Marshall E. Bloom

A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.

Tick-borne flaviviruses (TBFV) are sustained in nature through cycling between mammalian and tick hosts. In this study, we used African green monkey kidney cells (Vero) and Ixodes scapularis tick cells (ISE6) to compare virus-induced changes in mammalian and arthropod cells. Using confocal microscopy, transmission electron microscopy (TEM), and electron tomography (ET), we examined viral protein distribution and the ultrastructural changes that occur during TBFV infection. Within host cells, flaviviruses cause complex rearrangement of cellular membranes for the purpose of virus replication. Virus infection was accompanied by a marked expansion in endoplasmic reticulum (ER) staining and markers for TBFV replication were localized mainly to the ER in both cell lines. TEM of Vero cells showed membrane-bound vesicles enclosed in a network of dilated, anastomosing ER cisternae. Virions were seen within the ER and were sometimes in paracrystalline arrays. Tubular structures or elongated vesicles were occasionally noted. In acutely and persistently infected ISE6 cells, membrane proliferation and vesicles were also noted; however, the extent of membrane expansion and the abundance of vesicles were lower and no viral particles were observed. Tubular profiles were far more prevalent in persistently infected ISE6 cells than in acutely infected cells. By ET, tubular profiles, in persistently infected tick cells, had a cross-sectional diameter of 60–100 nm, reached up to 800 nm in length, were closed at the ends, and were often arranged in fascicle-like bundles, shrouded with ER membrane. Our experiments provide analysis of viral protein localization within the context of both mammalian and arthropod cell lines as well as both acute and persistent arthropod cell infection. Additionally, we show for the first time 3D flavivirus infection in a vector cell line and the first ET of persistent flavivirus infection.

Differential salivary gland transcript expression profile in Ixodes scapularis nymphs upon feeding or flavivirus infection.

Ixodid ticks are vectors of human diseases such as Lyme disease, babesiosis, anaplasmosis, and tick-borne encephalitis. These diseases cause significant morbidity and mortality worldwide and are transmitted to humans during tick feeding. The tick-host-pathogen interface is a complex environment where host responses are modulated by the molecules in tick saliva to enable the acquisition of a blood meal. Disruption of host responses at the site of the tick bite may also provide an advantage for pathogens to survive and replicate. Thus, the molecules in tick saliva not only aid the tick in securing a nutrient-rich blood meal, but can also enhance the transmission and acquisition of pathogens. To investigate the effect of feeding and flavivirus infection on the salivary gland transcript expression profile in ticks, a first-generation microarray was developed using ESTs from a cDNA library derived from Ixodes scapularis salivary glands. When the salivary gland transcript profile in ticks feeding over the course of 3 days was compared to that in unfed ticks, a dramatic increase in transcripts related to metabolism was observed. Specifically, 578 transcripts were up-regulated compared to 151 down-regulated transcripts in response to feeding. When specific time points post attachment were analyzed, a temporal pattern of gene expression was observed. When Langat virus-infected ticks were compared to mock-infected ticks, transcript expression changes were observed at all 3 days of feeding. Differentially regulated transcripts include putative secreted proteins, lipocalins, Kunitz domain-containing proteins, anti-microbial peptides, and transcripts of unknown function. These studies identify salivary gland transcripts that are differentially regulated during feeding or in the context of flavivirus infection in Ixodes scapularis nymphs, a medically important disease vector. Further analysis of these transcripts may identify salivary factors that affect the transmission or replication of tick-borne flaviviruses.

Caspase activation during virus infection: more than just the kiss of death?

To facilitate their replication, viruses utilize many host cell factors including receptors, enzymes, transcription factors, organelles, and membranes. In short, they commandeer the cellular environment. At the same time, viruses must circumvent cellular responses that have evolved to limit virus replication. Apoptosis, the genetically and biochemically controlled process of cell death, is one such cellular response that limits both the time and cellular machinery available for virus replication. Viruses employ a variety of strategies to evade apoptosis, and this is the subject of many good review articles (e.g., Cuconati and White, 2002; Everett and McFadden, 2002). The existence of multiple viral anti-apoptotic gene products across diverse groups of viruses suggests that apoptosis is something viruses should avoid at all costs. However, as is often the case in biological systems, the relationship between virus replication and apoptosis is actually much more complex. It is now recognized that some viruses directly utilize components of the apoptotic pathway to facilitate their replication. This novel and seemingly paradoxical strategy of virus replication, in the face of a very effective anti-viral response, is the subject of this review.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study.

Molecular characterization of the small nonstructural proteins of parvovirus Aleutian mink disease virus (AMDV) during infection.

Aleutian mink disease virus (AMDV) is the only member in genus Amdovirus of the family Parvoviridae. During AMDV infection, six species of viral transcripts are generated from one precursor mRNA through alternative splicing and alternative polyadenylation. In addition to the large non-structural protein NS1, two small non-structural proteins, NS2 and NS3, are putatively encoded (Qiu J, et al., 2006. J. Virol. 80 654-662). However, these two proteins have not been experimentally demonstrated during virus infection, and nothing is known about their function. Here, we studied the nonstructural protein expression profile of AMDV, and for the first time, confirmed expression of NS2 and NS3 during infection, and identified their intracellular localization. More importantly, we provided evidence that both NS2 and NS3 are necessary for AMDV replication.

Internal polyadenylation of parvoviral precursor mRNA limits progeny virus production

Aleutian Mink Disease Virus (AMDV) is the only virus in the genus Amdovirus of family Parvoviridae. In adult mink, AMDV causes a persistent infection associated with severe dysfunction of the immune system. Cleavage of AMDV capsid proteins has been previously shown to play a role in regulating progeny virus production (Fang Cheng et al., J. Virol. 84:2687-2696, 2010). The present study shows that AMDV has evolved a second strategy to limit expression of capsid proteins by preventing processing of the full-length capsid protein-encoding mRNA transcripts. Characterization of the cis-elements of the proximal polyadenylation site [(pA)p] in the infectious clone of AMDV revealed that polyadenylation at the (pA)p site is controlled by an upstream element (USE) of 200 nts in length, the AAUAAA signal, and a downstream element (DSE) of 40 nts. A decrease in polyadenylation at the (pA)p site, either by mutating the AAUAAA signal or the DSE, which does not affect the encoding of amino acids in the infectious clone, increased the expression of capsid protein VP1/VP2 and thereby increased progeny virus production approximately 2-3-fold. This increase was accompanied by enhanced replication of the AMDV genome. Thus, this study reveals correlations among internal polyadenylation, capsid production, viral DNA replication and progeny virus production of AMDV, indicating that internal polyadenylation is a limiting step for parvovirus replication and progeny virus production.

A comparison between permissive and restricted infections with Aleutian mink disease parvovirus (ADV): characterization of the viral protein composition at nuclear sites of virus replication

We used three-color fluorescent labeling and confocal microscopy to compare the permissive and the antibody-mediated, restricted replication of Aleutian mink disease parvovirus (ADV). In both permissive (CRFK cells) and restricted (K562 cells) situations, both ADV non-structural proteins (NS1 and NS2) concentrated at focal sites in the nucleus, which also contained viral DNA. Bromodeoxyuridine labeling demonstrated that these sites also supported active ADV single-strand DNA synthesis, indicating that they were replication compartments. ADV capsid proteins were located in intranuclear shells surrounding the replication compartments. At later time points, NS2 was readily detected in the cytoplasm of permissively infected CRFK cells, whereas the cytoplasmic presence of NS2 was much less pronounced in the K562 cells. These results showed that both permissive and restricted ADV replication are associated with a tight nuclear subcompartmentalization of viral products. Furthermore, differences between the permissive and restricted virus-cell interactions were noted, suggesting that there may be a morphological basis for examining the outcome of ADV infection.

PARVOVIRUS INFECTIONS: FEATURES REMINISCENT OF AIDS

An infectious agent, possibly a virus, may be responsible for initiating the acquired immune deficiency syndrome (AIDS).’ In 1981 AIDS was recognized as a distinct syndrome of severe opportunistic infection and Kaposi’s Sarcoma in young previously healthy homosexual males.’. ’ Since then, cases have also been found in intravenous drug users,’ Haitians,’ hemophiliacs,6 and close contacts of AIDS patient^.^, The epidemiology has suggested spread by intimate, often sexual, contact: lo contaminated blood products lo or drug paraphernalia.” The underlying problem is a profound cellular immune deficiency characterized by decreased T4 helper lymphocytes and reversed helper-suppressor T-lymphocyte ratios,’-’’ but in addition, abnormalities of B-lymphocyte function ”* I’ have also been detected (TABLE 1). A condition characterized by unexplained lymphadenopathy and similar alterations of the immune system, but without severe opportunistic infection or Kaposi’s sarcoma, has also been described.’)-” This syndrome, which occurs in the same populations at risk for AIDS, may be a prodrome to or an attenuated form of AIDS.”. A number of viruses have been identified in AIDS patients, including hepatitis A virus,”. l6 hepatitis B virus,ll, 16, 19 herpes simplex virus,17 Epstein-Barr virus,’8 cytomegalovirus,’. ‘E-” papovavirus,” adenovirus,22 and human T-cell leukemia virus,2’ but in this setting of compromised immune function, it is uncertain that any recognized agent is etiologic. The parvoviruses 26 are one group of viruses that has as yet received little attention in AIDS. These small nonenveloped viruses” contain single stranded DNA genomes ’‘” encoding information for two or three structural polypeptides ”* 26 and at least one nonvirion protein of undetermined function?’ The defective parvoviruses (adeno-associated viruses or AAV) are absolutely dependent on coinfecting adenoviruses ’* or herpes viruses for replication, whereas the nondefective members of the group can replicate autonomously.” A number of models exist for parvovirus infections in vivo, and findings from several of these systems include features that in composite bear resemblance to features of AIDS. The purpose of this paper is to describe several parvoviral disease models and to point out the aspects that make these viruses attractive as potential etiologic agents in AIDS.

Modern dosimetric tools for (60)Co irradiation at high containment laboratories.

Abstract Purpose: To evaluate an innovative photo-fluorescent film as a routine dosimetric tool during 60Co irradiations at a high containment biological research laboratory, and to investigate whether manufacturer-provided chamber exposure rates can be used to accurately administer a prescribed dose to biological specimens. Materials and methods: Photo-fluorescent, lithium fluoride film dosimeters and National Institutes of Standards and Technology (NIST) transfer dosimeters were co-located in a self-shielded 60Co irradiator and exposed to γ-radiation with doses ranging from 5–85 kGy. Film dose-response relationships were developed for varying temperatures simulating conditions present when irradiating infectious biological specimens. Dose measurement results from NIST transfer dosimeters were compared to doses predicted using manufacturer-provided irradiator chamber exposure rates. Results: The film dosimeter exhibited a photo-fluorescent response signal that was consistent and nearly linear in relationship to γ-radiation exposure over a wide dose range. The dosimeter response also showed negligible effects from dose fractionization and humidity. Significant disparities existed between manufacturer-provided chamber exposure rates and actual doses administered. Conclusion: This study demonstrates the merit of utilizing dosimetric tools to validate the process of exposing dangerous and exotic biological agents to γ-radiation at high containment laboratories. The film dosimeter used in this study can be utilized to eliminate potential for improperly administering γ-radiation doses.

The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research

Each year there are more than 15u202f000 cases of human disease caused by infections with tick-borne viruses (TBVs). These illnesses occur worldwide and can range from very mild illness to severe encephalitis and hemorrhagic fever. Although TBVs are currently identified as neglected vector-borne pathogens and receive less attention than mosquito-borne viruses, TBVs are expanding into new regions, and infection rates are increasing. Furthermore, effective vaccines, diagnostic tools, and other countermeasures are limited. The application of contemporary technologies to TBV infections presents an excellent opportunity to develop improved, effective countermeasures. Experimental tick and mammal models of infection can be used to characterize determinants of infection, transmission, and virulence and to test candidate countermeasures. The use of ex vivo tick cultures in TBV research provides a unique way to look at infection in specific tick organs. Mammal ex vivo organ slice and, more recently, organoid cultures are additional models that can be used to elucidate direct tissue-specific responses to infection. These ex vivo model systems are convenient for testing methods involving transcript knockdown and small molecules under tightly controlled conditions. They can also be combined with in vitro and in vivo studies to tease out possible host factors and potential vaccine or therapeutic candidates. In this brief perspective, we describe how ex vivo cultures can be combined with modern technologies to advance research on TBV infections.