Department of Veterinary Medicine

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A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus
(MDPI, 2016-08-18) Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin
Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology.
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.
Advances in Hepatitis E Virus Biology and Pathogenesis
(MDPI, 2021-02-09) Lin, Shaoli; Zhang, Yan-Jin
Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.
Alternatively activated lung alveolar and interstitial macrophages promote fungal growth
(Elsevier, 2023-05-19) Strickland, Ashley B.; Chen, Yanli; Sun, Donglei; Shi, Meiqing
How lung macrophages, especially interstitial macrophages (IMs), respond to invading pathogens remains elusive. Here, we show that mice exhibited a rapid and substantial expansion of macrophages, especially CX3CR1+ IMs, in the lung following infection with Cryptococcus neoformans, a pathogenic fungus leading to high mortality among patients with HIV/AIDS. The IM expansion correlated with enhanced CSF1 and IL-4 production and was affected by the deficiency of CCR2 or Nr4a1. Both alveolar macrophages (AMs) and IMs were observed to harbor C. neoformans and became alternatively activated following infection, with IMs being more polarized. The absence of AMs by genetically disrupting CSF2 signaling reduced fungal loads in the lung and prolonged the survival of infected mice. Likewise, infected mice depleted of IMs by the CSF1 receptor inhibitor PLX5622 displayed significantly lower pulmonary fungal burdens. Thus, C. neoformans infection induces alternative activation of both AMs and IMs, which facilitates fungal growth in the lung.
An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission
(Springer Nature, 2023-11-06) Li, Weizhong; Wang, Tao; Rajendrakumar, Arunraj M.; Acharya, Gyanada; Miao, Zizhen; Varghese, Berin P.; Yu, Hailiang; Dhakal, Bibek; LeRoith, Tanya; Karunakaran, Athira; Tuo, Wenbin; Zhu, Xiaoping
SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission.
Anaphylatoxin signaling activates macrophages to control intracellular Rickettsia proliferation
(American Society for Microbiology, 2023-10) Dahmani, Mustapha; Zhu, Jinyi C.; Cook, Jack H.; Riley, Sean P.
Pathogenic Rickettsia species proliferate within the cytoplasm of permissive host cells in vivo. The cytoplasm of these host cells is adequate to support the complex metabolic and physiological needs for Rickettsia growth. However, a dramatic host/pathogen interplay occurs when Rickettsia encounter innate immune cells, whereby the bacteria can proliferate as normal or the host can restrict bacterial growth. This interplay is most divergent within myeloid host cells, where intra- and extracellular factors can produce either successful Rickettsia parasitism or innate immune control of bacterial proliferation. With the prior knowledge that the mammalian complement system is activated during mammalian infection, we sought to determine if extracellular complement activation and anaphylatoxin signaling can modify the fate of Rickettsia within mononuclear host cells. Results indicate that supplementation of growth media with either C3a or C5a anaphylatoxin peptides is sufficient for many myeloid cells to control the proliferation of multiple different Rickettsia species. Chemical or genetic disruption of anaphylatoxin signaling or anaphylatoxin receptors eliminates complement-induced restriction of bacterial proliferation. Finally, anaphylatoxin signaling modifies macrophage physiology by inducing inflammatory phenotypes that ultimately control the intracellular proliferation of these pathogens.
Antagonizing JAK-STAT signaling by porcine reproductive and respiratory syndrome virus
(2018) Yang, Liping; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by numerous cytokines. JAK-STAT pathways involve in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of their significance in immune response, they are often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and severe respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One possible reason for the weak protective immune response is that PRRSV interferes with innate immunity and modulates cytokine signaling, including JAK-STAT pathways. The objective of this project was to elucidate the mechanisms of PRRSV interference with JAK-STAT2 and JAK-STAT3 signaling. This study demonstrates that PRRSV antagonizes interferon (IFN)-activated JAK-STAT2 signaling and oncostatin M (OSM)-activated JAK-STAT3 pathway via inducing STAT2 and STAT3 degradation. Mechanistically, PRRSV non-structural protein 11 (nsp11) and nsp5 induce the degradation of STAT2 and STAT3, respectively, via the ubiquitin-proteasome pathway. Notably, PRRSV manipulates karyopherin alpha 6 (KPNA6), an importin that is responsible for STAT3 nuclear translocation in the JAK-STAT signaling, to facilitate viral replication. Knockdown of KPNA6 expression led to significant reduction in PRRSV replication. These data demonstrate that PRRSV interferes with different JAK-STAT pathways to evade host antiviral response while harnessing cellular factors for its own replication. These findings provide new insights into PRRSV-cell interactions and its molecular pathogenesis in interference with the host immune response, and facilitate the development of novel antiviral therapeutics.
AVIAN PARAMYXOVIRUS-VECTORED VACCINES AGAINST INFECTIOUS BRONCHITIS VIRUS AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS
(2019) Shirvani, Edris; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines. Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV. Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts. Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions. In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.
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.
Biological Significance of selected Ixodes scapularis Transcription Factors regulating Tick Hematophagy and Development
(2023) Antara, Kazi Rifat; Pal, Utpal Professor; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lyme disease is one of the most prominent vector-borne diseases, which is transmitted by the Ixodes scapularis tick and related species, and the causative agent is the bacterial pathogen Borrelia burgdorferi. Besides I. scapularis, many other tick species are also prolific vectors of several bacterial, viral, or eukaryotic pathogens affecting humans and animals. I. scapularis possess a large genome of 2.26 Gbp, predominantly featuring repetitive DNA or transposomal elements. Although many orthologous genes are present in other arthropods and blood-borne vectors, the genome also encodes numerous unique tick-specific genes. Despite many advances in Ixodes biology and genomics, the molecular basis of their hematophagy and development remains unknown. During feeding on the host, a major tick organ like the gut undergoes remarkable yet poorly understood episodes of cell division and differentiation, accommodating a huge blood meal that can be up to 100-fold greater than their body weight. The gut, therefore, plays a vital role in blood meal acquisition, digestion, and storage, supporting the long-term survival of ticks during prolonged off-host periods of nutrient deprivation. Understanding the molecular mechanism of gut physiology, including cell division and differentiation, is an essential area of research. As transcription factors are central to the biology and development of metazoan organisms yet remain largely uncharacterized in ticks, the goal of this dissertation is to decipher the biological significance of representative groups of major development-associated transcription factors in I. scapularis that are expressed in the gut, especially during blood meal engorgement process. Among them, two of the highly upregulated transcription factors in the gut were chosen for further characterization. We show that both transcription factors, Immunoglobin-fold transcription factor (SuH) and POU domain transcription factor (Nubbin), play essential roles in tick physiology, as their knockdowns impart phenotypic defects, impacting tick feeding, development and life cycle. The latter part of the dissertation will highlight the molecular mechanism of their functions. A fundamental understanding of the molecular basis of tick biology, hematophagy, and development may contribute to developing novel strategies to curb the spread of tick-infection.
The C proteins of human parainfluenza virus type 1 exert broad control over the host innate immune response
(2010) Boonyaratanakornkit, Jim; Samal, Siba; Collins, Peter; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Human parainfluenza virus type 1 (HPIV1) is an important pediatric respiratory pathogen, and its virulence in vivo can be attenuated by introducing mutations into the C gene, a strategy that has been used to design live attenuated candidate vaccines. By tracking gene expression over time, we found that a HPIV1 mutant with a single point mutation in the C gene, referred to as C(F170S), and a HPIV1 mutant with complete deletion of the C gene, referred to as P(C-), altered the expression of over 1000 genes, in sharp contrast to wild-type (WT) HPIV1. Using functional bioinformatics, we found that binding sites for the IRF and NF-kB family of transcription factors were over-represented in many of the C protein targeted pathways. By examining the activation of the major components of the type I interferon (IFN) enhanceosome, we found that the C mutant viruses, but not WT HPIV1, activated IRF3 phosphorylation and IkBB degradation, steps integral to the formation of the interferon enhanceosome. To investigate the basis for the observed antagonism of the host response by the WT C proteins, which are expressed from the C open reading frame as a nested set of carboxy-coterminal proteins, we searched for but were unable to identify C-interacting proteins among members of the RIG-I/MDA5 pathway. Furthermore, we also found that the WT C proteins supplied in trans could block IFNB induced by P(C-) HPIV1 infection but not by heterologous inducers of RIG-I and MDA5, namely RSV and poly I:C, respectively. These two lines of evidence suggested that the HPIV1 C proteins do not directly block type I IFN production, such as by interacting with a host factor of the RIG-I/MDA5 pathway. Using knockout mouse embryonic fibroblasts, we found that HPIV1-induced IFNB production relied mainly on MDA5. Consistent with this observation, a striking amount of intracellular dsRNA was detected during infection with the C mutant HPIV1 viruses but not with WT HPIV1. A marked increase in viral genome, antigenome, and mRNA as well as a decrease in viral protein accumulation provided compelling evidence for dysregulated viral RNA synthesis and an inhibition of viral protein synthesis in the absence of WT C proteins. We suggest that this resulted in an imbalance in the N protein-to-genomic RNA ratio, leading to incomplete encapsidation and an intracellular environment permissive for the generation of dsRNA. PKR activation followed the kinetics of dsRNA accumulation and contributed to both IFNB induction and the reduction in viral protein levels. This study establishes the profound effects that the C proteins of wild-type HPIV1 play in evading the host response to HPIV1 infection, and it extends our current understanding of the innate immune response to HPIV1 infection.
CD23 MEDIATED IGE TRANSCYTOSIS IN AIRWAY INFLAMMATION
(2012) Palaniyandi, Senthilkumar; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
CD23 (FceRII), a C-type lectin type II membrane glycoprotein, plays an important role in IgE homeostasis and development of allergic inflammation. I showed that CD23 was constitutively expressed in the established or primary human airway epithelial cells and its expression was significantly up-regulated by IL-4 stimulation. In a transcytosis assay, human IgE or IgE derived immune complex was transported and enhanced by IL-4 stimulation across a polarized Calu-3 monolayer. A CD23 specific antibody or soluble CD23 significantly reduced the transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed antigen-IgE complexes were competent in inducing degranulation of the cultured human mast cells. This study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation. To verify the above results in a mouse model, CD23 expression was detected in epithelial cells lining mouse airway and enhanced by IL-4 exposure as well as in ovalbumin (OVA) sensitized mouse. I showed that CD23 transported IgE and OVA-IgE derived immune complex across airway epithelial cells in wild-type, but not CD23 knockout (KO), mice. The chimeric CD23KO mice repopulated with wild-type myeloid cells, sensitized and challenged with OVA showed significant reduction in siglec-F+ cells, eosinophils, macrophages and IL-4 in bronchoalveolar lavage fluid recovered 24 hours later compared to the wild-type mice. Our finding of CD23-mediated IgE transport in airway epithelial cells suggest a possibility of CD23 transporting an IgE Fc-fused protein for immunotherapy. CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) which competitively binds CD80 and CD86 expressed on antigen presenting cells and inhibits CD28 mediated co-stimulation of T cell activation. A CTLA4-Fc (IgE) fusion protein produced in Chinese hamster ovary cells was intranasally administrated into mouse airway for assessing its specific transport by CD23. The effect of this fusion protein on the development of allergic inflammation is being fully investigated in wild-type, CD23-KO, and chimeric mouse model.
Challenge for One Health: Co-Circulation of Zoonotic H5N1 and H9N2 Avian Influenza Viruses in Egypt
(MDPI, 2018-03-09) Kim, Shin-Hee
Highly pathogenic avian influenza (HPAI) H5N1 viruses are currently endemic in poultry in Egypt. Eradication of the viruses has been unsuccessful due to improper application of vaccine-based control strategies among other preventive measures. The viruses have evolved rapidly with increased bird-to-human transmission efficacy, thus affecting both animal and public health. Subsequent spread of potentially zoonotic low pathogenic avian influenza (LPAI) H9N2 in poultry has also hindered efficient control of avian influenza. The H5N1 viruses acquired enhanced bird-to-human transmissibility by (1) altering amino acids in hemagglutinin (HA) that enable binding affinity to human-type receptors, (2) loss of the glycosylation site and 130 loop in the HA protein and (3) mutation of E627K in the PB2 protein to enhance viral replication in mammalian hosts. The receptor binding site of HA of Egyptian H9N2 viruses has been shown to contain the Q234L substitution along with a H191 mutation, which can increase human-like receptor specificity. Therefore, co-circulation of H5N1 and H9N2 viruses in poultry farming and live bird markets has increased the risk of human exposure, resulting in complication of the epidemiological situation and raising a concern for potential emergence of a new influenza A virus pandemic. For efficient control of infection and transmission, the efficacy of vaccine and vaccination needs to be improved with a comprehensive control strategy, including enhanced biosecurity, education, surveillance, rapid diagnosis and culling of infected poultry.
CHARACTERIZATION AND DEVELOPMENT OF REVERSE GENETICS SYSTEM FOR AVIAN PARAMYXOVIRUS TYPE-3 AND ITS EVALUATION AS A LIVE VIRAL VECTOR
(2010) Kumar, Sachin; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avian Paramyxovirus (APMV) serotype 3 is one of the nine serotypes of APMV that infect a variety of avian species around the world. In chickens and turkeys, APMV-3 causes respiratory illness and drop in egg production. To understand the molecular characteristics of APMV-3, the complete genome sequences of prototype strain Netherlands and strain Wisconsin were determined. The genome length of APMV-3 strain Netherlands is 16,272 and for strain Wisconsin is 16,181 nucleotides (nt). Each genome consists of six non-overlapping genes in the order 3'N-P/V/W-M-F-HN-L5' similar to most of APMVs. Comparison of the APMV-3 strain Wisconsin nt and the aggregate predicted amino acid (aa) sequences with those of APMV-3 strain Netherlands revealed 67 and 78%, identity, respectively. The phylogenetic and serological analyses of APMV-3 strains Netherlands and Wisconsin indicated the existence of two subgroups within the same serotype. Both the strains were found to be avirulent for chickens by mean death time and intracerebral pathogenicity test. To further study the molecular biology and pathogenesis of APMV-3, a reverse genetics system for strain Netherlands was established in which infectious recombinant APMV-3 was recovered from a cloned APMV-3 antigenomic cDNA. The recovered recombinant virus showed in vitro growth characteristics and in vivo pathogenicity similar to parental virus. A recombinant APMV-3 expressing enhanced green fluorescent protein was also recovered, suggesting its potential use as a vaccine vector. Furthermore, generation and characterization of recombinant APMV-3 expressing Newcastle disease virus (NDV) F and HN proteins demonstrated that the F protein plays a major role in protection against virulent NDV challenge. Overall, the study conducted here has several downstream applications. The complete genome sequence of APMV-3 is useful in designing diagnostic reagents and in epidemiological studies. The reverse genetics system for APMV-3 would be of considerable utility for introducing defined mutations into the genome of this virus and developing a vaccine vector for animal and human pathogens.
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 BORRELIA BURGDORFERI GENE PRODUCTS CRITICAL FOR PATHOGEN PERSISTENCE AND TRANSMISSION
(2010) KUMAR, MANISH; PAL, UTPAL; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The pathogen of Lyme disease, Borrelia burgdorferi, persists in a natural tick-rodent infection cycle. An assessment of microbial transcriptome in vivo encoding selected membrane proteins shows that bba52 is upregulated during pathogen transmission. Deletion of bba52 in infectious B. burgdorferi did not alter the outcome of murine inflammation or long-term pathogen persistence in mice or ticks. However, the bba52 mutant was impaired for transmission between feeding ticks and mice, phenotypic defects that could be rescued when bba52 was genetically restored to the original genomic locus. I show that BBA52 is a surface-exposed outer membrane protein. As BBA52 is highly immunogenic, I next assessed whether BBA52 could serve as a potential candidate for transmission-blocking vaccines against spirochete infection. Passive transfer of BBA52 antibodies into the ticks did not interfere with microbial persistence in unfed ticks but blocked pathogen transmission from feeding ticks to murine hosts. More importantly, active immunization of mice with recombinant BBA52 protein significantly blocked B. burgdorferi transmission from ticks to naïve murine hosts. As BBA52 antibodies lacked detectable borreliacidal activities, their interference with spirochete survival in vivo could result from the inhibition of BBA52 function, such as vector-pathogen interaction. By far-western analysis using tick gut proteins, I found that BBA52 interacts with a ~ 35 kDa tick gut protein. I also show that BBA52 forms distinct homo-oligomer in borrelial cells and also interacts with two proteins of B. burgdorferi. These BBA52 interacting proteins possess approximate molecular weight of 33 kDa and 25 kDa and are located in the outer membrane and protoplasm, respectively. Taken together, these studies suggested the remarkable existence of B. burgdorferi surface antigens that are differentially expressed in vivo and support microbial transitions between hosts and the vector. Identification and characterization of novel vector-specific and feeding induced borrelial antigens could contribute to the development of transmission-blocking vaccines against Lyme disease.
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 LysBC17, a Lytic Endopeptidase from Bacillus cereus
(MDPI, 2019-09-19) Swift, Steven M.; Etobayeva, Irina V.; Reid, Kevin P.; Waters, Jerel J.; Oakley, Brian B.; Donovan, David M.; Nelson, Daniel C.
Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.