Differences In Tissue Tropism Research Articles

Hunting Drosophila viruses from wild populations: A novel isolation approach and characterisation of viruses

Metagenomic studies have demonstrated that viruses are extremely diverse and abundant in insects, but the difficulty of isolating them means little is known about the biology of these newly discovered viruses. To overcome this challenge in Drosophila, we created a cell line that was more permissive to infection and detected novel viruses by the presence of double-stranded RNA. We demonstrate the utility of these tools by isolating La Jolla virus (LJV) and Newfield virus (NFV) from several wild Drosophila populations. These viruses have different potential host ranges, with distinct abilities to replicate in five Drosophila species. Similarly, in some species they cause high mortality and in others they are comparatively benign. In three species, NFV but not LJV caused large declines in female fecundity. This sterilization effect was associated with differences in tissue tropism, as NFV but not LJV was able to infect Drosophila melanogaster follicular epithelium and induce follicular degeneration in the ovary. We saw a similar effect in the invasive pest of fruit crops Drosophila suzukii, where oral infection with NFV caused reductions in the fecundity, suggesting it has potential as a biocontrol agent. In conclusion, a simple protocol allowed us to isolate new viruses and demonstrate that viruses identified by metagenomics have a large effect on the fitness of the model organism D. melanogaster and related species.

IRE1-Mediated Unfolded Protein Response Promotes the Replication of Tick-Borne Flaviviruses in a Virus and Cell-Type Dependent Manner.

Tick-borne flaviviruses (TBFV) can cause severe neurological complications in humans, but differences in tissue tropism and pathogenicity have been described for individual virus strains. Viral protein synthesis leads to the induction of the unfolded protein response (UPR) within infected cells. The IRE1 pathway has been hypothesized to support flavivirus replication by increasing protein and lipid biogenesis. Here, we investigated the role of the UPR in TBFV infection in human astrocytes, neuronal and intestinal cell lines that had been infected with tick-borne encephalitis virus (TBEV) strains Neudoerfl and MucAr-HB-171/11 as well as Langat virus (LGTV). Both TBEV strains replicated better than LGTV in central nervous system (CNS) cells. TBEV strain MucAr-HB-171/11, which is associated with gastrointestinal symptoms, replicated best in intestinal cells. All three viruses activated the inositol-requiring enzyme 1 (IRE1) pathway via the X-box binding protein 1 (XBP1). Interestingly, the neurotropic TBEV strain Neudoerfl induced a strong upregulation of XBP1 in all cell types, but with faster kinetics in CNS cells. In contrast, TBEV strain MucAr-HB-171/11 failed to activate the IRE1 pathway in astrocytes. The low pathogenic LGTV led to a mild induction of IRE1 signaling in astrocytes and intestinal cells. When cells were treated with IRE1 inhibitors prior to infection, TBFV replication in astrocytes was significantly reduced. This confirms a supporting role of the IRE1 pathway for TBFV infection in relevant viral target cells and suggests a correlation between viral tissue tropism and the cell-type dependent induction of the unfolded protein response.

K-Mer Analyses Reveal Different Evolutionary Histories of Alpha, Beta, and Gamma Papillomaviruses.

Papillomaviruses (PVs) are a heterogeneous group of DNA viruses that can infect fish, birds, reptiles, and mammals. PVs infecting humans (HPVs) phylogenetically cluster into five genera (Alpha-, Beta-, Gamma-, Mu- and Nu-PV), with differences in tissue tropism and carcinogenicity. The evolutionary features associated with the divergence of Papillomaviridae are not well understood. Using a combination of k-mer distributions, genetic metrics, and phylogenetic algorithms, we sought to evaluate the characteristics and differences of Alpha-, Beta- and Gamma-PVs constituting the majority of HPV genomes. A total of 640 PVs including 442 HPV types, 27 non-human primate PV types, and 171 non-primate animal PV types were evaluated. Our analyses revealed the highest genetic diversity amongst Gamma-PVs compared to the Alpha and Beta PVs, suggesting reduced selective pressures on Gamma-PVs. Using a sequence alignment-free trimer (k = 3) phylogeny algorithm, we reconstructed a phylogeny that grouped most HPV types into a monophyletic clade that was further split into three branches similar to alignment-based classifications. Interestingly, a subset of low-risk Alpha HPVs (the species Alpha-2, 3, 4, and 14) split from other HPVs and were clustered with non-human primate PVs. Surprisingly, the trimer-constructed phylogeny grouped the Gamma-6 species types originally isolated from the cervicovaginal region with the main Alpha-HPV clade. These data indicate that characterization of papillomavirus heterogeneity via orthogonal approaches reveals novel insights into the biological understanding of HPV genomes.

Tick‐Borne Flaviviruses Activate the IRE1 Stress Pathway in a Virus‐ and Cell‐Type Dependent Manner Promoting Virus Replication

Tick-borne flaviviruses (TBFV) are transmitted by either tick-bite or consumption of raw milk products from infected ruminants. After crossing the blood-brain barrier, the virus enters the central nervous system (CNS) leading to neural inflammation and causing neurological complications. Individual viruses within this group, show differences in tissue tropism and pathogenicity. The mechanisms behind these differences are poorly understood, but likely result from a mix of direct viral replication and cellular responses to infection. Flavivirus replication is ER-associated and uses the cellular machinery for viral protein synthesis. The increased protein burden results in the activation of the unfolded protein response (UPR). The IRE1 pathway is hypothesized to support flavivirus replication by increasing protein and lipid biogenesis. We investigated the ER stress response following flavivirus infection in relevant target cell types as well as the effect of ER stress inhibition on virus replication. For this purpose, human astrocyte, neuronal and intestinal cell lines were infected with tick-borne encephalitis (TBEV) strains Neudörfl and HB171 as well as Langat virus (LGTV) and the rate of virus replication and activated UPR pathways in these cells were investigated. All three viruses activated the ER stress response in a virus- and cell-type dependent manner with IRE1 being the prominent pathway. The upregulation of X-box binding protein 1 (XBP1) as marker protein for the IRE1 pathway, differed between the cell lines and showed varying levels of intensities. The levels of activation of the IRE1 pathway among the three viruses correlated with their described levels of pathogenicity. As we found IRE1 to be the most strongly induced pathway, we further investigated the effect of three different IRE1 inhibitors on virus replication and cell viability during TBFV infection. Our data showed that TBFV replication was significantly reduced upon inhibition of the kinase and RNAse activity of IRE1, either combined or alone. These results confirm the supporting role of the IRE1 pathway for TBFV infection in relevant viral target cells and suggest that virus and cell-type dependent differences in UPR induction may affect the outcome of the infection.

Genome Sequencing of Leishmania infantum Causing Cutaneous Leishmaniosis from a Turkish Isolate with Next-Generation Sequencing Technology.

Leishmania subgenus Leishmania causes leishmaniosis, which is a chronic systemic disease in humans and animals, in which the skin and visceral organs can be affected. The disease generally consists of three different clinical types in humans: visceral (kala-azar, VL), cutaneous (CL) and mucocutaneous leishmaniosis (MCL). According to the World Health Organization (WHO), leishmaniosis is still one of the world's most neglected diseases. It has been nearly 13-14years since the completion of the first complete genome sequence of a Leishmania parasite. However, much information about these parasites remains to be elucidated, such as the causes of differences in tissue tropism. The aim of this study is to perform the whole-genome sequencing of Leishmania infantum causing cutaneous leishmaniosis from a Turkish isolate with next-generation sequencing technology. Genomic sequencing was performed on the Illumina HiSeq 2500 platform. The TruSeq Nano DNA Low Throughput Library Prep Kit, compatible with the Illumina HiSeq 2500 platform, was used to generate the library. Synthesis sequencing (SBS) was performed with a HiSeq Rapid SBS Kit v2 to generate single-fragment reads (2 × 150bp; PE) with two fragment end-to-end assemblies. Bioinformatics analyses were performed on the Geneious 11.0.5. ( www.genius.com ) platform. In our study, a high-quality whole-genome sequence (WGS) of L. infantum was successfully generated, and a total of 32,009,137 base pairs of genomic DNA from 36 chromosomes were obtained. The resulting genomic DNA sequence was submitted to the US National Center for Biotechnology Information (NCBI) GenBank ( www.ncbi.nlm.nih.gov ) database and registered under the name Leishmania infantum_TR01 (Lin_TR01). The following accession numbers were assigned by NCBI to the 36 chromosomes of the Lin_TR01 genome: CP027807, CP027810, CP027808, CP027811, CP027809, CP027812, CP027813, CP027814, CP027817, CP027818, CP027819, CP027815, CP027821, CP027816, CP027823, CP027820, CP027822, CP027824, CP027825, CP027826, CP027827, CP027828, CP027829, CP027830, CP027831, CP027832, CP027833, CP027834, CP027835, CP027836, CP027837, CP027838, CP027839, CP027840, CP027841, CP027842. As a result of the annotation of the Lin_TR01 genome, 3153 polymorphisms, 8324 genes, 8199 CDSs, 8109 mRNAs, 67 tRNAs, 11 rRNAs and 58 ncRNA were identified. Among the 8199 CDS obtained, 5278 encode hypothetical proteins. In this study, a high-quality WGS of Leishmania infantum was successfully obtained for the first time in Turkey. According to a review of WGS studies on this subject, the Lin_TR01 strain is the first strain to be isolated from cutaneous leishmaniosis. The reference genome of L. infantum JPCM5 (Peacock et al., 2007) was obtained from a visceral leishmaniosis case, in accordance with the classical tissue and organ tropism of the species. Lin_TR01 is the second whole-genome-sequenced strain in the world after the JPCM5 strain. The Lin_TR01 genome is the only L. infantum whole-genome sequence that is completed assembly level from 36 chromosomes among the genomes obtained thus far ( https://www.ncbi.nlm.nih.gov/genome/genomes/249 ).

Strain-specific joint invasion and colonization by Lyme disease spirochetes is promoted by outer surface protein C.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.

Roles of the 5′ Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication

The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Factors responsible for the development of Lyme carditis

Abstract Borrelia burgdorferi sensu lato spirochetes are unique in many aspects. They are the etiological agents of Lyme borreliosis, meta-zoonotic, tick-borne disease of mammals, including humans. Ixodes spp. ticks are the vector. With the exception of erythema chronicum migrant (EM), manifestations of the disease may vary depending on the genospecies of Borrelia burgdorferi sensu lato. One of the symptoms is Lyme carditis. To date, the causative factors and the mechanisms of pathogenesis have not been well-described. Borrelia burgdorferi spirochetes are considered as one of the most invasive mammalian pathogen. They are able to move through the skin, as well as break into and out of blood vessels, easily crossing the blood-brain barrier. Genes encoding various motility forms are bound with chemotaxis signaling system which leads and coordinates motion functions. The attachment of bacteria to host cells or extracellular matrix may promote colonization and disease development. Lyme disease spirochetes encode several surface proteins including decorin binding adhesion (DbpA), which varies among strains contributing to strain-specific differences in tissue tropism. The strains demonstrating the greatest decorin-binding activity promote the greatest colonization of heart and cause the most severe carditis. Moreover, the manifestation of Lyme carditis in certain hosts may be a result of an autoimmunological reaction due to molecular mimicry between B. burgdorferi and host self-components. In mammals, infection with B. burgdorferi induces the development of antibodies which may cross-react with myosin and neural tissue. 1. Introduction. 2. Lyme carditis – symptoms, recognition and treatment. 3. Patho-mechanism of Lyme carditis. 3.1. Spirochetes motility. 3.2. Chemotaxis and adhesion. 3.3. Autoimmunological reactions. 4. Summary 1. Wstęp. 2. Lyme carditis – objawy, rozpoznanie i leczenie. 3. Patomechanizm zapalenia serca. 3.1. Ruch krętków. 3.2. Chemotaksja i adhezja. 3.3. Reakcje autoimmunologiczne. 4. Podsumowanie

Enteroviral proteases: structure, host interactions and pathogenicity.

SummaryEnteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2Apro and 3Cpro, are important mediators of pathology. These proteases perform the post‐translational proteolytic processing of the viral polyprotein, but they also cleave several host‐cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus‐associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2Apro‐mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus‐induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus‐associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease‐specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

Sequence analysis of the 5' third of glycoprotein C gene of South American bovine herpesviruses 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5′ region of the gC (5′ gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5′ gC1 compared to 5′ gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5′ gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

Strain-specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete.

Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.

Chinese highly pathogenic porcine reproductive and respiratory syndrome virus exhibits more extensive tissue tropism for pigs

BackgroundThe highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) emerging in China exhibits high fatality to pigs. However, the mechanism related to the increased pathogenicity of the virus remains unclear. In the present study, the differences in tissue tropism between the highly pathogenic PRRSV strain (JXwn06) and the low pathogenic PRRSV strain (HB-1/3.9) were investigated using PRRSV-specific immunohistochemistry (IHC) staining to provide evidence for elucidating possible mechanism of the pathogenicity of Chinese highly pathogenic PRRSV.FindingsIHC examination showed that PRRSV antigen in the tissues including spleen, tonsil, thymus, kidney, cerebellum, stomach, small intestine, large intestine, turbinal bone and laryngeal cartilage was positive in more pigs inoculated with JXwn06 than HB-1/3.9, and the tissues including trachea, esophagus, liver, mandibular gland and thyroid gland were positive for viral antigen in the pigs inoculated with JXwn06, but not in the pigs inoculated with HB-1/3.9. Meanwhile, we observed that epithelium in tissues including interlobular bile duct in liver, distal renal tubule of kidney, esophageal gland and tracheal gland were positive for viral antigen only in JXwn06-inoculated pigs, and epithelium of gastric mucosa and fundic gland, and intestinal gland were positive for viral antigen in both JXwn06- and HB-1/3.9-inoculated pigs, using monoclonal antibodies to N and Nsp2 proteins.ConclusionsTaken together, these findings indicate that the highly pathogenic PRRSV JXwn06 displays an expanded tissue tropism in vivo, suggesting this may contribute to its high pathogenicity to pigs.

Murine Noroviruses Bind Glycolipid and Glycoprotein Attachment Receptors in a Strain-Dependent Manner

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.

Binding of Avian Coronavirus Spike Proteins to Host Factors Reflects Virus Tropism and Pathogenicity

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.

Tissue Tropism of SV40 Transformation of Human Cells: Role of the Viral Regulatory Region and of Cellular Oncogenes

SV40 has been detected prevalently in a limited panel of human tumors: mesothelioma, bone and brain tumors, and lymphoma. These are the same tumor types that are specifically induced by SV40 when injected into hamsters, a finding that has raised concerns about the possible pathogenic role of SV40 in humans. Two different SV40 isolates differing in the number of 72-bp elements in the virus regulatory region, archetypal SV40 (1ESV40), which contains one 72 bp, and nonarchetypal SV40 (wtSV40), which contains two 72 bp, have been detected in human tumors. 1ESV40 has been prevalently detected in brain tumors, with wtSV40 prevalently in mesothelioma. The apparent different cell tropism could be related to the virus (i.e., possibly to the number of 72-bp elements) and to different expression of cellular genes, known to play a critical role in SV40-mediated transformation of human cells, such as Notch-1 and c-Met. To test for possible differences in tissue tropism, we infected primary human mesothelial cells (HM) and primary human astrocytes (Ast) with 1ESV40 and with wtSV40 from 2 different SV40 strains, 776 and Baylor. All viruses transformed astrocytes; only wtSV40 transformed HM. Intracellular signaling of c-Met and Notch-1 was differently induced by these 2 viruses in HM and Ast. Differences in Notch-1 expression and signaling (i.e., downstream effectors, c-Myc, HEY-1, HES-1, and HEY-L) appeared to influence SV40-mediated transformation of primary astrocytes and mesothelial cells. Our results provide a biological rationale to the observation that 1ESV40 is prevalently detected in brain tumors and wtSV40 in mesotheliomas.

Designer Bugs: Structural Engineering to Build a Better Mouse Model

Many animal models of bacterial diseases are hampered by differences in tissue tropism and the course of pathogenesis. In a recent issue of Cell, by rationally mutating a surface invasion protein (InlA) to have higher binding affinity for its cognate host receptor (E-cadherin), Wollert et al. were able to "murinize"Listeria monocytogenes, creating a strain capable of invading intestinal epithelial cells in mice, mimicking the route of infection in humans.

Distinct gene expression in human Vδ1 and Vδ2 γδ T cells following non-TCR agonist stimulation

The two major human γδ T cell subsets, Vδ1 and Vδ2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vδ1 and Vδ2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vδ2 cells being more inflammatory and Vδ1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of γδT cells to the innate immune system. Our results indicate that circulating Vδ1 and Vδ2 subsets in humans have considerable inherent differences in gene expression following treatment with the same agonist. The patterns of differentially expressed genes are consistent with unique functional roles for these cells in vivo.

Experimental systemic infection withCryptococcus neoformansvar.grubiiandCryptococcusgattiiin normal and immunodeficient mice

Cryptococcus neoformans (Cn) var. grubii or Cryptococcus neoformans var. neoformans infection is usually associated with immunocompromised hosts, whereas Cryptococcusgattii more frequently causes disease in immunocompetent hosts. We examined the effects of immunodeficiency and glucocorticoid-induced immunosuppression on systemic murine infection induced by i.v. inoculation with these pathogens. SCID and immunocompetent BALB/c and C57BL/6 mice were infected with <or=107 yeast of Cn var. grubii or C. gattii; immunosuppressed BALB/c mice were infected with <or=106 yeast. Mortality was inoculum size-dependent in each model system, for both organisms. Following infection with 106 CFU of either Cn var. grubii or C. gattii immunocompetent BALB/c mice survived longer than immunosuppressed mice (P<0.0001 in both cases); no differences were found using lower inocula. SCID mice infected with Cn var. grubii or C. gattii died sooner than BALB/c mice (P<0.0013, all comparisons). Unexpectedly, BALB/c mice infected with C. gattii developed external lesions. Immunocompetent mice developed rectal prolapse more frequently whereas immunosuppressed mice developed more frequent skin lesions, predominantly on the tail. The fungal burden was especially high in rectum, skin and lung tissues. Histologic examination showed extensive infection of the rectum and skin and pneumonitis. Determination of CFU from various organs of immunocompetent BALB/c mice infected i.v. with 105 CFU of C. gattii or Cn var. grubii showed significant temporal increase of burdens of Cn var. grubii in brain and liver (P<0.003); other organs showed decreasing fungal burden. C.gattii was recovered only from liver and lungs, no CFU were detected in the other organs. As opposed to epidemiologic observations, our results demonstrate no predilection by C. gattii for infection of immunocompetent over immunosuppressed hosts; immunosuppression increased the risk of severe cryptococcosis by both varieties, especially at high inocula. This is the first report of C. gattii inducing experimental cutaneous and intestinal mucosal infection; Cn var. grubii did not affect these tissues, indicating differences in tissue tropism of these pathogens.

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.