Department of Veterinary Medicine Research Works

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A proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication
(PLoS, 2022-10-28) Moghimi, Seyedehmahsa; Viktorova, Ekaterina G.; Gabaglio, Samuel; Zimina, Anna; Budnik, Bogdan; Wynn, Bridge G.; Sztul, Elizabeth; Belov, George A.
As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.
Activation of the RpoN-RpoS regulatory pathway during the enzootic life cycle of Borrelia burgdorferi
(Springer Nature, 2012-03-23) Ouyang, Zhiming; Narasimhan, Sukanya; Neelakanta, Girish; Kumar, Manish; Pal, Utpal; Fikrig, Erol; Norgard, Michael V
The maintenance of Borrelia burgdorferi in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in B. burgdorferi plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of B. burgdorferi, has been lacking. Herein, we examined the expression of rpoS and key lipoprotein genes regulated by RpoS, including ospC, ospA, and dbpA, throughout the entire tick-mammal infectious cycle of B. burgdorferi. Our data revealed that transcription of rpoS, ospC, and dbpA is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of rpoS and dbpA in B. burgdorferi within mouse tissues following borrelial dissemination. However, dbpA transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the dbpBA operon. Our results also provide further evidence for the downregulation of ospA expression during mammalian infection, and the repression of ospC at later phases of mammalian infection by B. burgdorferi. Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of B. burgdorferi to its mammalian host, and is sustained during mammalian infection.
A balanced game: chicken macrophage response to ALV-J infection
(Springer Nature, 2019-03-06) Feng, Min; Xie, Tingting; Li, Yuanfang; Zhang, Nan; Lu, Qiuyuan; Zhou, Yaohong; Shi, Meiqing; Sun, Jingchen; Zhang, Xiquan
Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes’ function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1β, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.
BB0324 and BB0028 are constituents of the Borrelia burgdorferi β-barrel assembly machine (BAM) complex
(Springer Nature, 2012-04-20) Lenhart, Tiffany R; Kenedy, Melisha R; Yang, Xiuli; Pal, Utpal; Akins, Darrin R
Similar to Gram-negative bacteria, the outer membrane (OM) of the pathogenic spirochete, Borrelia burgdorferi, contains integral OM-spanning proteins (OMPs), as well as membrane-anchored lipoproteins. Although the mechanism of OMP biogenesis is still not well-understood, recent studies have indicated that a heterooligomeric OM protein complex, known as BAM (β-barrel assembly machine) is required for proper assembly of OMPs into the bacterial OM. We previously identified and characterized the essential β-barrel OMP component of this complex in B. burgdorferi, which we determined to be a functional BamA ortholog. In the current study, we report on the identification of two additional protein components of the B. burgdorferi BAM complex, which were identified as putative lipoproteins encoded by ORFs BB0324 and BB0028. Biochemical assays with a BamA-depleted B. burgdorferi strain indicate that BB0324 and BB0028 do not readily interact with the BAM complex without the presence of BamA, suggesting that the individual B. burgdorferi BAM components may associate only when forming a functional BAM complex. Cellular localization assays indicate that BB0324 and BB0028 are OM-associated subsurface lipoproteins, and in silico analyses indicate that BB0324 is a putative BamD ortholog. The combined data suggest that the BAM complex of B. burgdorferi contains unique protein constituents which differ from those found in other proteobacterial BAM complexes. The novel findings now allow for the B. burgdorferi BAM complex to be further studied as a model system to better our understanding of spirochetal OM biogenesis in general.
Characterization of a Chikungunya virus strain isolated from banked patients’ sera
(Springer Nature, 2016-09-02) Chalaem, Pattra; Chusri, Sarunyou; Fernandez, Stefan; Chotigeat, Wilaiwan; Anguita, Juan; Pal, Utpal; Promnares, Kamoltip
Chikungunya virus (CHIKV) is a prevalent mosquito-borne pathogen that is emerging in many parts of the globe causing significant human morbidity. Here, we report the isolation and characterization of an infectious CHIKV from banked serum specimens of suspected patients from the 2009 epidemic in Thailand. Standard plaque assay was used for CHIKV isolation from the banked serum specimens. Isolated CHIKV was identified base on E1 structural gene sequence. Growth kinetic, infectivity, cell viability and cytokine gene expression throughout CHIKV infection in a permissive cell line, 293T cells, was performed using several approaches, including standard plaque assay, immunofluorescence assay, classical MTT assay, and quantitative real-time PCR. Two tailed Student’s t test was used for evaluation statistically significance between the mean values of the groups. Based on the E1 structural gene sequence and phylogenetic analysis, we identified the virus as the CHIK/SBY8/10 isolate from Indonesia. Assessment of the growth kinetics, cytopathic effects as well as its ability to induce cellular immune responses suggested that the currently isolated CHIK/SBY8/10 virus is relatively more virulent than a known CHIKV vaccine strain, which also induces more dramatic proinflammatory responses.
Characterization of influenza virus sialic acid receptors in minor poultry species
(2010-12-09) Kimble, Brian; Ramirez Nieto, Gloria; Perez, Daniel R
It is commonly accepted that avian influenza viruses (AIVs) bind to terminal a2,3 sialic acid (SA) residues whereas human influenza viruses bind to a2,6 SA residues. By a series of amino acid changes on the HA surface protein, AIVs can switch receptor specificity and recognize a2,6 SA positive cells, including human respiratory epithelial cells. Animal species, like pigs and Japanese quail, that contain both a2,3 and a2,6 SA become ideal environments for receptor switching. Here, we describe the SA patterns and distributions in 6 common minor domestic poultry species: Peking duck, Toulouse geese, Chinese ring-neck pheasant, white midget turkey, bobwhite quail, and pearl guinea fowl. Lectins specific to a2,3 and a2,6 SA (Maakia amurensis agglutinin and Sambuca nigra agglutinin, respectively) were used to detect SA by an alkaline phosphotase-based method and a fluorescent-based method. Differences in SA moieties and their ability to bind influenza viruses were visualized by fluorescent labeling of 4 different H3N2 influenza viruses known to be specific for one receptor or the other. The geese and ducks showed a2,3 SA throughout the respiratory tract and marginal a2,6 SA only in the colon. The four other avian species showed both a2,3 and a2,6 SA in the respiratory tract and the intestines. Furthermore, the turkey respiratory tract showed a positive correlation between age and a2,6 SA levels. The fact that these birds have both avian and human flu receptors, combined with their common presence in backyard farms and live bird markets worldwide, mark them as potential mixing bowl species and necessitates improved surveillance and additional research about the role of these birds in influenza host switching.
Characterization of tick organic anion transporting polypeptides (OATPs) upon bacterial and viral infections
(Springer Nature, 2018-11-14) Taank, Vikas; Zhou, Wenshuo; Zhuang, Xuran; Anderson, John F.; Pal, Utpal; Sultana, Hameeda; Neelakanta, Girish
Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV). Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites. This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.
CXCR2 is essential for cerebral endothelial activation and leukocyte recruitment during neuroinflammation
(Springer Nature, 2015-05-21) Wu, Fengjiao; Zhao, Yawei; Jiao, Tian; Shi, Dongyan; Zhu, Xingxing; Zhang, Mingshun; Shi, Meiqing; Zhou, Hong
Chemokines and chemokine receptors cooperate to promote immune cell recruitment to the central nervous system (CNS). In this study, we investigated the roles of CXCR2 and CXCL1 in leukocyte recruitment to the CNS using a murine model of neuroinflammation. Wild-type (WT), CXCL1−/−, and CXCR2−/− mice each received an intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). Esterase staining and intravital microscopy were performed to examine neutrophil recruitment to the brain. To assess endothelial activation in these mice, the expression of adhesion molecules was measured via quantitative real-time polymerase chain reaction (PCR) and Western blotting. To identify the cellular source of functional CXCR2, chimeric mice were generated by transferring bone marrow cells between the WT and CXCR2−/− mice. Expression levels of the chemokines CXCL1, CXCL2, and CXCL5 were significantly increased in the brain following the i.c.v. injection of LPS. CXCR2 or CXCL1 deficiency blocked neutrophil infiltration and leukocyte recruitment in the cerebral microvessels. In the CXCR2−/− and CXCL1−/− mice, the cerebral endothelial expression of adhesion molecules such as P-selectin and VCAM-1 was dramatically reduced. Furthermore, the bone marrow transfer experiments demonstrated that CXCR2 expression on CNS-residing cells is essential for cerebral endothelial activation and leukocyte recruitment. Compared with microglia, cultured astrocytes secreted a much higher level of CXCL1 in vitro. Astrocyte culture conditioned medium significantly increased the expression of VCAM-1 and ICAM-1 in cerebral endothelial cells in a CXCR2-dependent manner. Additionally, CXCR2 messenger RNA (mRNA) expression in cerebral endothelial cells but not in microglia or astrocytes was increased following tumor necrosis factor-α (TNF-α) stimulation. The intravenous injection of the CXCR2 antagonist SB225002 significantly inhibited endothelial activation and leukocyte recruitment to cerebral microvessels. CXCL1 secreted by astrocytes and endothelial CXCR2 play essential roles in cerebral endothelial activation and subsequent leukocyte recruitment during neuroinflammation.
CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection
(PLOS, 2021-10-06) Liu, Gongguan; Abas, Osama; Strickland, Ashley B.; Chen, Yanli; Shi, Meiqing
Liver macrophages internalize circulating bloodborne parasites. It remains poorly understood how this process affects the fate of the macrophages and T cell responses in the liver. Here, we report that infection by Trypanosoma brucei induced depletion of macrophages in the liver, leading to the repopulation of CXCL16-secreting intrahepatic macrophages, associated with substantial accumulation of CXCR6+CD4+ T cells in the liver. Interestingly, disruption of CXCR6 signaling did not affect control of the parasitemia, but significantly enhanced the survival of infected mice, associated with reduced inflammation and liver injury. Infected CXCR6 deficient mice displayed a reduced accumulation of CD4+ T cells in the liver; adoptive transfer experiments suggested that the reduction of CD4+ T cells in the liver was attributed to a cell intrinsic property of CXCR6 deficient CD4+ T cells. Importantly, infected CXCR6 deficient mice receiving wild-type CD4+ T cells survived significantly shorter than those receiving CXCR6 deficient CD4+ T cells, demonstrating that CXCR6+CD4+ T cells promote the mortality. We conclude that infection of T. brucei leads to depletion and repopulation of liver macrophages, associated with a substantial influx of CXCR6+CD4+ T cells that mediates mortality.
Detection of NP, N3 and N7 antibodies to avian influenza virus by indirect ELISA using yeast-expressed antigens
(Springer Nature, 2009-10-07) Upadhyay, Chitra; Ammayappan, Arun; Vakharia, Vikram N
Avian influenza viruses, belonging to the family Orthomyxoviridae, possess distinct combinations of hemagglutinin (H) and the neuraminidase (N) surface glycoproteins. Typing of both H and N antigens is essential for the epidemiological and surveillance studies. Therefore, it is important to find a rapid, sensitive, and specific method for their assay, and ELISA can be useful for this purpose, by using recombinant proteins. The nucleoprotein (NP) and truncated neuraminidase subtype 3 and 7 of avian influenza virus (AIV) were expressed in Saccharomyces cerevisiae and used to develop an indirect enzyme-linked immunosorbent assay for antibody detection. The developed assays were evaluated with a panel of 64 chicken serum samples. The performance of NP-ELISA was compared with the commercially available ProFlok® AIV ELISA kit. The results showed comparable agreement and sensitivity between the two tests, indicating that NP-ELISA assay can be used for screening the influenza type A antibody in AIV infected birds. The N3 and N7- ELISAs also reacted specifically to their type specific sera and did not exhibit any cross-reaction with heterologous neuraminidase subtype specific sera. The study demonstrates the expression of the NP, N3, and N7 proteins of AIV in yeast (S. cerevisiae) and their application in developing an indirect ELISA for detecting NP, N3 and N7 antibodies from AIV-infected chicken sera. The described indirect ELISAs are rapid, sensitive, specific and can be used as promising tests during serological surveillance.
Development of a recombinant Newcastle disease virus-vectored vaccine for infectious bronchitis virus variant strains circulating in Egypt
(Springer Nature, 2019-02-11) Abozeid, Hassanein H.; Paldurai, Anandan; Varghese, Berin P.; Khattar, Sunil K.; Afifi, Manal A.; Zouelfakkar, Sahar; El-Deeb, Ayman H.; El-Kady, Magdy F.; Samal, Siba K.
Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.
Evaluation of Candidate Reference Genes Stability for Gene Expression Analysis by Reverse Transcription QPCR in Clostridium Perfringens
(Springer Nature, 2022-11-13) Williams, Michele L.; Ghanem, Mostafa
Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase + . These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-h period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.
Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9
(Springer Nature, 2011-02-23) Samuel, Arthur S; Subbiah, Madhuri; Shive, Heather; Collins, Peter L; Samal, Siba K
Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.
Fungal dissemination is limited by liver macrophage filtration of the blood
(Springer Nature, 2019-10-08) Sun, Donglei; Sun, Peng; Li, Hongmei; Zhang, Mingshun; Liu, Gongguan; Strickland, Ashley B.; Chen, Yanli; Fu, Yong; Xu, Juan; Yosri, Mohammed; Nan, Yuchen; Zhou, Hong; Zhang, Xiquan; Shi, Meiqing
Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions.
Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor
(Nature Publishing Group, 2019-07-09) Liu, Xiaoyang; Palaniyandi, Senthilkumar; Zhu, Iowis; Tang, Jin; Li, Weizhong; Wu, Xiaoling; Ochsner, Susan Park; Pauza, C. David; Cohen, Jeffrey I.; Zhu, Xiaoping
Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgGFcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.
IL-27 Negatively Regulates Tip-DC Development during Infection
(American Society for Microbiology, 2021-02-16) Liu, Gongguan; Abas, Osama; Fu, Yong; Chen, Yanli; Strickland, Ashley B.; Sun, Donglei; Shi, Meiqing
Tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cells (Tip-DCs) have profound impacts on host immune responses during infections. The mechanisms regulating Tip-DC development remain largely unknown. Here, using a mouse model of infection with African trypanosomes, we show that a deficiency in interleukin-27 receptor (IL-27R) signaling results in escalated intrahepatic accumulation of Ly6C-positive (Ly6C1) monocytes and their differentiation into Tip-DCs. Blocking Tip-DC development significantly ameliorates liver injury and increases the survival of infected IL-27R2/2 mice. Mechanistically, Ly6C1 monocyte differentiation into pathogenic Tip-DCs in infected IL-27R2/2 mice is driven by a CD41 T cell-interferon gamma (IFN-g) axis via cell-intrinsic IFN-g signaling. In parallel, hyperactive IFN-g signaling induces cell death of Ly6C-negative (Ly6C2) monocytes in a cell-intrinsic manner, which in turn aggravates the development of pathogenic Tip-DCs due to the loss of the negative regulation of Ly6C2 monocytes on Ly6C1 monocyte differentiation into Tip-DCs. Thus, IL-27 inhibits the dual-track exacerbation of Tip-DC development induced by a CD41 T cell–IFN-g axis. We conclude that IL-27 negatively regulates Tip-DC development by preventing the cell-intrinsic effects of IFN-g and that the regulation involves CD41 T cells and Ly6C2 monocytes. Targeting IL-27 signaling may manipulate Tip-DC development for therapeutic intervention.
IL-27 Signaling Promotes Th1 Responses and Is Required to Inhibit Fungal Growth in the Lung during Repeated Exposure to Aspergillus fumigatus
(American Association of Immunologists, 2022-01-01) Strickland, Ashley B.; Sun, Donglei; Sun, Peng; Chen, Yanli; Liu, Gongguan; Shi, Meiqing
Aspergillus fumigatus is an opportunistic fungal pathogen that causes a wide spectrum of diseases in humans, including life-threatening invasive infections as well as several hypersensitivity respiratory disorders. Disease prevention is predicated on the host’s ability to clear A. fumigatus from the lung while also limiting inflammation and preventing allergic responses. IL-27 is an important immunoregulatory cytokine, but its role during A. fumigatus infection remains poorly understood. In contrast to most infection settings demonstrating that IL-27 is anti-inflammatory, in this study we report that this cytokine plays a proinflammatory role in mice repeatedly infected with A. fumigatus. We found that mice exposed to A. fumigatus had significantly enhanced secretion of IL-27 in their lungs. Genetic ablation of IL-27Rα in mice resulted in significantly higher fungal burdens in the lung during infection. The increased fungal growth in IL-27Rα−/− mice was associated with reduced secretion of IL-12, TNF-α, and IFN-γ, diminished T-bet expression, as well as a reduction in CD4+ T cells and their activation in the lung, demonstrating that IL-27 signaling promotes Th1 immune responses during repeated exposure to A. fumigatus. In addition, infected IL-27Rα−/− mice displayed reduced accumulation of dendritic cells and exudate macrophages in their lungs, and these cells had a lower expression of MHC class II. Collectively, this study suggests that IL-27 drives type 1 immunity and is indispensable for inhibiting fungal growth in the lungs of mice repeatedly exposed to A. fumigatus, highlighting a protective role for this cytokine during fungal infection.
Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses
(2011-01-21) Cai, Yibin; Song, Haichen; Ye, Jianqiang; Shao, Hongxia; Padmanabhan, Rangarajan; Sutton, Troy C; Perez, Daniel R
Mass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18- day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 10 6 EID50 of LAIVs improved hatchability to ≥90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.
Inducing Autophagic Cell Death by Nsp5 of Porcine Reproductive and Respiratory Syndrome Virus
(Austin Publishing Group, 2015-11-10) Yang, Liping; Wang, Rong; Ma, Zexu; Wang, Yu; Zhang, Yanjin
Porcine Reproductive and Respiratory Syndrome (PRRS) leads to severe economic losses to the swine-producing industry. Many unclear questions remain on pathogenesis of PRRS virus (PRRSV), including the mechanism of PRRSV-induced cell death. In this study, we cloned and expressed a PRRSV non-structural protein, nsp5, and discovered that it induced cell death in cultured cells. The nsp5 protein localized in cytoplasm and majority of the protein concentrated in perinuclear region. Along with extension of incubation time, the nsp5 tended to form puncta and polarized besides nucleus. An interesting observation was that the nsp5 expression induced cell death. Cell viability assay showed that the cells with nsp5 expression had over 2-fold more cell death than cells with empty vector. Further study indicated that the nsp5 induced cell death via autophagy. Treatment with 3-MA, an autophagy inhibitor, blocked the nsp5- induced cell death. These results suggest that nsp5 might play an important role in PRRSV-induced cell death. Further examination on the mechanism is warranted.
Interference of Apoptosis by Hepatitis B Virus
(MDPI, 2017-08-18) Lin, Shaoli; Zhang, Yan-Jin
Hepatitis B virus (HBV) causes liver diseases that have been a consistent problem for human health, leading to more than one million deaths every year worldwide. A large proportion of hepatocellular carcinoma (HCC) cases across the world are closely associated with chronic HBV infection. Apoptosis is a programmed cell death and is frequently altered in cancer development. HBV infection interferes with the apoptosis signaling to promote HCC progression and viral proliferation. The HBV-mediated alteration of apoptosis is achieved via interference with cellular signaling pathways and regulation of epigenetics. HBV X protein (HBX) plays a major role in the interference of apoptosis. There are conflicting reports on the HBV interference of apoptosis with the majority showing inhibition of and the rest reporting induction of apoptosis. In this review, we described recent studies on the mechanisms of the HBV interference with the apoptosis signaling during the virus infection and provided perspective.

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