When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (... more When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (ADV), 100% of both Aleutian and non-Aleutian genotype mink died of interstitial pneumonia characterized by permissive ADV infection of alveolar type H cells. Treatment of infected kits with either mink anti-ADV gamma globulin or mouse monoclonal antibodies against ADV structural proteins reduced mortality by 50 to 75% and * Corresponding author. sen, unpublished results), the accumulated information suggested that perhaps maternally transferred antibodies masked the development of pneumonia. By using immunofluorescence, Southern blot analysis, and strand-specific in situ hybridization, we previously showed that the acute pneumonia is caused by cytopathic replication of ADV in lung alveolar type II cells of newborn ADV antibody-negative mink kits. This infection is associated with high levels of viral antigen, viral DNA, the viral replicative forms (RFs) of DNA, and viral mRNA in each infected cell (7). In contrast, in mink infected as adults, viral replication and transcription are dramatically altered. The target cells lie within lymphoid organs (probably lymphocytes), and ADV expression is markedly restricted at the
ABSTRACT The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death ... more ABSTRACT The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.
• TBEV is the most medically important member of the tick-borne serocomplex group within the genu... more • TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. • Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). Additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalaya subtype (Himalayan and “178-79” group; TBEV-HIM). • TBEV virions are small enveloped spherical particles about 50 nm in diameter. • The TBEV genome consists of a single-stranded positive sense RNA molecule. • The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). • The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. • The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). • TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.
The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TB... more The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ϳ10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets. IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.
Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) pos... more Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.
Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian d... more Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens. Aleutian disease (AD) is a parvovirus-induced disease of mink characterized by viral persistence, hypergammaglobulinemia, and immune complex-mediated renal and arterial lesions. Affected mink usually die of terminal glomerulonephritis after a mostly subclinical course lasting weeks, months, or years (6, 12, 18, 22, 29, 31). Immune complexes play a prominent role in the pathogenesis of the renal disease, and although some of the immune complexes contain infectious virus, it is clear that many complexes are too small to contain intact virus (4, 23, 30). Consequently, it is uncertain what viral components actually participate in the generation of immune complexes. Sera from infected animals contain antibody to both structural and nonstructural viral proteins (7). Furthermore, the proteins of AD virus (ADV) seem to be degraded during in vivo infection (3). Thus, a variety of viral proteins could be involved in immune complex development. Furthermore, antibodies to DNA are present in the sera of infected mink, and DNA-anti-DNA complexes may also have a role in the pathogenesis (16, 17). In addition, it is not clear whether virus detected earlier (30) was within cells replicating virus or rather identified cells that had phagocytized viral antigen, and since the reagent used previously to detect virus is polyclonal, it is not clear that only viral components were recognized. The ability to distinguish viral polypeptides immunologically would make it possible to define which viral products were present, both in soluble immune complexes and complexes deposited in tissues. Another major problem in the study of AD has been the difficulty in differentiating virus strains. Although the relative pathogenicity of various ADV isolates suggests that
Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I... more Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I strain, but not the cell culture-adapted ADV-G strain, infects mink peritoneal macrophage cultures and the human macrophage cell line U937 in vitro. However, preincubation of ADV-G with ADV-infected mink serum enhanced its infectivity for U937 cells. The enhancing activity was present in the protein A-binding immunoglobulin G fraction in the serum, but F(ab')2 fragments failed to enhance the infection. On the other hand, the same sera inhibited ADV-G infection of Crandell feline kidney (CRFK) cells. Although U937 cells were not fully permissive for antibody-enhanced ADV-G infection, ADV mRNA expression, genome amplification, and protein expression were identical to those found previously for ADV-Utah I infection of U937 cells. Preincubation of ADV-Utah I with soluble protein A partly inhibited the infection of U937 cells but did not affect infection of CRFK cells. In mink peritoneal macrophages, preincubation with the infected mink serum did not make ADV-G infectious. However, the infectivity for mink macrophages of antibody-free ADV-Utah I prepared from the lungs of infected newborn mink kits was enhanced by ADV-infected mink serum. Moreover, protein A partly blocked ADV-Utah I infection of mink macrophage cultures. These results suggested that ADV-Utah I enters mink macrophages and U937 cells via an Fc receptor-mediated mechanism. This mechanism, antibodydependent enhancement, may also contribute to ADV infection in vivo. Furthermore, since ADV infection in mink is characterized by overproduction of anti-ADV immunoglobulins, antibody-dependent enhancement may play a critical role in the establishment of persistent infection with ADV in vivo. Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with a severe disorder of the immune systcm. The discase in adult mink, known as classical Aleutian disease (AD), is characterized by marked hypergammaglobulinemia, plasmacytosis, arteritis, and fatal immune complex glomerulonephritis (37, 38). Moreover, serum immune complexes that contain ADV from infected adult mink are still infectious (35). Studies on adult mink infected with ADV indicate that macrophages are target cells for ADV replication but that infection is restricted (25, 32) in that the levels of replicative intermediates are greatly reduced compared with those in permissive infections (4, 10, 14). In order to study the virus-cell interactions in a more controlled system, we recently developed an in vitro system of ADV infection with the human macrophage lineage cell line U937 (26). In these studies, we found that the pathogenic ADV-Utah I strain could infect the U937 cells and that the infection was restricted in a fashion similar to that observed for mink macrophages in vivo (4, 25, 26, 32). However, the nonpathogenic cell culture-adapted ADV-G strain (12) was unable to initiate infection in U937 cells (26). On the other hand, both ADV-G and ADV-Utah I can infect Crandell feline kidney (CRFK) cells (12). These differences suggest that there may be unique features about the virus-cell interactions. U937 cells are macrophage lineage cells and express Fc receptors (44), and although they are not mink cells, their infection with ADV-Utah I bears similarities to the in vivo situation (4, 25, 26, 32). ADV-Utah I
A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned i... more A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.
Journal of Veterinary Diagnostic Investigation, 1994
A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and V... more A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1-infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23-25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log 2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.
We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK ... more We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.
Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. ... more Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. The progression from acute to chronic Powassan disease is not well understood, severely limiting treatment and prevention options.
Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Seve... more Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, sitedirected mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease. MATERIALS AND METHODS Viruses, cells, and plasmids. Replication-competent mutant viruses were propagated and assayed in CrFK cells as previously described (8). The in vivo propagation and assay of ADV-Utah in adult Aleutian genotype (sapphire) mink have also been described previously (37). Cloning and mutagenesis. Full-length molecular clones were transformed and amplified in Escherichia coli JC8111 (recBCsbcrecF) by standard techniques (7,
A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) ... more A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, W:ADSP. Immunohistochemical staining of W:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of W:ADSP-infected cell lysates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified W:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with W:ADSP contained ADV-specific antibodies and two of these
Zika Virus Biology, Transmission, and Pathology, 2021
Abstract The testicle surprisingly emerged as important for Zika virus (ZIKV) infection, viral pe... more Abstract The testicle surprisingly emerged as important for Zika virus (ZIKV) infection, viral persistence, and sexual transmission. Systemic infection traffics ZIKV to the cells of the testes via the hematogenous or lymphogenous routes. Studies in mice and humans have shown that Sertoli cells, spermatogonia, and primary spermatocytes are preferred targets of ZIKV infection, but Leydig cells in the interstitium are infected sparingly. ZIKV-infected testicular cells may harbor virus/viral RNA for months, suggesting viral persistence. ZIKV destroys infected testicular cells leading to atrophy, which is associated with low testosterone and inhibins levels, and may thus negatively impact fertility in men. Testicular atrophy appears to be a slow process because ZIKV infection of the testis does not cause a dramatic induction of genes that cause apoptotic cell death. Nevertheless, ZIKV infection of the testis is important for persistence and sexual transmission. The ZIKV-host cell relationship is intriguingly complex and requires further study.
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co... more Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
Each year there are more than 15,000 cases of human disease caused by infections with tick-borne ... more Each year there are more than 15,000 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 vectorborne 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 in 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.
When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (... more When mink kits were infected neonatally with a highly virulent strain of Aleutian disease virus (ADV), 100% of both Aleutian and non-Aleutian genotype mink died of interstitial pneumonia characterized by permissive ADV infection of alveolar type H cells. Treatment of infected kits with either mink anti-ADV gamma globulin or mouse monoclonal antibodies against ADV structural proteins reduced mortality by 50 to 75% and * Corresponding author. sen, unpublished results), the accumulated information suggested that perhaps maternally transferred antibodies masked the development of pneumonia. By using immunofluorescence, Southern blot analysis, and strand-specific in situ hybridization, we previously showed that the acute pneumonia is caused by cytopathic replication of ADV in lung alveolar type II cells of newborn ADV antibody-negative mink kits. This infection is associated with high levels of viral antigen, viral DNA, the viral replicative forms (RFs) of DNA, and viral mRNA in each infected cell (7). In contrast, in mink infected as adults, viral replication and transcription are dramatically altered. The target cells lie within lymphoid organs (probably lymphocytes), and ADV expression is markedly restricted at the
ABSTRACT The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death ... more ABSTRACT The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.
• TBEV is the most medically important member of the tick-borne serocomplex group within the genu... more • TBEV is the most medically important member of the tick-borne serocomplex group within the genus Flavivirus, family Flaviviridae. • Three antigenic subtypes of TBEV correspond to the 3 recognized genotypes: European (TBEV-EU), also known as Western, Far Eastern (TBEV-FE), and Siberian (TBEV-SIB). Additional 2 genotypes have been identified in the Irkutsk region of Russia, currently named TBE virus Baikalian subtype (TBEV-BKL) and TBE virus Himalaya subtype (Himalayan and “178-79” group; TBEV-HIM). • TBEV virions are small enveloped spherical particles about 50 nm in diameter. • The TBEV genome consists of a single-stranded positive sense RNA molecule. • The genome encodes one open reading frame (ORF), which is flanked by untranslated (non-coding) regions (UTRs). • The 5′-UTR end has a methylated nucleotide cap for canonical cellular translation. The 3′-UTR is not polyadenylated and is characterized by extensive length and sequence heterogeneity. • The ORF encodes one large polyprotein, which is co- and post-translationally cleaved into 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). • TBEV replicates in the cytoplasm of the host cell in close association with virus-induced intracellular membrane structures. Virus assembly occurs in the endoplasmic reticulum. The immature virions are transported to the Golgi complex, and mature virions pass through the host secretory pathway and are finally released from the host cell by fusion of the transport vesicle membrane with the plasma membrane.
The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TB... more The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ϳ10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets. IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.
Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) pos... more Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.
Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian d... more Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens. Aleutian disease (AD) is a parvovirus-induced disease of mink characterized by viral persistence, hypergammaglobulinemia, and immune complex-mediated renal and arterial lesions. Affected mink usually die of terminal glomerulonephritis after a mostly subclinical course lasting weeks, months, or years (6, 12, 18, 22, 29, 31). Immune complexes play a prominent role in the pathogenesis of the renal disease, and although some of the immune complexes contain infectious virus, it is clear that many complexes are too small to contain intact virus (4, 23, 30). Consequently, it is uncertain what viral components actually participate in the generation of immune complexes. Sera from infected animals contain antibody to both structural and nonstructural viral proteins (7). Furthermore, the proteins of AD virus (ADV) seem to be degraded during in vivo infection (3). Thus, a variety of viral proteins could be involved in immune complex development. Furthermore, antibodies to DNA are present in the sera of infected mink, and DNA-anti-DNA complexes may also have a role in the pathogenesis (16, 17). In addition, it is not clear whether virus detected earlier (30) was within cells replicating virus or rather identified cells that had phagocytized viral antigen, and since the reagent used previously to detect virus is polyclonal, it is not clear that only viral components were recognized. The ability to distinguish viral polypeptides immunologically would make it possible to define which viral products were present, both in soluble immune complexes and complexes deposited in tissues. Another major problem in the study of AD has been the difficulty in differentiating virus strains. Although the relative pathogenicity of various ADV isolates suggests that
Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I... more Aleutian mink disease parvovirus (ADV) infects macrophages in adult mink. The virulent ADV-Utah I strain, but not the cell culture-adapted ADV-G strain, infects mink peritoneal macrophage cultures and the human macrophage cell line U937 in vitro. However, preincubation of ADV-G with ADV-infected mink serum enhanced its infectivity for U937 cells. The enhancing activity was present in the protein A-binding immunoglobulin G fraction in the serum, but F(ab')2 fragments failed to enhance the infection. On the other hand, the same sera inhibited ADV-G infection of Crandell feline kidney (CRFK) cells. Although U937 cells were not fully permissive for antibody-enhanced ADV-G infection, ADV mRNA expression, genome amplification, and protein expression were identical to those found previously for ADV-Utah I infection of U937 cells. Preincubation of ADV-Utah I with soluble protein A partly inhibited the infection of U937 cells but did not affect infection of CRFK cells. In mink peritoneal macrophages, preincubation with the infected mink serum did not make ADV-G infectious. However, the infectivity for mink macrophages of antibody-free ADV-Utah I prepared from the lungs of infected newborn mink kits was enhanced by ADV-infected mink serum. Moreover, protein A partly blocked ADV-Utah I infection of mink macrophage cultures. These results suggested that ADV-Utah I enters mink macrophages and U937 cells via an Fc receptor-mediated mechanism. This mechanism, antibodydependent enhancement, may also contribute to ADV infection in vivo. Furthermore, since ADV infection in mink is characterized by overproduction of anti-ADV immunoglobulins, antibody-dependent enhancement may play a critical role in the establishment of persistent infection with ADV in vivo. Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with a severe disorder of the immune systcm. The discase in adult mink, known as classical Aleutian disease (AD), is characterized by marked hypergammaglobulinemia, plasmacytosis, arteritis, and fatal immune complex glomerulonephritis (37, 38). Moreover, serum immune complexes that contain ADV from infected adult mink are still infectious (35). Studies on adult mink infected with ADV indicate that macrophages are target cells for ADV replication but that infection is restricted (25, 32) in that the levels of replicative intermediates are greatly reduced compared with those in permissive infections (4, 10, 14). In order to study the virus-cell interactions in a more controlled system, we recently developed an in vitro system of ADV infection with the human macrophage lineage cell line U937 (26). In these studies, we found that the pathogenic ADV-Utah I strain could infect the U937 cells and that the infection was restricted in a fashion similar to that observed for mink macrophages in vivo (4, 25, 26, 32). However, the nonpathogenic cell culture-adapted ADV-G strain (12) was unable to initiate infection in U937 cells (26). On the other hand, both ADV-G and ADV-Utah I can infect Crandell feline kidney (CRFK) cells (12). These differences suggest that there may be unique features about the virus-cell interactions. U937 cells are macrophage lineage cells and express Fc receptors (44), and although they are not mink cells, their infection with ADV-Utah I bears similarities to the in vivo situation (4, 25, 26, 32). ADV-Utah I
A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned i... more A 7- to 95-map unit segment of DNA from Aleutian disease of mink parvovirus (ADV) was subcloned into a bacteriophage SP6 based transcription vector and used to produce radiolabeled viral RNA transcripts corresponding to either 'plus' or 'minus' sense. The radiolabeled transcripts were reacted against Southern blots of whole cell DNA from ADV infected cell cultures as hybridization probes. The 'plus' sense RNA probe hybridized both to duplex replicative forms (RFs) as well as to single-stranded virion DNA (SS DNA), which is 'minus' in sense. In contrast, the 'minus' sense RNA probe reacted preferentially with the duplex RFs. When these probes were tested against DNA extracted from mink infected with the virulent ADV-Utah I strain, RFs were detected at 10 days after infection in mesenteric lymph node, liver, spleen and gut, but only in gut and mesenteric lymph node at 43 days. SS DNA was noted in these tissues at 10, 43 and 60 days, and was more abundant than RFs. Only SS DNA at very low levels was observed in bone marrow cells. Serum contained large amounts of SS DNA (probably in virions) at 10 days, less at 43 days, and no detectable DNA at 60 days. These findings suggest that ADV replication may have occurred in the gut as well as lymphoreticular tissues, and that bone marrow was not a major site of ADV replication.
Journal of Veterinary Diagnostic Investigation, 1994
A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and V... more A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1-infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23-25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log 2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.
We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK ... more We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.
Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. ... more Half of Powassan infection survivors experience long-term, mild to severe neurological symptoms. The progression from acute to chronic Powassan disease is not well understood, severely limiting treatment and prevention options.
Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Seve... more Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, sitedirected mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease. MATERIALS AND METHODS Viruses, cells, and plasmids. Replication-competent mutant viruses were propagated and assayed in CrFK cells as previously described (8). The in vivo propagation and assay of ADV-Utah in adult Aleutian genotype (sapphire) mink have also been described previously (37). Cloning and mutagenesis. Full-length molecular clones were transformed and amplified in Escherichia coli JC8111 (recBCsbcrecF) by standard techniques (7,
A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) ... more A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, W:ADSP. Immunohistochemical staining of W:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of W:ADSP-infected cell lysates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified W:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with W:ADSP contained ADV-specific antibodies and two of these
Zika Virus Biology, Transmission, and Pathology, 2021
Abstract The testicle surprisingly emerged as important for Zika virus (ZIKV) infection, viral pe... more Abstract The testicle surprisingly emerged as important for Zika virus (ZIKV) infection, viral persistence, and sexual transmission. Systemic infection traffics ZIKV to the cells of the testes via the hematogenous or lymphogenous routes. Studies in mice and humans have shown that Sertoli cells, spermatogonia, and primary spermatocytes are preferred targets of ZIKV infection, but Leydig cells in the interstitium are infected sparingly. ZIKV-infected testicular cells may harbor virus/viral RNA for months, suggesting viral persistence. ZIKV destroys infected testicular cells leading to atrophy, which is associated with low testosterone and inhibins levels, and may thus negatively impact fertility in men. Testicular atrophy appears to be a slow process because ZIKV infection of the testis does not cause a dramatic induction of genes that cause apoptotic cell death. Nevertheless, ZIKV infection of the testis is important for persistence and sexual transmission. The ZIKV-host cell relationship is intriguingly complex and requires further study.
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co... more Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
Each year there are more than 15,000 cases of human disease caused by infections with tick-borne ... more Each year there are more than 15,000 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 vectorborne 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 in 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.
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