pinecone/core-2020-05-10-deduplication huggingface dataset:
TNFα, Interferon, and Stress Response Induction as a Function of Age-Related Susceptibility to Fatal Sindbis Virus Infection of Mice

AbstractThe age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-α/β and TNF-α and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73–83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions
Molecular Responses of Macrophages to Porcine Reproductive and Respiratory Syndrome Virus Infection

AbstractThe detailed mechanism(s) by which porcine reproductive and respiratory syndrome virus (PRRSV) impairs alveolar Mø homeostasis and function remains to be elucidated. We used differential display reverse-transcription PCR (DDRT-PCR) to identify molecular genetic changes within PRRSV-infected Mø over a 24 h post infection period. From over 4000 DDRT-PCR amplicons examined, 19 porcine-derived DDRT-PCR products induced by PRRSV were identified and cloned. Northern blot analysis confirmed that four gene transcripts were induced during PRRSV infection. PRRSV attachment and penetration alone did not induce these gene transcripts. DNA sequence revealed that one PRRSV-induced expressed sequence tag (EST) encoded porcine Mx1, while the remaining 3 clones represented novel ESTs. A full-length cDNA clone for EST G3V16 was obtained from a porcine blood cDNA library. Sequence data suggests that it encodes an ubiquitin-specific protease (UBP) that regulates protein trafficking and degradation. In pigs infected in vivo, upregulated transcript levels were observed for Mx1 and Ubp in lung and tonsils, and for Mx1 in tracheobronchial lymph node (TBLN). These tissues correspond to sites for PRRSV persistence, suggesting that the Mx1 and Ubp genes may play important roles in clinical disease during PRRSV infection
Ebola Virus Defective Interfering Particles and Persistent Infection

AbstractEbola virus (Zaire subtype) is associated with high mortality disease outbreaks that commonly involve human to human transmission. Surviving patients can show evidence of prolonged virus persistence. The potential for Ebola virus to generate defective interfering (DI) particles and establish persistent infections in tissue culture was investigated. It was found that serial undiluted virus passages quickly resulted in production of an evolving population of virus minireplicons possessing both deletion and copyback type DI genome rearrangements. The tenth undiluted virus passage resulted in the establishment of virus persistently infected cell lines. Following one or two crises, these cells were stably maintained for several months with continuous shedding of infectious virus. An analysis of the estimated genome lengths of a selected set of the Ebola virus minireplicons and standard filoviruses revealed no obvious genome length rule, such as “the rule of six” found for the phylogenetically related Paramyxovirinae subfamily viruses. Minimal promoters for Ebola virus replication were found to be contained within 156 and 177 nucleotide regions of the genomic and antigenomic RNA 3′ termini, respectively, based on the length of authentic termini retained in the naturally occurring minireplicons analyzed. In addition, using UV-irradiated preparations of virus released from persistently infected cells, it was demonstrated that Ebola virus DI particles could potentially be used as natural minireplicons to assay standard virus support functions
Increased Neurovirulence of Polytropic Mouse Retroviruses Delivered by Inoculation of Brain with Infected Neural Stem Cells

AbstractFollowing intraperitoneal (IP) inoculation of neonatal mice, the polytropic recombinant murine leukemia virus (MuLV), Fr98, induces a severe brain disease characterized by ataxia, seizures and death. In contrast, no apparent clinical neurological disease is seen after IP infection with Fr54, a polytropic MuLV differing from Fr98 in its envelope gene sequences. In the brain both Fr98 and Fr54 infect primarily capillary endothelial cells and microglia. However, the level of microglial infection by Fr98 is twofold higher than by Fr54, which might account for the difference in neurovirulence. In the present study, in order to test directly whether an increase in the number of microglia infected by Fr54 would be sufficient to induce clinical disease, we attempted to increase the level of Fr54 in the brain by changing the route of infection. After intraventricular inoculation with Fr54-infected neural stem cells (clone C17.2), a well-established vehicle for delivery of viruses and genes to the brain, mice became ataxic and died 4 weeks postinfection. In these mice induction of brain disease was correlated with a higher level of viral antigen in the cerebrum and an increase in the number of infected microglial cells in all brain regions examined compared with mice inoculated IP. In contrast, mice inoculated with neural stem cells infected with an ecotropic nonneurovirulent murine leukemia virus, FB29, developed no clinical disease in spite of evidence for widespread infection of microglia in brain. Since the main differences between Fr54 and FB29 are in the SU (gp70) region of the envelope gene, this region is most likely to account for the differences in induction of CNS disease seen in the current experiments
Evaluation of Tick-Borne Encephalitis DNA Vaccines in Monkeys

AbstractTick-borne encephalitis is usually caused by infection with one of two flaviviruses: Russian spring summer encephalitis virus (RSSEV) or Central European encephalitis virus (CEEV). We previously demonstrated that gene gun inoculation of mice with naked DNA vaccines expressing the prM and E genes of these viruses resulted in long-lived homologous and heterologous protective immunity (Schmaljohn et al., 1997). To further evaluate these vaccines, we inoculated rhesus macaques by gene gun with the RSSEV or CEEV vaccines or with both DNA vaccines and compared resulting antibody titers with those obtained by vaccination with a commercial, formalin-inactivated vaccine administered at the human dose. Vaccinations were given at days 0, 30, and 70. All of the vaccines elicited antibodies detected by ELISA and by plaque-reduction neutralization tests. The neutralizing antibody responses persisted for at least 15 weeks after the final vaccination. Because monkeys are not uniformly susceptible to tick-borne encephalitis, the protective properties of the vaccines were assessed by passive transfer of monkey sera to mice and subsequent challenge of the mice with RSSEV or CEEV. One hour after transfer, mice that received 50 μl of sera from monkeys vaccinated with both DNA vaccines had circulating neutralizing antibody levels <20–80. All of these mice were protected from challenge with RSSEV or CEEV. Mice that received 10 μl of sera from monkeys vaccinated with the individual DNA vaccines, both DNA vaccines, or a commercial vaccine were partially to completely protected from RSSEV or CEEV challenge. These data suggest that DNA vaccines may offer protective immunity to primates similar to that obtained with a commercial inactivated-virus vaccine
Early Steps of Replication of Moloney Murine Leukemia Virus in Resting Lymphocytes

AbstractWe explored the role of cell type in the early steps of replication of Moloney murine leukemia virus (Mo-MLV) by comparing viral entry and reverse transcription in physiologically quiescent peripheral blood B and T lymphocytes. Virus entry was identical in both cell types. In contrast to previous results, full-length viral DNA was synthesized in resting B lymphocytes, but in agreement with earlier reports, reverse transcription was abortive in resting T lymphocytes. The addition of exogenous nucleosides in the culture medium of resting T lymphocytes allowed reverse transcription to proceed in these cells, without inducing cell cycling. These data suggest that the difference in the ability of quiescent T and B lymphocytes to sustain reverse transcription of Mo-MLV can be explained by a difference in the dNTP pool sizes of these two populations of quiescent cells
Comparisons of Defective HTLV-I Proviruses Predict the Mode of Origin and Coding Potential of Internally Deleted Genomes

AbstractCell lines infected with a variety of HTLV-I isolates were examined for the presence of defective proviruses that contain deletions spanning the gag, pol, and env genes. Internally deleted proviruses were identified by Southern blotting and by PCR amplification with 5′ and 3′ primers complementary to gag and tax sequences, respectively. PCR products representing eight defective proviruses from seven different cell lines were subsequently cloned and sequenced. The objectives of this study were twofold: first, we sought to determine whether nucleotide sequences surrounding sites of deletion shared common features that might reveal the mechanisms by which the defective genomes originated. Second, we asked whether deleted proviruses encode Gag fusion proteins with related C-terminal residues derived from open reading frames in the pX region. While most of the defective proviruses had incurred a single, large deletion, two of them displayed a more complex pattern of multiple rearrangements. Alignments of bases flanking the 5′ and 3′ deletion endpoints within each provirus showed tracts of sequence identity consistent with a mechanism involving aberrant intramolecular strand-transfer events during replication. We suggest that the amount or activity of HTLV-I polymerase in virions may contribute both to the poor infectivity of the virus and to the high deletion frequency. Two of the eight proviruses that were examined encoded a gag gene joined to an extended open reading frame; the other six had very short open reading frames (one to six amino acids) derived from pX or env regions joined to gag that showed no apparent amino acid sequence similarity
Protection of Mice against Challenge with Foot and Mouth Disease Virus (FMDV) by Immunization with Foliar Extracts from Plants Infected with Recombinant Tobacco Mosaic Virus Expressing the FMDV Structural Protein VP1

AbstractA tobacco mosaic virus (TMV)-based vector has been used to express in plants the complete open reading frame coding for VP1, the major immunogenic protein of foot and mouth disease virus (FMDV). In vitro RNA transcripts were inoculated into Nicotiana benthamiana plants and detectable amounts of recombinant VP1 were identified by Western blot as soon as 4 days postinfection. Foliar extracts prepared from infected leaves were injected intraperitoneally into mice and all of the immunized animals developed a specific antibody response to both the complete virus particle and the major immunogenic region as determined by ELISA and Western blot analysis. Most importantly, all immunized mice developed a protective immune response against experimental challenge with virulent FMDV. To our knowledge, this is the first report showing the expression of a complete open reading frame of an antigenic foreign protein in plants, using a recombinant plant virus, in sufficient quantity to permit use of the crude plant extract as an experimental immunogen to protect animals against virus challenge
Time-Related Changes in Full-Length Hepatitis C Virus Sequences and Hepatitis Activity

AbstractThe activity of hepatitis varies in chronic hepatitis C virus (HCV) infection. Some patients show persistently normal serum alanine aminotransferase (ALT) values, whereas the others show high-ALT values and progress to liver cirrhosis or hepatocellular carcinoma. However, virological mechanisms causing hepatitis have not fully been elucidated. We analyzed serial changes in full-length HCV sequences in 10 patients with various profiles of hepatitis activity. In the nonstructural 5A (NS5A) and NS5B, the rate of amino acid changes, as well as the proportion of nonsilent ones, was low in patients with normal ALT values compared with those with abnormal ALT (for the rate of amino acid changes, 0 × 10−3vs 3.19 × 10−3 changes/site/year (P = 0.037) in NS5A and 0 × 10−3vs 1.22 × 10−3 changes/site/year (P = 0.023) for NS5B, for the proportion of nonsilent changes, 4 vs 22% (P = 0.017) in NS5A and 0 vs 16% (P = 0.039) in NS5B). Also, the flare-up of hepatitis coincided with higher nucleotide/amino acid substitution rates in NS5B. In conclusion, the genomic structures of the NS5A and NS5B regions may correlate with hepatitis activity in chronic hepatitis C
The Tomato Golden Mosaic Virus Transactivator (TrAP) is a Single-Stranded DNA and Zinc-Binding Phosphoprotein with an Acidic Activation Domain

AbstractThe AL2 gene found in members of the genus Begomovirus of the Geminiviridae encodes a transcriptional activator protein (TrAP; also known as AL2, AC2, or C2 protein). TrAP activates expression from the viral coat protein (CP) and BR1 movement gene promoters in mesophyll cells and protoplasts and acts to derepress the CP promoter in vascular tissue. The experiments presented here were designed to elucidate some of the biochemical properties of this multifunctional regulatory protein and to define its activation domain. The results indicate that TrAP from tomato golden mosaic virus (TGMV) binds single-stranded DNA in a sequence nonspecific manner and only weakly interacts with double-stranded DNA, confirming earlier results obtained with TrAP from other begomoviruses. In addition, evidence is presented that indicates that TrAP binds zinc and that zinc is necessary for optimal interaction with ssDNA. We also show that TrAP is phosphorylated when expressed in insect cells and that it contains a transcriptional activation domain of the acidic type. The minimal activation domain is quite small; the region comprising only the 15 C-terminal amino acids of the protein is capable of activating transcription in mouse fibroblasts (NIH3T3 cells) when fused to a heterologous DNA-binding domain
Biochemical and Genetic Evidence for Interactions between Potato A Potyvirus-Encoded Proteins P1 and P3 and Proteins of the Putative Replication Complex

AbstractInteractions of the first and third proteins (P1 and P3) of the potato A potyvirus (PVA) with the other six main proteins of PVA were studied using Escherichia coli-expressed recombinant proteins in two in vitro interaction assays and a genetic assay yeast two-hybrid system (YTHS). In overlay blotting and binding assays in liquid, P1 and P3 interacted with each other and with proteins of the putative replication complex of potyvirus: RNA-helicase (CI), viral protein genome-linked (VPg), NIa proteinase part (NIaPro), and RNA-dependent-RNA-polymerase (NIb). In addition, P1 self-interaction and interaction with helper-component proteinase (HC-Pro) also were detected. Neither P1 nor P3 interact with coat protein (CP) or with various control proteins. In the YTHS, P1 interacted only with CI and P3 with NIb. The different results obtained using the two test systems may reflect changes in interactions at different stages of potyvirus infection: in the virus genome replication and the virion accumulation stages when nonstructural proteins form inclusions. Our data are consistent with previous functional data, indicating that P1 and P3 proteins are involved in potyvirus genome amplification and provide the first direct evidence that these proteins interact with the proteins that have been shown to be part of the replication complex
Protein 2A of Grapevine Fanleaf Nepovirus Is Implicated in RNA2 Replication and Colocalizes to the Replication Site

AbstractRNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2BMP, and the coat protein 2CCP. Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5′ and 3′ noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2BMP and/or 2CCP or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1Dpro and 1CVPg, two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5′ triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins
Internal Initiation of Translation Directed by the 5′-Untranslated Region of the Tobamovirus Subgenomic RNA I2

AbstractPreviously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRESCP,148CR). The equivalent 148-nt sequence from TMV U1 RNA (U1CP,148SP) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRESMP,228CR) contained an IRES element that directed in vitro translation of the 3′-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcripts as well. Consequently this sequence was termed IRESMP,228U1. It was shown that IRESMP,228CR, IRESMP,228U1, and IRESCP,148CR could mediate expression of the 3′-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRESMP,75), which corresponded closely to the length of the 5′UTR of TMV subgenomic RNA (sgRNA) I2. The RNA transcripts structurally equivalent to I2 sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRESMP,75 (HIRESMP,75CR-MP-CP-3′UTR; HIRESMP,75U1-MP-CP-3′UTR), were able to express the MP gene in vitro. The capacity of HIRESMP,75CR sequence for mediating internal translation of the 3′-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I2 sgRNAs might proceed via internal ribosome entry pathway mediated by IRESMP element contained in the 75-nt 5′UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I2 sgRNA operates concurrently
Phage-Displayed Peptides Mimicking the Discontinuous Neutralization Sites of Puumala Hantavirus Envelope Glycoproteins

AbstractWe selected peptide ligands mimicking the surface structure of discontinuous binding sites of Puumala hantavirus-neutralizing monoclonal antibodies from a random 18-amino acid peptide library containing a disulfide bridge in a fixed position and displayed on a filamentous phage. The varying of selection conditions, either by shortening of the association time or by competitive elution with antigen, was crucial for the selection of peptide inserts that could be aligned with the primary sequences of the envelope glycoproteins G1 and G2. Correspondingly, when the envelope glycoprotein sequences were synthesized as overlapping peptides as spots on membrane, the same site in primary structure was found as with phage display, which corroborates the use of the two methods in mapping of conformational epitopes. Also, epitopes reactive with early-phase sera from Puumala virus infection were defined with the pepspot assay in the amino-terminal region of G1. Similarities of the selected phage clones to a monoclonal antibody–escape mutant site and to a linear early-phase epitope were found
Genetic Analysis Reveals That Both Haemagglutinin and Neuraminidase Determine the Sensitivity of Naturally Occurring Avian Influenza Viruses to Zanamivir in Vitro

AbstractThe basis of differential sensitivity of replication of influenza viruses to the neuraminidase-specific inhibitor zanamivir was examined using four avian influenza viruses and reassortants produced between them. IC50 values for inhibition of neuraminidase activity by zanamivir were similar for each of the four viruses, whereas the haemagglutinating activity of each of the viruses was relatively insensitive to zanamivir. However, the four viruses showed distinct zanamivir-sensitivity profiles in tissue culture. Analysis of the reassortant viruses showed that sensitivity was determined by the haemagglutinin gene (segment 4) and the neuraminidase gene (segment 6) and was independent of the remaining six RNA segments. Decreased sensitivity to zanamivir was associated with possession of a haemagglutinin that is released from cells with decreased dependence on neuraminidase and with possession of a neuraminidase that has a short stalk region
Mutations in Conserved Domain II of the Large (L) Subunit of the Sendai Virus RNA Polymerase Abolish RNA Synthesis

AbstractThe large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I–VI) thought to be responsible for the enzymatic activities. Charged-to-alanine mutagenesis was carried out in a highly charged, conserved region (amino acids 533–569) within domain II to test the hypothesis of Müller et al. [J. Gen. Virol. 75, 1345–1352 (1994)] that this region may contribute to the template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral RNA in vitro and in vivo, and protein–protein interactions. Five of the seven mutants were completely defective in all viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. One of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the replication assays and thus the mutation uncoupled transcription and replication. Because the completely inactive L mutants can bind to the P protein to form the polymerase complex and the polymerases bind to the viral nucleocapsid template, these amino acids are essential for the activity of the L protein