Nonpermissive Host Research Articles

Molecular cloning and characterization of a gene encoding methionine aminopeptidase 2 of Schistosoma japonicum

Methionine aminopeptidase 2 (MAP2) is an essential enzyme that is involved in protein maturation. It plays a key role in the removal of the initiating methionine residue from nascent polypeptide chains. In the present study, a gene encoding methionine aminopeptidase 2 of Schistosoma japonicum (SjMAP2) was cloned and characterized. Real-time RT-PCR and Western blot analysis revealed that this was expressed in each testing developmental stage in S. japonicum, but more highly expressed at 42 days in male adult worm, suggesting this gene as male differentially expressed. The results also showed that the gene was differentially expressed in worms from three different host species. It was highly expressed in worms from the schistosome-susceptible mouse, expressed at a lower level in worms from the less susceptible rat, and at an even lower level in worms from the non-permissive host Microtus fortis. The expression of the gene was affected significantly when the hosts were treated with praziquantel: Expression was down-regulated by 92.17% and 49.01%, respectively, in treated male and female adult worms in comparison with untreated worms. An immuno-experiment in mice indicated that vaccination with recombinant SjMAP2 could induce partial protective efficacy against schistosome infection. The data presented here suggest that SjMAP2 is an important molecule in the development of the schistosome and that it may be a potential new drug target or vaccine candidate for schistosomiasis.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Egf1.5 is a second phenoloxidase cascade inhibitor encoded by Microplitis demolitor bracovirus

The three-member Egf gene family from the polydnavirus Microplitis demolitor bracovirus (MdBV) encodes novel proteins distinguished by a shared cysteine-rich motif. Prior studies determined that one family member, Egf1.0, inhibits melanization of hemolymph from the moth Manduca sexta by disabling phenoloxidase activating proteinases (PAPs). Here we characterized a second family member, Egf1.5, which shares an identical cysteine-rich motif with Egf1.0, but possesses an extended C-terminal repeat domain. Similar to Egf1.0, Egf1.5 inhibited processing and the amidolytic activity of PAP1 and PAP3 from M. sexta. Egf1.5 also bound PAP1, PAP3 and serine proteinase homolog 2 (SPH2). Comparative studies indicated that Egf1.5 and Egf1.0 similarly inhibited melanization of plasma from two lepidopterans ( Pseudoplusia includens and Helicoverpa zea) that are permissive hosts for M. demolitor and MdBV, and two lepidopterans ( M. sexta and Bombyx mori) that are nonpermissive hosts. Expression studies showed that transcript abundance of egf1.5 and egf1.0 was also similar in MdBV-infected P. includens and H. zea. Taken together, our results indicate that Egf1.5 and Egf1.0 are functionally similar paralogs.

Abstract 2327: Functional investigation of glioblastoma pathology in the context of matrix metalloproteinase-1 tumor expression

Abstract Features emblematic to glioblastoma multiforme (GBM) include diffuse tumor cell invasion into normal brain and hyperplastic tumor-associated endothelium. Previously we reported the over-expression of MMP-1 in human GBM and demonstrated the importance of MMP-1 over-expression in GBM cell motility. Herein we report on our current investigations into the potential consequences of MMP-1 expression in GBM cells as related to: proteomic shifts with changes in MMP-1, tumor take in a non-permissive environment in vivo, and endothelial proliferation and differentiation in vitro. METHODS: The T98G cell line produces a detectable level of MMP-1. These cells were stably transduced with a MMP-1 specific shRNA lentivirus. Conversely U251 cells do not produce MMP-1 and these were stably transfected with a cDNA vector containing MMP-1. Cell lysates were applied to proteomic arrays containing antibodies for angiogenesis related proteins. To assess tumor cell growth in a non-permissive setting, athymic nude mice were inoculated in the hind flanks with these cells. Palpable tumor growth was observed for up to 41 days using external caliper measurement. In vitro endothelial assays consisted of microcarrier beads coated with endothelial cells and immobilized in a three-dimensional fibrin gel culture system. The gels were overlaid with either soluble factors or glioma cells. After 2-5 days of incubation, beads were randomly assessed for endothelial sprout number as a sign of proliferation and sprout length as differentiation. RESULTS: Angiogenesis antibody arrays revealed a balance between MMP-1 expression and anti-angiogenic proteins. With the over-expression of MMP-1 the detection of endostatin and CXCL4 decreased. Yet most notable is an induction of tissue inhibitor of metalloproteinases (TIMP)-4 in the absence of MMP-1. This was confirmed with immunoblot analysis. Tumor growth data indicated a significant increase in tumor volume with MMP-1 over-expression and decrease with knockdown (p<0.05). This was during the initial growth delay phase of either tumor type indicating that tumor take in a non-permissive environment was impacted. There was a 62.5% reduction in tumor take with MMP-1 knockdown, and a 44.4% increase with over-expression (p<0.05). Three-dimensional culture data indicated increased endothelial proliferation and differentiation with either recombinant MMP-1 or co-cultured over-expressers, and the converse with MMP-1 knockdowns (p<0.05). Altogether these and prior data implicate MMP-1 as an important factor in the hallmark GBM pathologies. MMP-1 positively influences the propagation of small population tumor foci in a non-permissive host environment, and it is integral to angiogenic induction. A key mechanism of MMP-1 mediated induction of angiogenesis may involve a coordinated inhibition of TIMP-4 and other anti-angiogenic proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2327.

The immune response to and immunomodulation byHymenolepis diminuta

Analyses of laboratory-based helminth-rodent model systems have been immensely useful in delineating the workings of the mammalian immune system. Investigations in the 1970s-1980s on the fate of the rat tapeworm, Hymenolepis diminuta, in rats and mice and the systemic and local responses evoked following infection have contributed directly to our knowledge of how permissive and non-permissive hosts respond to the challenge of infection with a helminth parasite. This convenient laboratory model system has, in the authors' opinion, regrettably received considerably less attention in recent years. With the goal of highlighting the utility of this model system, data is presented on: (1) the immune and enteric responses of rats and mice to infection with H. diminuta; (2) the ability of excretory or secretory products derived from H. diminuta to significantly reduce T cell and macrophage activation in vitro; and (3) how assessment of H. diminuta-rodent models can be used to identify immune effector or regulatory mechanisms that can be translated into novel treatments for inflammatory and autoimmune disorders.

Differences of proteolytic enzymes and pathological changes in permissive and nonpermissive animal hosts for Angiostrongylus cantonensis infection

In this study, mice (nonpermissive hosts) infected with Angiostrongylus cantonensis showed significant worm degeneration and eosinophil degranulation, whereas infected rats (permissive hosts) showed lower worm degeneration and eosinophil degranulation. Pathophysiological changes developed to a lesser extent in rat than in the mouse strains. Neurological evaluation of A. cantonensis-infected mice showed mechanical damage caused by worms migrating to the brain. A significant correlation between the proteolytic enzymes, plasminogen activator (PA) and matrix metalloproteinase-9 (MMP-9), and pathological changes was found in hosts with eosinophilic meningitis or meningoencephalitis. Also, the ratio of cerebrospinal fluid (CSF) to serum albumin was consistently increased in hosts with angiostrongyliasis as compared with control. These data clearly indicate that PA and MMP-9 proteolytic enzymes as well as pathological changes are different in permissive and nonpermissive hosts.

Erratum: The Classic: Integration of Deoxyribonucleic Acid Specific for Rous Sarcoma Virus after Infection of Permissive and Nonpermissive Hosts

Erratum to: Clin Orthop Relat Res DOI 10.1007/s11999–008–0355–8 In the online version of this article, the affiliations of Harold E. Varmus, Peter K. Vogt, and J. Michael Bishop are not current. They are current as displayed here. Note that Dr. Varmus's correct email address is [email protected]. In the original publication of Varmus HE, Vogt PK, Bishop JM. Integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts (RNA tumor viruses/reassociation kinetics/duck cells). Proc Nat Acad Sci USA. 1973;70:3067–3071, the authors’ affiliations were listed as follows: Harold E. Varmus, J. Michael Bishop Department of Microbiology, University of California, San Francisco, Calif. 94122 Peter K. Vogt Department of Microbiology, University of Southern California Medical School, 2025 Zonal Ave., Los Angeles, Calif. 90033

A simple method for evaluating dengue vaccine effectiveness in mice based on levels of viremia caused by intraperitoneal injection of infected culture cells

A simple dengue vaccine evaluation system was established using a model of dengue type 2 virus (DENV2) infection in immunocompetent mice. Mice are usually non-permissive hosts, and artificial viremia was therefore created by intraperitoneal injection of K562 cells infected with DENV2. Plasma virus titers were approximately 4–5 log 10 focus-forming units/ml at 10 h after injection of 1 × 10 7 K562 cells into ICR, ddY and BALB/c mice. ICR mice immunized with an experimental vaccine against DENV2 showed reduced levels of viremia, associated with neutralizing antibody titers. Similarly, ICR mice passively immunized with purified IgG fractions of monoclonal antibodies possessing neutralizing activities also had reduced levels of viremia. However, the degree of viremia reduction differed according to the antibody species. Although some mice developed neurologic symptoms and/or died within 21 days of K562 injection, viremia reduction was considered to be a reliable indicator of the protective capacities of candidate dengue vaccines.

The classic: integration of deoxyribonucleic acid specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts: (RNA tumor viruses/reassociation kinetics/duck cells). 1973.

A relatively simple but stringent technique was developed to detect the integration of virus-specific DNA into the genomes of higher organisms. In both permissive (duck) and nonpermissive (mammalian) cells which normally contain no nucleotide sequences specific for Rous sarcoma virus, transformation by the virus results in the appearance of DNA specific for Rous sarcoma virus covalently integrated into strands of host-cell DNA containing reiterated sequences. Early after infection of mouse or duck cells by Rous sarcoma virus, unintegrated DNA specific for the virus can be demonstrated.

Antigenicity and recombination of VlsE, the antigenic variation protein ofBorrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s) from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

Characterization of the immuno-regulatory response to the tapeworm Hymenolepis diminuta in the non-permissive mouse host

Hymenolepis diminuta is spontaneously expelled from mice; concomitant with worm expulsion was protection against colitis induced by dinitrobenzene sulphonic acid (DNBS). Here we examined the immune response mobilized by Balb/c and C57Bl/6 male mice in response to H. diminuta and assessed the requirement for CD4 + cells (predominantly T cells) in worm expulsion and the anti-colitic effect. Wild-type (CD4 +) or CD4 knock-out (CD4 −/−) mice received five H. diminuta cysticercoids and segments of jejunum and mesenteric lymph nodes (MLNs), or spleen, were excised 5, 8 and 1l days later for mRNA analysis and cytokine production, respectively. In separate experiments uninfected and infected mice received DNBS by intra-rectal infusion and indices of inflammation were assessed 3 days later (i.e. 11 days p.i.). Infection of Balb/c mice resulted in a time-dependent increase in intestinal mRNA for Foxp3, a marker of natural regulatory T cells, and markers of alternatively activated macrophages (arginase-1, FIZZ1), while concanavalin-A activation of MLN cells revealed a significant increase in T helper 2 (TH2) type cytokines: IL-4, -5, -9, -10, -13. MLN cells showed a reduced ability to induce Foxp3 expression upon stimulation. CD4 −/− mice did not display this response to infection, but surprisingly did expel H. diminuta. Moreover, DNBS-induced colitis in CD4 −/− mice (wasting, tissue damage, elevated myeloperoxidase) was not reduced by H. diminuta infection, whereas time-matched infected CD4 + C57Bl/6 mice had significantly less DNBS-induced inflammation. In conclusion: (i) in addition to stereotypical TH2 events, H. diminuta-infected Balb/c mice develop a local immuno-regulatory response; and (ii) CD4 + cells are not essential for H. diminuta expulsion from mice but are critical in mediating the anti-colitic effect that accompanies infection in this model.

The influence of host suitability on the range of grasshopper species utilized by Blaesoxipha atlanis (Diptera: Sarcophagidae) in the field

Blaesoxipha atlanis (Aldrich) is a common parasitic fly of agriculturally important grasshoppers in Canada. The suitability of Camnula pellucida (Scudder), Melanoplus bivittatus (Say), Melanoplus packardii Scudder, and Melanoplus sanguinipes (Fabricius) as hosts was studied in the laboratory. Grasshoppers were singly-parasitized or left unparasitized and reared for 9 days. Melanoplus bivittatus and M. packardii did not support parasite development, i.e. were non-permissive hosts. In both species, parasite larvae were melanized and encapsulated, but development proceeded further in M. packardii. Melanoplus sanguinipes and C. pellucida were permissive host species with, respectively, 70% and 35% of the implanted larvae emerging from their hosts of which 86% and 50% developed into adults. Parasite development time was longer in C. pellucida. Adult B. atlanis dry mass varied with host species and host mass at parasitism, but not with host sex. Parasites developing in M. sanguinipes were larger in terms of dry mass than counterparts developing in C. pellucida. In permissive species, unparasitized grasshoppers gained in body mass while parasitized insects lost mass during the 9-day observation period. In non-permissive species, all insects gained in body mass, but parasitized females gained less mass than unparasitized conspecifics. All unparasitized grasshoppers survived while 75-95% of permissive and 30-40% of non-permissive hosts died. Variation in the intensity of field parasitism among grasshopper species may be explained, at least in part, by qualitative differences in suitability between potential host species. Novel pest management strategies emphasize preservation of a small proportion of the pest population for natural enemies. Consideration of the outcome of specific host-parasite interactions should improve the understanding of grasshopper population dynamics and increase the predictive value of models that assess potential crop losses.

Interferon Induction by Viruses. XXV.

Chicken embryonic cells (CEC) are nonpermissive hosts for the replication of human adenoviruses, yet they respond to infection by producing interferon (IFN). The nature of the IFN inducer moiety in these viruses has been elusive since its initial study by Ilona Béládi and colleagues some 40 years ago. We tested the hypothesis that viral dsRNA was the IFN inducer molecule--for two reasons: (i) dsRNA has been identified as a potent inducer of IFN, and (ii) developmentally mature CEC cells as cultured in vitro can develop a hyper-responsive state to dsRNA such that a single molecule of dsRNA per cell constitutes the threshold of detection. Furthermore, the number of particles in a virus population capable of inducing-IFN, irrespective of their replication capacity, can be quantified through the analysis of dose (multiplicity)-response (IFN yield) curves, thus allowing a determination of the number particles in virus populations that possess the capacity to induce IFN. This study demonstrates that type 5 wild type adenovirus (Ad5) and mutants dl312, dl334, and ts19 induce from 8,000 to 80,000 IFN U per 10(7) CEC. UV irradiation showed that transcription of about 20-50% of the Ad5 genome was required to produce the IFN inducer moiety. The ratio of IFN-inducing particles to plaque-forming particles (IFP : PFP) was as low as 1:6, indicating that only a small fraction of the total particles in a virus population ever function as IFP. We conclude that adenovirus dsRNA produced during symmetric transcription of some regions of the viral genome, coupled with fine-tuning of the IFN-induction pathway, account for the IFN-inducing capacity of adenoviruses in the non-permissive chicken cell.

Prevention of Virus Persistence and Protection against Immunopathology after Borna Disease Virus Infection of the Brain by a Novel Orf Virus Recombinant

The Parapoxvirus Orf virus represents a promising candidate for novel vector vaccines due to its immune modulating properties even in nonpermissive hosts such as mouse or rat. The highly attenuated Orf virus strain D1701 was used to generate a recombinant virus (D1701-VrVp40) expressing nucleoprotein p40 of Borna disease virus, which represents a major antigen for the induction of a Borna disease virus-specific humoral and cellular immune response. Infection with Borna disease virus leads to distinct neurological symptoms mediated by the invasion of activated specific CD8+ T cells into the infected brain. Usually, Borna disease virus is not cleared from the brain but rather persists in neural cells. In the present study we show for the first time that intramuscular application of the D1701-VrVp40 recombinant protected rats against Borna disease, and importantly, virus clearance from the infected brain was demonstrated in immunized animals. Even 4 and 8 months after the last immunization, all immunized animals were still protected against the disease. Initial characterization of the immune cells attracted to the infected brain areas suggested that D1701-VrVp40 mediated induction of B cells and antibody-producing plasma cells as well as T cells. These findings suggest the induction of various defense mechanisms against Borna disease virus. First studies on the role of antiviral cytokines indicated that D1701-VrVp40 immunization did not lead to an enhanced early response of gamma or alpha interferon or tumor necrosis factor alpha. Collectively, this study describes the potential of the Orf virus vector system in mediating long-lasting, protective antiviral immunity and eliminating this persistent virus infection without provoking massive neuronal damage.

Experimental schistosomiasis in the common marmoset Callithrix jacchus.

In order to evaluate Callithrix jacchus as an animal model for mansoni schistosomiasis, a group of 10 male animals were once percutaneously exposed to 250 cercariae of the Schistosoma mansoni SLM (São Lourenço da Mata) strain. Animals were periodically bled for measuring serum level of enzymes and proteins and for blood cell counting. When comparing pre-infection to post-infection values, a significant increase was found for alkaline phosphatase at 15 to 120 days p.i., differential counts of eosinophil at 45 and 60 days, and total protein and global eosinophil counts at 120 days. No Schistosoma mansoni eggs were found in stools. Adult worms of small size were recovered from five animals. At day 120, the number of Schistosoma mansoni eggs/g of tissue was 0-289.7 (liver), 0-30.1 (large intestine) and 0-171.4 (small intestine). These findings lead us to classify Callithrix jacchus as a non-permissive host to the SLM strain of Schistosoma mansoni.

Eosinophils Chemoattracted by Eotaxin from Cerebrospinal Fluid of Mice Infected with Angiostrongylus Cantonensis Assayed in a Microchamber

When non-permissive hosts are infected with Angiostrongylus cantonensis, the migration of the worms to the brain and their subsequent development manifests as marked eosinophilic pleocytosis. We used microchambers to demonstrate direct eosinophil chemotactic activity by adding a variety of antibodies into cerebrospinal fluid (CSF) of BALB/c mice 21 days post-infection with A. cantonensis. The antibodies were directed to neutralize eotaxin, RANTES (regulated on activation, normal T-cells expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and platelet-activating factor (PAF), respectively. Eosinophil migration into the polycarbonate membrane covering CSF with anti-eotaxin or anti-MIP-1alpha antibodies was significantly lower than that for antibody-free CSF (Student's t test: p < 0.01, p < 0.05). We also collected CSF from mice 21 days after infection with 10, 20, 30, 40, and 50 third-stage larvae (L3) respectively for dose-dependent testing, and 40 L3 at days 7, 14, and 21 after infection for time-dependent testing. Chemokine production in CSF was affected by A. cantonensis infection intensity and post-infection time. In conclusion, eotaxin and MIP-1alpha released in the CSF of A. cantonensis-infected mice have eosinophil chemotactic activity in this in vitro assay.

Oral transfer of adult Ancylostoma ceylanicum hookworms into permissive and nonpermissive host species.

Syrian hamsters become anemic and exhibit delayed growth following oral infection with third-stage Ancylostoma ceylanicum hookworm larvae. Here we describe experiments designed to determine the feasibility of adult worm transfer (AWT) between hosts, a technique that would facilitate the specific study of bloodfeeding hookworms in vivo without prior exposure of the host to larva-specific antigens, permit the ex vivo manipulation of adult parasites prior to reimplantation, and also allow for cross-species transfer of worms. Weanling hamsters given an oral AWT of 40 or 60 mixed-sex A. ceylanicum worms rapidly developed anemia; in the higher-dose group, hemoglobin levels declined from prechallenge levels by 44% within 4 days following AWT. Long-term survival of transferred worms was demonstrated by recovery of parasites from the intestines 42 days after AWT. AWT hamsters acquired humoral immune responses against soluble adult hookworm extracts and excretory-secretory products that were comparable in magnitude to those of animals given a typical infection with larvae. In AWT experiments employing the nonpermissive murine model, C57BL/6 mice given adult worms rapidly became anemic and lost weight in a manner similar to AWT hamsters. Infection of additional mouse strains demonstrated that while C57BL/10 and CD-1 mice also developed anemia following AWT, BALB/c mice were resistant. The technique of AWT to mice may further our understanding of hookworm pathogenesis by allowing the study of adult hookworm infections in a species with well-characterized genetics and an abundance of available reagents.

Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines

The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) share about 90% identity at the genomic level but they have non-overlapping host range and show a high degree of host specificity. We have demonstrated here that AcMNPV undergoes DNA replication and early gene expression in Bombyx-derived BmN cells but fails to show very late gene expression or produce budded virion (BV) particles. Coinfection with BmNPV supported BV production from AcMNPV in BmN cells at low levels but not very late gene expression or polyhedral inclusion body formation. BV production and very late gene expression from BmNPV, on the contrary, were adversely affected in coinfections. In Spodoptera frugiperda-derived Sf21 cell lines, BmNPV DNA replication, BV production, and very late gene expression took place only when coinfected with AcMNPV. BmNPV exerted a less profound effect on AcMNPV multiplication and very late gene expression in permissive host cell lines. AcMNPV shuts down cellular and viral protein synthesis completely when infected alone or coinfected with BmNPV in BmN cells, whereas BmNPV infection did not affect cellular and viral protein synthesis in Sf21 cells. Overall, AcMNPV showed a more dominant effect by complementing the multiplication of BmNPV in nonpermissive host cells while inhibiting it in BmN cells.

Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules.

Cathepsin D aspartic proteases of hookworms were recently implicated in the host-specific digestion of haemoglobin by adult parasites. Ac-APR-1 from the dog hookworm, Ancylostoma caninum and Na-APR-1 from the human hookworm, Necator americanus, were shown to be expressed in the infective larval stage (L3) as well as adult worms. We now show that both proteases degraded skin macromolecules and serum proteins, some of which were cleaved more readily from permissive definitive hosts as opposed to non-permissive hosts. Na-APR-1 degraded human collagens more efficiently than did Ac-APR-1, and Ac-APR-1 degraded canine serum albumin more efficiently than did Na-APR-1. On the other hand, both enzymes degraded human serum proteins (albumin and fibrinogen) with approximately equal efficiency under the conditions of our assays in vitro. Molecular models of these 2 orthologous, aspartic proteases showed that, despite having active site clefts with identical primary sequences, residues in the S3 pocket adopted different conformations, likely accounting for different substrate preferences reported previously. Antisera raised to both proteases partially inhibited (16-26%) migration of hookworm L3 through hamster skin in vitro, further implying a connective tissue invasive role for these enzymes in addition to digestion of serum and erythrocyte proteins for nutrition.