bluuebunny/crossref_metadata_2025_split huggingface dataset:
African Swine Fever Virus Multigene Family 360 and 530 Genes Are Novel Macrophage Host Range Determinants
Authors: Zsak L. , Lu Z. , Burrage T. G. , Neilan J. G. , Kutish G. F. , Moore D. M. , Rock D. L. 
Published: July 2002

ABSTRACT
Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.
Brome Mosaic Virus Protein 1a Recruits Viral RNA2 to RNA Replication through a 5′ Proximal RNA2 Signal
Authors: Chen Jianbo , Noueiry Amine , Ahlquist Paul 
Published: July 2002

ABSTRACT
Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5′-proximal third of RNA2. The RNA2 5′ untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5′-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TΨC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.
Regression of a Murine Gammaherpesvirus 68-Positive B-Cell Lymphoma Mediated by CD4 T Lymphocytes
Authors: Robertson Kevin A. , Usherwood Edward J. , Nash Anthony A. 
Published: July 2002

ABSTRACT
Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.
Generation of a Highly Pathogenic Avian Influenza A Virus from an Avirulent Field Isolate by Passaging in Chickens
Authors: Ito Toshihiro , Goto Hideo , Yamamoto Eiji , Tanaka Hiroko , Takeuchi Mutsuko , Kuwayama Masaru , Kawaoka Yoshihiro , Otsuki Koichi 
Published: July 2002

ABSTRACT
Highly virulent avian influenza viruses can arise from avirulent strains maintained in poultry, but evidence to support their generation from viruses in wild birds is lacking. The most likely mechanism for the acquisition of virulence by benign avian viruses is the introduction of mutations by error-prone RNA polymerase, followed by the selection of virulent viruses. To investigate whether this mechanism could apply to wild waterfowl, we studied an avirulent wild-swan virus that replicates poorly in chickens. After 24 consecutive passages by air sac inoculation, followed by five passages in chicken brain, the avirulent virus became highly pathogenic in chickens, producing a 100% mortality rate. Sequence analysis at the hemmaglutinin cleavage site of the original isolate revealed a typical avirulence type of sequence, R-E-T-R, which progressed incrementally to a typical virulence type of sequence, R-R-K-K-R, during repeated passages in chickens. These results demonstrate that avirulent viruses maintained in wild waterfowl in nature and bearing the consensus avirulence type sequence R-E-T-R have the potential to become highly pathogenic while circulating in chickens.
Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display
Authors: de Carvalho Nicacio Cristina , Williamson R. Anthony , Parren Paul W. H. I. , Lundkvist Åke , Burton Dennis R. , Björling Ewa 
Published: July 2002

ABSTRACT

Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an ⊘80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an ⊘60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between ≈2 and 7 μg ml
−1
. Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries.
Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs
Authors: Chang C.-C. , Yoon K.-J. , Zimmerman J. J. , Harmon K. M. , Dixon P. M. , Dvorak C. M. T. , Murtaugh M. P. 
Published: July 2002

ABSTRACTPorcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.
Primary Immune Responses by Cord Blood CD4+T Cells and NK Cells Inhibit Epstein-Barr Virus B-Cell Transformation In Vitro
Authors: Wilson A. Douglas , Morgan Andrew J. 
Published: July 2002

ABSTRACTEpstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 105.9. We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4+T cells and CD16+CD56+NK cells. After virus challenge both CD4+T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16+CD69+cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4+T cells and CD16+NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4+T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4+T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4+T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.
Expression of a Membrane-Anchored Glycoprotein, the Influenza Virus Hemagglutinin, by Dicistronic Replicons Derived from the Poliovirus Genome
Authors: Vignuzzi Marco , Gerbaud Sylvie , van der Werf Sylvie , Escriou Nicolas 
Published: July 2002

ABSTRACT
Mono- and dicistronic poliovirus replicons were constructed to express the influenza virus hemagglutinin, retaining its signal peptide and transmembrane region. Picornavirus genomes do not normally encode glycoproteins, and only the dicistronic replicon, in which the foreign and poliovirus sequences were separated by the encephalomyocarditis virus internal ribosomal entry site, replicated and expressed glycosylated hemagglutinin.
Bone Marrow Chimeras Reveal Non-H-2Hematopoietic Control of Susceptibility to Theiler's Virus Persistent Infection
Authors: Aubagnac Stéphanie , Brahic Michel , Bureau Jean-François 
Published: July 2002

ABSTRACTThe DA strain of Theiler's murine encephalomyelitis virus persists in the white matter of the spinal cords of susceptible mice. Previous results showed that the difference in susceptibility to viral persistence between the susceptible SJL/J strain and the resistant B10.S strain was due to multiple non-H-2loci. The respective roles of hematopoietic and nonhematopoietic cells in this difference have been evaluated with bone marrow chimeras. The results show that non-H-2loci with a major effect on susceptibility are expressed in hematopoietic cells. However, the study of the SJL.B10-D10Mit180-D10Mit74congenic line suggests that other loci expressed in nonhematopoietic cells also play a role.
Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease
Authors: Someya Yuichi , Takeda Naokazu , Miyamura Tatsuo 
Published: July 2002

ABSTRACT

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in
Escherichia coli
, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.
A Tyrosine-Based Motif in the Cytoplasmic Tail of Pseudorabies Virus Glycoprotein B Is Important for both Antibody-Induced Internalization of Viral Glycoproteins and Efficient Cell-to-Cell Spread
Authors: Favoreel Herman W. , Van Minnebruggen Geert , Nauwynck Hans J. , Enquist Lynn W. , Pensaert Maurice B. 
Published: July 2002

ABSTRACTPseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXΦ motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.
K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function
Authors: Sharp Tyson V. , Wang Hsei-Wei , Koumi Andrew , Hollyman Daniel , Endo Yoshio , Ye Hongtao , Du Ming-Qing , Boshoff Chris 
Published: July 2002

ABSTRACTThe Kaposi’s sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, ∼50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman’s disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.
Herpes Simplex Virus Type 1 Immediate-Early Protein ICP0 and Its Isolated RING Finger Domain Act as Ubiquitin E3 Ligases In Vitro
Authors: Boutell Chris , Sadis Seth , Everett Roger D. 
Published: July 2002

ABSTRACT
Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.
Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication
Authors: Li Feng , Chen Chaoping , Puffer Bridget A. , Montelaro Ronald C. 
Published: July 2002

ABSTRACT
We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.
PrP Expression and Replication by Schwann Cells: Implications in Prion Spreading
Authors: Follet Jérôme , Lemaire-Vieille Catherine , Blanquet-Grossard Françoise , Podevin-Dimster Valérie , Lehmann Sylvain , Chauvin Jean-Paul , Decavel Jean-Pierre , Varea Ruth , Grassi Jacques , Fontès Michel , Cesbron Jean-Yves 
Published: July 2002

ABSTRACT

Prion infection relies on a continuous chain of PrP
c
-expressing tissues to spread from peripheral sites to the central nervous system (CNS). Direct neuroinvasion via peripheral nerves has long been considered likely. However, the speed of axonal flow is incompatible with the lengthy delay prior to the detection of PrP
Sc
in the brain. We hypothesized that Schwann cells could be the candidate implicated in this mechanism; for that, it has to express PrP
c
and to allow PrP
Sc
conversion. We investigated in vivo localization of PrP
c
in sciatic nerve samples from different strains of mice. We demonstrated that PrP
c
is mainly localized at the cell membrane of the Schwann cell. We also studied in vitro expression of PrP
c
in the Schwann cell line MSC-80 and demonstrated that it expresses PrP
c
at the same location. More specifically, we demonstrated that this glial cell line, when infected in vitro with the mouse Chandler prion strain, both produces the PrP
Sc
till after 18 passages and is able to transmit disease to mice, which then develop the typical signs of prion diseases. It is the first time that infection and replication of PrP
Sc
are shown in a peripheral glial cell line.