (PBMCs) a strong transcriptional response. CD8 antiviral factor activity plays a role in the control or prevention of HIV-1 infection by a non-cytolytic mechanism. The ability of Tα1 to modulate the release of antiretroviral soluble factors by CD8+ cells was investigated.Methods: Supernatants from lipopolysaccharide (LPS) stimulated CD8+-isolated cells treated with Tα1 were screened on in vitro infection of human monocyte-derived macrophages (MDMs) and PBMCs with HIV-1, and of PBMCs with human T lymphotropic virus 1 (HTLV-1). In CD8+ cells, as well as in PBMCs of healthy donors as from HIV+ individuals, a microarray analysis to assess the transcriptional response after treatment was performed.Results: Tα1 potentiates the release, in LPS-stimulated CD8+ cells, of soluble factors able to inhibit both in vitro HIV-1 infection of MDMs and PBMCs and in vitro HTLV-1 infection of PBMCs. A distinctive transcriptional profile was induced by Tα1 in PBMCs from HIV+ donors.Conclusions: These findings suggest that Tα1 would represent a re-evaluated approach to antiretroviral therapy in combination with innovative treatments and with vaccine administration.

Ex vivo host and parasite response to antileishmanial drugs and immunomodulators.

BackgroundTherapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.MethodologyTo achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg SbV/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg SbV/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.Principal FindingsAnti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL -10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.Conclusions and SignificanceHuman PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.

Transgenic cattle produced by nuclear transfer of fetal fibroblasts carrying Ipr1 gene at a specific locus

This study aimed to assess the effects of the intracellular pathogen resistance 1 (Ipr1) transgene on preventing infection of Mycobacterium bovis in cattle. A specific expression vector for the Ipr1 gene was constructed and inserted in the genome between surfactant protein A and methionine adenosyltransferase I of bovine fetal fibroblasts. After SCNT, cleavage (86.9% vs. 87.4%, P > 0.05) and blastocyst developmental rates (34.6% vs. 33.5%, P > 0.05) were similar between transgenic and nontransgenic bovine fetal fibroblasts. Four surviving and one dead Ipr1-transgenic female cattle were produced by transfer of the SCNT blastocysts. Polymerase chain reaction and Southern blot analyses confirmed that the Ipr1 transgene of the cattle was located at the expected site. Inserting Ipr1 gene did not affect the expression of the surrounding genes. Main death modality of M bovis–infected peripheral blood mononuclear cells (PBMCs) derived from Ipr1-transgenic cattle was apoptosis, whereas that of PBMCs from control cattle was necrosis. In addition, the number of colony-forming units in PBMCs of Ipr1-transgenic cattle was significantly lower than that of the control cattle (P < 0.05). The finding that expression of Ipr1 transgene in PBMCs significantly increased anti–M bovis activity suggested breeding anti–M bovis cattle population by the transgenic SCNT technique could be a feasible strategy.

The mRNA expression of immune-related genes in crossbred and Tharparkar cattle in response to in vitro infection with Theileria annulata.

Tropical theileriosis is a major protozoan disease of cattle and is associated with high rates of morbidity and mortality. Indigenous cattle (Bos indicus) are less affected by this disease than exotic and crossbred cattle. Genetic basis of resistance to tropical theileriosis in indigenous cattle is not well studied. Recent reports suggest that number of immune response genes expressed differentially in exotic and indigenous breeds play an important role in breed specific resistance to tropical theileriosis. Such studies comparing expression of these genes in crossbred cattle and indigenous cattle are lacking. The present study compares the mRNA expression of immune-related genes in response to Theileria annulata infection in indigenous and crossbred cattle. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of indigenous (Tharparkar) and crossbred (HF/BS/Jersey×Hariana) cattle and challenged with prepared ground-up tick supernatant carrying Theileria annulata sporozoites in vitro. qPCR was employed to measure relative mRNA expression of toll-like receptor 10 (TLR10), signal-regulatory protein alpha (SIRPA), MHC class II DQα (BoLA-DQA), musculoaponeurotic fibrosarcoma (MAF) and prion protein (PRNP) genes in infected and control PBMCs from crossbred and indigenous cattle. On the basis of comparative fold change analysis, significant up-regulation in SIRPA, PRNP and MHC DQα genes and significant down-regulation in TLR10, cMAF and MAFB genes in crossbreds as compared to indigenous cattle was observed. Results of the present study suggest that breed specific differential expression of the genes under study may contribute to the breed specific resistance to Theileria annulata infection in indigenous cattle compared to crossbred cattle.

Transcriptome analysis of feline infectious peritonitis virus infection.

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal’s Z test statistical analysis is used to analyze the differentially expressed genes from the infected CRFK cells. In addition, RT-qPCR analysis is used for further transcriptional profiling of selected genes in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diagnosed cats.

Compartmentalization of hepatitis B virus: Looking beyond the liver.

Hepatitis B virus (HBV) is classically considered to be hepatotropic, but accumulating evidences strongly support its extra-hepatotropic nature too. HBV nucleic acids and proteins have long been reported in a variety of extra-hepatic tissues. Of these, HBV has been studied in details in the peripheral blood mononuclear cells (PBMCs), due to its accessibility. From these studies, it is now well established that PBMCs are permissive to HBV infection, replication, transcription and production of infective virions. Furthermore, molecular evolutionary studies have provided definite evidences towards evolution of HBV genome in PBMCs, which is independent of evolution occurring in the liver, leading to the emergence and selection of compartment specific escape variants or drug resistant strains. These variants/resistant strains of HBV remain restricted within the PBMCs and are rarely detected in the serum/plasma. In addition, HBV infected PBMCs have been reported to be directly transmitted through intrauterine modes, and this infection does not correlate significantly with serum HBV surface antigen or HBV DNA markers. This editorial briefly reviews the current knowledge on this topic, emphasizes and delineates the gaps that are required to be filled to properly understand the biological and clinical relevance of extrahepatic tropism of HBV.

Bos taurus papillomavirus activity in peripheral blood mononuclear cells: demonstrating a productive infection.

Bovine papillomavirus (BPV) is an oncogenic virus with mucous and epithelial tropism. Possible productive virus infection in other tissues, such as blood, has been hypothesized. In order to investigate this possibility, three samples of skin papillomas and blood were collected from bovines with BPV infection and five samples of peripheral blood and one sample of normal tissue were collected from a calf without BPV infection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and examined by reverse transcription-polymerase chain reaction, immunofluorescence, in situ hybridization, and electron microscopy. The tissue samples were examined for histopathological and immunohistochemical features. The skin papillomas showed the presence of DNA sequences of BPV-2, BPV-11, and a putative virus type. The blood samples showed DNA sequences of BPV-1, 2, and 4 simultaneously. Immunohistochemistry showed BPV L1 protein in both epithelium and stroma and BPV E2 protein in koilocytes. In situ hybridization confirmed the presence of BPV DNA in PBMCs and immunofluorescence showed nuclear labeling of E2 and L1 BPV proteins in PBMCs. The transcription analysis revealed transcripts of BPV-1 L1, BPV-2 L2, and BPV-4 E7 in blood and papilloma samples of BPV-infected cattle. The comet assay revealed high levels of host cell DNA damage upon BPV infection. Electron microscopy analysis of PBMCs identified the presence of particles in the cytoplasm that are consistent with papillomavirus in size and shape. The productive infection of PBMCs with BPV has been previously discussed and this study provides evidence indicating that PBMCs are a target of BPV.

Kaposi's sarcoma-associated herpesvirus genome programming during the early stages of primary infection of peripheral blood mononuclear cells.

ABSTRACTThe early period of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs.