bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of Bovine Spongiform Encephalopathy-Specific PrP
Sc
by Treatment with Heat and Guanidine Thiocyanate
Authors: Meyer Rudolf K. , Oesch Bruno , Fatzer Rosmarie , Zurbriggen Andreas , Vandevelde Marc 
Published: December 2019

ABSTRACT

The conversion of a ubiquitous cellular protein (PrP
C
), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP
Sc
is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP
Sc
has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP
Sc
from PrP
C
in bovine brain homogenates. PrP
Sc
was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP
Sc
is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP
C
in cattle is similar to that in hamsters, PrP
Sc
sparse in comparison. The detection of PrP
Sc
by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.
Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants
Authors: Gavin Denise K. , Young Samuel M. , Xiao Weidong , Temple Brenda , Abernathy Corinne R. , Pereira Daniel J. , Muzyczka Nicholas , Samulski Richard Jude 
Published: December 2019

ABSTRACT

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (
ts
) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32°C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37°C and was undetectable at 39°C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a
ts
phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 × 10
6
and 3 × 10
3
, respectively), demonstrating that this is a relatively tight mutant. In addition to the
ts
Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg
2+
-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep
ts
mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.
Mucosal Immunization of Cynomolgus Macaques with Two Serotypes of Live Poliovirus Vectors Expressing Simian Immunodeficiency Virus Antigens: Stimulation of Humoral, Mucosal, and Cellular Immunity
Authors: Crotty Shane , Lohman Barbara L. , Lü Fabien X.-S. , Tang ShenBei , Miller Christopher J. , Andino Raul 
Published: December 2019

ABSTRACTPoliovirus live virus vectors are a candidate recombinant vaccine system. Previous studies using this system showed that a live poliovirus vector expressing a foreign antigen between the structural and nonstructural proteins generates both antibody and cytotoxic T-lymphocyte responses in mice. Here we describe a novel in vitro method of cloning recombinant polioviruses involving a hybrid-PCR approach. We report the construction of recombinant vectors of two different serotypes of poliovirus-expressing simian immunodeficiency virus (SIV) antigens and the intranasal and intravenous inoculations of four adult cynomolgus macaques with these poliovirus vectors expressing the SIV proteins p17gagand gp41env. All macaques generated a mucosal anti-SIV immunoglobulin A (IgA) response in rectal secretions. Two of the four macaques generated mucosal antibody responses detectable in vaginal lavages. Strong serum IgG responses lasting for at least 1 year were detected in two of the four monkeys. SIV-specific T-cell lymphoproliferative responses were detected in three of the four monkeys. SIV-specific cytotoxic T lymphocytes were detected in two of the four monkeys. This is the first report of poliovirus-elicited vaginal IgA or cytotoxic T lymphocytes in any naturally infectable primate, including humans. These findings support the concept that a live poliovirus vector is a potentially useful delivery system that elicits humoral, mucosal, and cellular immune responses against exogenous antigens.
Temporal and Spatial Analysis of Sin Nombre Virus Quasispecies in Naturally Infected Rodents
Authors: Feuer Ralph , Boone John D. , Netski Dale , Morzunov Sergey P. , St. Jeor Stephen C. 
Published: December 2019

ABSTRACT

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (
Peromyscus maniculatus
), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3′ small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.
Markers for
trans
-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells
Authors: Mackenzie Jason M. , Jones Malcolm K. , Westaway Edwin G. 
Published: December 2019

ABSTRACT

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650–6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the
trans
-Golgi membranes and the
trans
-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying
trans
-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.
Observation of Measles Virus Cell-to-Cell Spread in Astrocytoma Cells by Using a Green Fluorescent Protein-Expressing Recombinant Virus
Authors: Duprex W. Paul , McQuaid Stephen , Hangartner Lars , Billeter Martin A. , Rima Bert K. 
Published: December 2019

ABSTRACT
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Pathogenesis of Experimental Rhesus Cytomegalovirus Infection
Authors: Lockridge Kristen M. , Sequar Getachew , Zhou Shan Shan , Yue Yujuan , Mandell Carol P. , Barry Peter A. 
Published: December 2019

ABSTRACT

Human cytomegalovirus (HCMV) establishes and maintains a lifelong persistence following infection in an immunocompetent host. The determinants of a stable virus-host relationship are poorly defined. A nonhuman primate model for HCMV was used to investigate virological and host parameters of infection in a healthy host. Juvenile rhesus macaques (
Macaca mulatta
) were inoculated with rhesus cytomegalovirus (RhCMV), either orally or intravenously (i.v.), and longitudinally necropsied. None of the animals displayed clinical signs of disease, although hematologic abnormalities were observed intermittently in i.v. inoculated animals. RhCMV DNA was detected transiently in the plasma of all animals at 1 to 2 weeks postinfection (wpi) and in multiple tissues beginning at 2 to 4 wpi. Splenic tissue was the only organ positive for RhCMV DNA in all animals. The location of splenic cells expressing RhCMV immediate-early protein 1 (IE1) in i.v. inoculated animals changed following inoculation. At 4 to 5 wpi, most IE1-positive cells were perifollicular, and at 25 wpi, the majority were located within the red pulp. All animals developed anti-RhCMV immunoglobulin M (IgM) antibodies within 1 to 2 wpi and IgG antibodies within 2 to 4 wpi against a limited number of viral proteins. Host reactivity to RhCMV proteins increased in titer (total and neutralizing) and avidity with time. These results demonstrate that while antiviral immune responses were able to protect from disease, they were insufficient to eliminate reservoirs of persistent viral gene expression.
Quasispecies of TT Virus (TTV) with Sequence Divergence in Hypervariable Regions of the Capsid Protein in Chronic TTV Infection
Authors: Nishizawa Tsutomu , Okamoto Hiroaki , Tsuda Fumio , Aikawa Tatsuya , Sugai Yoshiki , Konishi Keiko , Akahane Yoshihiro , Ukita Masato , Tanaka Takeshi , Miyakawa Yuzo , Mayumi Makoto 
Published: December 2019

ABSTRACT
Three hypervariable regions were identified in a central portion of open reading frame 1 of TT virus DNA, which codes for a putative capsid protein of 770 amino acids. TT virus circulates as quasispecies, with many amino acid substitutions in hypervariable regions, to evade immune surveillance of the hosts and to establish a persistent infection.
Persistent Zoonotic Infection of a Human with Simian Foamy Virus in the Absence of an Intactorf-2Accessory Gene
Authors: Callahan Margaret E. , Switzer William M. , Matthews Aprille L. , Roberts Beverly D. , Heneine Walid , Folks Thomas M. , Sandstrom Paul A. 
Published: December 2019

ABSTRACTAlthough foamy viruses (FVs) are endemic among nonhuman primates, FV infection among humans is rare. Recently, simian foamy virus (SFV) infection was reported in 4 of 231 individuals occupationally exposed to primates (1.8%). Secondary transmission to spouses has not been seen, suggesting that while FV is readily zoonotic, humans may represent dead-end hosts. Among different simian species, SFV demonstrates significant sequence diversity within the U3 region of the long terminal repeat (LTR) and 3′ accessory open reading frames (ORFs). To examine if persistent human SFV infection and apparent lack of secondary transmission are associated with genetic adaptations in FV regulatory regions, we conducted sequence analysis of the LTR, internal promoter, ORF-1, and ORF-2 on a tissue culture isolate and peripheral blood mononuclear cell samples from a human infected with SFV of African green monkey origin (SFV-3). Compared to the prototype SFV-3 sequence, the LTR, internal promoter, and FV transactivator (ORF-1) showed sequence conservation, suggesting that FV zoonosis is not dependent on host-specific adaptation to these transcriptionally important regions. However, ORF-2 contains a number of deleterious mutations predicted to result in premature termination of protein synthesis. ORF-2 codes in part for the 60-kDa Bet fusion protein, proposed to be involved in the establishment of persistent cellular SFV infections. These results suggest that persistent human infection by SFV and reduced transmissibility may be influenced by the absence of a functional ORF-2.
Minimal Incidence of Serum Antibodies Reactive with Intact Primary Isolate Virions in Human Immunodeficiency Virus Type 1-Infected Individuals
Authors: Cavacini Lisa A. , Peterson J. Elaine , Nappi Erica , Duval Mark , Goldstein Robert , Mayer Kenneth , Posner Marshall R. 
Published: December 2019

ABSTRACTImmunoglobulin G reactive with primary isolate virions was detected in 36% of serum samples from individuals infected with human immunodeficiency virus type 1. Of these individuals, serum samples from only 7% captured significant quantities of virus. Virion-specific antibody correlated with CD4 counts and, of more significance, primary isolate neutralization. Further dissection of this response should lead to the identification of antibodies and antigenic epitopes for vaccine purposes.
Herpes Simplex Virus Type 1 Serum Neutralizing Antibody Titers Increase during Latency in Rabbits Latently Infected with Latency-Associated Transcript (LAT)-Positive but Not LAT-Negative Viruses
Authors: Perng Guey-Chuen , Slanina Susan M. , Yukht Ada , Ghiasi Homayon , Nesburn Anthony B. , Wechsler Steven L. 
Published: December 2019

ABSTRACT

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation in the rabbit ocular model of HSV-1 latency and reactivation. LAT is also the only viral gene abundantly expressed during latency. Rabbits were ocularly infected with the wild-type HSV-1 strain McKrae or the McKrae-derived LAT null mutant
d
LAT2903. Serum neutralizing antibody titers were determined at various times during acute and latent infection. The neutralizing antibody titers induced by both viruses increased and were similar throughout the first 45 days after infection (
P
> 0.05). However, by day 59 postinfection (approximately 31 to 45 days after latency had been established), the neutralizing antibody titers induced by wild-type virus and
d
LAT2903 diverged significantly (
P
= 0.0005). The
d
LAT2903-induced neutralizing antibody titers decreased, while the wild-type virus-induced neutralizing antibody titers continued to increase. A rescuant of
d
LAT2903, in which spontaneous reactivation was fully restored, induced wild-type neutralizing antibody levels on day 59 postinfection. A second LAT mutant with impaired spontaneous reactivation had neutralizing antibody levels comparable to those of
d
LAT2903. In contrast to the results obtained in rabbits, in mice, neutralizing antibody titers did not increase over time during latency with any of the viruses. Since LAT is expressed in both rabbits and mice during latency, the difference in neutralizing antibody titers between these animals is unlikely to be due to expression of a LAT protein during latency. In contrast, LAT-positive (LAT
+
), but not LAT-negative (LAT
−
), viruses undergo efficient spontaneous reactivation in rabbits, while neither LAT
+
nor LAT
−
viruses undergo efficient spontaneous reactivation in mice. Thus, the increase in neutralizing antibody titers in rabbits latently infected with LAT
+
viruses may have been due to continued restimulation of the immune system by spontaneously reactivating virus.
Wild-Derived Inbred Mice Have a Novel Basis of Susceptibility to Polyomavirus-Induced Tumors
Authors: Velupillai Palanivel , Yoshizawa Izumi , Dey Dilip C. , Nahill Sharon R. , Carroll John P. , Bronson Roderick T. , Benjamin Thomas L. 
Published: December 2019

ABSTRACTPolyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing theH-2khaplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vβ deletion. C57BR/cdJ (BR) mice, which areH-2kbut lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F1-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F1progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.
Subtype-Independent Immature Secretion and Subtype-Dependent Replication Deficiency of a Highly Frequent, Naturally Occurring Mutation of Human Hepatitis B Virus Core Antigen
Authors: Yuan Thomas Ta-Tung , Tai Pei-Ching , Shih Chiaho 
Published: December 2019

ABSTRACTThe most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtypeaywhas been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtypeaywmutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtypeadr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, theadrvariant I97L replicates as well as its parentaladrwild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by thetrans-acting effect of a mutant core protein, while the latter by acis-acting effect of a mutated nucleotide on theaywgenome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.
Screening of Protective Antigens of Japanese Encephalitis Virus by DNA Immunization: a Comparative Study with Conventional Viral Vaccines
Authors: Chen Hsin-Wei , Pan Chien-Hsiung , Liau Ming-Yi , Jou Ruwen , Tsai Chiao-Jung , Wu Hsin-Jung , Lin Yi-Ling , Tao Mi-Hua 
Published: December 2019

ABSTRACTIn this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.