bluuebunny/crossref_metadata_2025 huggingface dataset:
Mosaic Structure of the Human Immunodeficiency Virus Type 1 Genome Infecting Lymphoid Cells and the Brain: Evidence for Frequent In Vivo Recombination Events in the Evolution of Regional Populations
Authors: Morris A. , Marsden M. , Halcrow K. , Hughes E. S. , Brettle R. P. , Bell J. E. , Simmonds P.
Published: December 2019

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17gag,pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17gagand V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.
Association of Nef with the Human Immunodeficiency Virus Type 1 Core
Authors: Kotov Alexander , Zhou Jing , Flicker Paula , Aiken Christopher
Published: December 2019

Highly conserved among primate lentiviruses, the human immunodeficiency virus type 1 (HIV-1) Nef protein enhances viral infectivity by an unknown mechanism. Nef-defective virions are blocked at a stage of the HIV-1 life cycle between entry and reverse transcription, possibly virus uncoating. Nef is present in purified HIV-1 particles; however, it has not been determined whether Nef is specifically recruited into HIV-1 particles or whether virion-associated Nef plays a functional role in HIV-1 replication. To address the specificity and potential functionality of virion-associated Nef, we determined the subviral localization of Nef. HIV-1 cores were isolated by detergent treatment of concentrated virions followed by equilibrium density gradient sedimentation. Relative to HIV-1 virions, HIV-1 cores contained equivalent amounts of reverse transcriptase and integrase, decreased amounts of the viral matrix protein, and trace quantities of the viral transmembrane glycoprotein gp41. Examination of the particles by electron microscopy revealed cone-shaped structures characteristic of lentiviral cores. Similar quantities of proteolytically processed Nef protein were detected in gradient fractions of HIV-1 cores and intact virions. In addition, detergent-resistant subviral complexes isolated from immature HIV-1 particles contained similar quantities of Nef as untreated virions. These results demonstrate that Nef stably associates with the HIV-1 core and suggest that virion-associated Nef plays a functional role in accelerating HIV-1 replication.
Requirement for Cell-to-Cell Contact in Epstein-Barr Virus Infection of Nasopharyngeal Carcinoma Cells and Keratinocytes
Authors: Chang Yao , Tung Che-Huang , Huang Yu-Tzu , Lu Jean , Chen Jen-Yang , Tsai Ching-Hwa
Published: December 2019

Two nasopharyngeal carcinoma (NPC) cell lines and one keratinocyte cell line could be infected with Epstein-Barr virus (EBV) by cocultivation with virus-producing cells but not by cell-free virus. Using porous culture inserts to manipulate the cell-to-cell contact, we demonstrated that contact between EBV donor B cells and EBV recipient epithelial cells was required for the infection. Cell-to-cell contact not only provided a CR2-independent route of infection but also enhanced CR2-mediated infection in a synergistic manner. Activity of two EBV promoters (Cp and Wp) and expression of EBNA2 were detected in the infected population. A small proportion of the infected cells spontaneously entered an EBV lytic state, which could be induced prominently by chemical treatment. This study provides information on how EBV may infect epithelial cells in vivo, such as at the onset of NPC development.
Strong Selective Pressure for Evolution of an Epstein-Barr Virus LMP2B Homologue in the Rhesus Lymphocryptovirus
Authors: Rivailler Pierre , Quink Carol , Wang Fred
Published: December 2019

Latent membrane protein 2B (LMP2B) is expressed during latent Epstein-Barr virus (EBV) infection, but little is known about its role. The goal of this study was to determine whether an LMP2B homologue is conserved in the rhesus monkey lymphocryptovirus (LCV). Both rhesus LCV LMP2A and LMP2B genes were cloned and sequenced. The rhesus LCV LMP2B gene is positionally conserved, and the EBNA-2 responsiveness and the bidirectional nature of the LMP1-LMP2B promoter have also been functionally conserved. However, this region of the genome encoding the LMP1, LMP1-LMP2B promoter, and LMP2B first exon demonstrates the most dramatic nucleotide sequence divergence between human and nonhuman LCV observed to date. Evolution of the rhesus LCV LMP2B promoter and transcript despite the dynamic nature of this genomic region reflects strong selective pressure for a yet-to-be-identified LMP2B function.
Inhibition of Influenza Virus Replication by Nitric Oxide
Authors: Rimmelzwaan Guus F. , Baars Marianne M. J. W. , de Lijster Pim , Fouchier Ron A. M. , Osterhaus Albert D. M. E.
Published: December 2019

Nitric oxide (NO) has been shown to contribute to the pathogenesis of influenza virus-induced pneumonia in mouse models. Here we show that replication of influenza A and B viruses in Mabin Darby canine kidney cells is severely impaired by the NO donor,S-nitroso-N-acetylpenicillamine. Reduction of productively infected cells and virus production proved to correlate with inhibition of viral RNA synthesis, indicating that NO affects an early step in the replication cycle of influenza viruses.
Assessment of Bovine Leukemia Virus Transcripts In Vivo
Authors: Rovnak Joel , Casey James W.
Published: December 2019

Reverse transcriptase PCR (RT-PCR) consistently detected bovine leukemia virus transcripts in fresh cells, and competitive RT-PCR enumerated these transcripts. The detection of transcripts in limited numbers of tumor cells indicated that expression occurs in a minority of cells. The data suggest that individual cells contain hundreds of copies of the
tax/rex
transcript in vivo.
An MΨ-Containing Heterologous RNA, but Not
env
mRNA, Is Efficiently Packaged into Avian Retroviral Particles
Authors: Banks Jennifer D. , Kealoha Bonnie O. , Linial Maxine L.
Published: December 2019

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MΨ, is located upstream of the 5′ splice site and therefore is present on both genomic and spliced RNAs. We now show that an MΨ-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MΨ. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MΨ is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3′ end of MΨ was found to reduce the packaging efficiency of heterologous RNAs.
Interleukin-4 Diminishes CD8+Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo
Authors: Aung Sandra , Tang Yi-Wei , Graham Barney S.
Published: December 2019

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using anH-2Kd-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct51Cr release assay and intracellular gamma interferon (IFN-γ) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-γ production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8+RSV-specific CTL responses in vivo.
Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles
Authors: Everly David N. , Read G. Sullivan
Published: December 2019

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157–7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.
Sendai Virus Gene Start Signals Are Not Equivalent in Reinitiation Capacity: Moderation at the Fusion Protein Gene
Authors: Kato Atsushi , Kiyotani Katsuhiro , Hasan Mohammad K. , Shioda Tatsuo , Sakai Yuko , Yoshida Tetsuya , Nagai Yoshiyuki
Published: December 2019

In paramyxovirus transcription, viral RNA polymerase synthesizes each monocistronic mRNA by recognizing the gene start (S) and end (E) signals flanking each gene. These signal sequences are well conserved in the virus family; nevertheless, they do exhibit some variations even within a virus species. In Sendai virus (SeV) Z strain, the E signals are identical for all six genes but there are four (N, P/M/HN, F, and L) different S signals with one or two nucleotide variations. The significance of these variations for in vitro and in vivo replication has been unknown. We addressed this issue by SeV reverse genetics. The luciferase gene was placed between the N and P gene so that recombinant SeVs expressed luciferase under the control of each of the four different S signals. The S signal for the F gene was found to drive a lower level of transcription than that of the other three, which exhibited comparable reinitiation capacities. The polar attenuation of SeV transcription thus appeared to be not linear but biphasic. Then, a mutant SeV whose F gene S signal was replaced with that used for the P, M, and HN genes was created, and its replication capability was examined. The mutant produced a larger amount of F protein and downstream gene-encoded proteins and replicated faster than wild-type SeV in cultured cells and in embryonated eggs. Compared with the wild type, the mutant virus also replicated faster in mice and was more virulent, requiring a dose 20 times lower to kill 50% of mice. On the other hand, the unique F start sequence as well as the other start sequences are perfectly conserved in all SeV isolates sequenced to date, including highly virulent fresh isolates as well as egg-adapted strains, with a virulence several magnitudes lower than that of the fresh isolates. This moderation of transcription at the F gene may therefore be relevant to viral fitness in nature.
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

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

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

Poliovirus 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

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

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

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

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

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

Although 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

Immunoglobulin 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

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

Polyomavirus 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

The 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

In 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.
Characterization of Primary Isolate-Like Variants of Simian-Human Immunodeficiency Virus
Authors: Crawford John M. , Earl Patricia L. , Moss Bernard , Reimann Keith A. , Wyand Michael S. , Manson Kelledy H. , Bilska Miroslawa , Zhou Jin Tao , Pauza C. David , Parren Paul W. H. I. , Burton Dennis R. , Sodroski Joseph G. , Letvin Norman L. , Montefiori David C.
Published: December 2019

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.
Lymphocyte Activation during Acute Simian/Human Immunodeficiency Virus SHIV89.6PDInfection in Macaques
Authors: Wallace Marianne , Waterman Paul M. , Mitchen Jacque L. , Djavani Mahmoud , Brown Charles , Trivedi Parul , Horejsh Douglas , Dykhuizen Marta , Kitabwalla Moiz , Pauza C. David
Published: December 2019

Host-virus interactions control disease progression in human immunodeficiency virus-infected human beings and in nonhuman primates infected with simian or simian/human immunodeficiency viruses (SHIV). These interactions evolve rapidly during acute infection and are key to the mechanisms of viral persistence and AIDS. SHIV89.6PDinfection in rhesus macaques can deplete CD4+T cells from the peripheral blood, spleen, and lymph nodes within 2 weeks after exposure and is a model for virulent, acute infection. Lymphocytes isolated from blood and tissues during the interval of acute SHIV89.6PDinfection have lost the capacity to proliferate in response to phytohemagglutinin (PHA). T-cell unresponsiveness to mitogen occurred within 1 week after mucosal inoculation yet prior to massive CD4+T-cell depletion and extensive virus dissemination. The lack of mitogen response was due to apoptosis in vitro, and increased activation marker expression on circulating T cells in vivo coincided with the appearance of PHA-induced apoptosis in vitro. Inappropriately high immune stimulation associated with rapid loss of mature CD4+T cells suggested that activation-induced cell death is a mechanism for helper T-cell depletion in the brief period before widespread virus dissemination. Elevated levels of lymphocyte activation likely enhance SHIV89.6PDreplication, thus increasing the loss of CD4+T cells and diminishing the levels of virus-specific immunity that remain after acute infection. The level of surviving immunity may dictate the capacity to control virus replication and disease progression. We describe this level of immune competence as the host set point to show its pivotal role in AIDS pathogenesis.
Experimental Infection of Rhesus and Pig-Tailed Macaques with Macaque Rhadinoviruses
Authors: Mansfield Keith G. , Westmoreland Susan V. , DeBakker Colin D. , Czajak Susan , Lackner Andrew A. , Desrosiers Ronald C.
Published: December 2019

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (
Macaca mulatta
) and pig-tailed macaques (
Macaca nemestrina
) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days;
P
< 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20
+
B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.
Dynamin Is Required for Recombinant Adeno-Associated Virus Type 2 Infection
Authors: Duan Dongsheng , Li Qiang , Kao Aimee W. , Yue Yongping , Pessin Jeffrey E. , Engelhardt John F.
Published: December 2019

Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.
In Vivo Regulation of Hepatitis B Virus Replication by Peroxisome Proliferators
Authors: Guidotti Luca G. , Eggers Carrie M. , Raney Anneke K. , Chi Susan Y. , Peters Jeffrey M. , Gonzalez Frank J. , McLachlan Alan
Published: December 2019

The role of the peroxisome proliferator-activated receptor α (PPARα) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARα. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARα but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice.