bluuebunny/crossref_metadata_2025 huggingface dataset:
TT Virus Infection in Nonhuman Primates and Characterization of the Viral Genome: Identification of Simian TT Virus Isolates
Authors: Abe Kenji , Inami Tomoko , Ishikawa Koichi , Nakamura Shin , Goto Shunji
Published: July 2002

Newly discovered TT virus (TTV) is widely distributed in human populations. To understand more about the relationship between TTV and its hosts, we tested 400 sera from various nonhuman primates for the presence of TTV DNA by PCR assay. We collected serum samples from 24 different species of nonhuman primates. TTV DNA was determined by PCR with primers designed from the 5′-end region of the TTV genome. Nucleotide sequencing and phylogenetic analysis of viral genomes were also performed. TTV DNA was detected in 87 of 98 (89%) chimpanzees and 3 of 21 (14%) crab-eating macaques. Nucleotide sequences of the PCR products obtained from both animals were 80 to 100% identical between two species. In contrast, the sequences differed from TTV isolates in humans by 24 to 33% at the nucleotide level and 36 to 50% at the amino acid level. Phylogenetic analysis demonstrated that all TTV isolates obtained from simians were distinct from the human TTV isolates. Furthermore, TTV in simians, but not in humans, was classified into three different genotypes. Our results indicate that TTV in simians represents a group different from, but closely related to, TTV in humans. From these results, we tentatively named this TTV simian TTV (s-TTV). The existence of the s-TTV will be important in determining the origin, nature, and transmission of human TTV and may provide useful animal models for studies of the infection and pathogenesis of this new DNA virus.
Infection of Primary Human Monocytes by Epstein-Barr Virus
Authors: Savard Martin , Bélanger Carole , Tardif Mélanie , Gourde Pierrette , Flamand Louis , Gosselin Jean
Published: July 2002

Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolated nuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This was supported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two early lytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with the replicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were also detected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negative results. Viral replication was also confirmed by the release of newly synthesized infectious viral particles in supernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reduced phagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads. Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functions might represent a new mechanism to disrupt the immune response and promote viral propagation during the early stages of infection.
Role of Hemagglutinin Surface Density in the Initial Stages of Influenza Virus Fusion: Lack of Evidence for Cooperativity
Authors: Günther-Ausborn Susanne , Schoen Pieter , Bartoldus Ingrid , Wilschut Jan , Stegmann Toon
Published: July 2002

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.
A Recombinant Human Parainfluenza Virus Type 3 (PIV3) in Which the Nucleocapsid N Protein Has Been Replaced by That of Bovine PIV3 Is Attenuated in Primates
Authors: Bailly Jane E. , McAuliffe Josephine M. , Durbin Anna P. , Elkins William R. , Collins Peter L. , Murphy Brian R.
Published: July 2002

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans.
Cospeciation and Horizontal Transmission of Avian Sarcoma and Leukosis Virus
gag
Genes in Galliform Birds
Authors: Dimcheff Derek E. , Drovetski Sergei V. , Krishnan Mallika , Mindell David P.
Published: July 2002

In a study of the evolution and distribution of avian retroviruses, we found avian sarcoma and leukosis virus (ASLV)
gag
genes in 26 species of galliform birds from North America, Central America, eastern Europe, Asia, and Africa. Nineteen of the 26 host species from whom ASLVs were sequenced were not previously known to contain ASLVs. We assessed congruence between ASLV phylogenies based on a total of 110
gag
gene sequences and ASLV-host phylogenies based on mitochondrial 12S ribosomal DNA and ND2 sequences to infer coevolutionary history for ASLVs and their hosts. Widespread distribution of ASLVs among diverse, endemic galliform host species suggests an ancient association. Congruent ASLV and host phylogenies for two species of
Perdix
, two species of
Gallus
, and
Lagopus lagopus
and
L. mutus
also indicate an old association with vertical transmission and cospeciation for these ASLVs and hosts. An inference of horizontal transmission of ASLVs among some members of the Tetraoninae subfamily (grouse and ptarmigan) is supported by ASLV monophyletic groups reflecting geographic distribution and proximity of hosts rather than host species phylogeny. We provide a preliminary phylogenetic taxonomy for the new ASLVs, in which named taxa denote monophyletic groups.
Transfer of Primer Binding Site-Mutated Simian Immunodeficiency Virus Vectors by Genetically Engineered Artificial and Hybrid tRNA-Like Primers
Authors: Hansen Anette Chemnitz , Grunwald Thomas , Lund Anders Henrik , Schmitz Alexander , Duch Mogens , Überla Klaus , Pedersen Finn Skou
Published: July 2002

Simian immunodeficiency viruses (SIV) harbor primer binding sites (PBS) matching tRNA

3

Lys

or tRNA

5

Lys

. To study determinants of primer usage in SIV, a SIVmac239-based vector was impaired by mutating the PBS to a sequence (PBS-X2) with no match to any tRNA. By cotransfection of a synthetic gene encoding a tRNA
Pro
-like RNA with a match to PBS-X2, the activity of this vector could be restored to a transduction efficiency slightly lower than that of the wild-type vector. A vector with a PBS matching tRNA
Pro
was functional at a level slightly below that of the wild-type vector, but higher transduction efficiency could be obtained by cotransfection of a gene for an engineered tRNA
Pro
-tRNA

3

Lys

hybrid with a match to PBS-Pro. The importance of tRNA backbone identity was further analyzed by complementing the PBS-X2 vector with a gene for a matching x2 primer with a tRNA

3

Lys

backbone, which led to three- to fourfold-higher titers than those observed for the x2 primer with the tRNA
Pro
backbone. In summary, our results demonstrate flexibility in PBS and primer usage for SIVmac239, with PBS-primer complementarity being the major determinant, in analogy with previous findings for murine leukemia viruses and human immunodeficiency virus type 1.
Regulation of mRNA Translation and Cellular Signaling by Hepatitis C Virus Nonstructural Protein NS5A
Authors: He Yupeng , Tan Seng-Lai , Tareen Semih U. , Vijaysri Sangeetha , Langland Jeffrey O. , Jacobs Bertram L. , Katze Michael G.
Published: July 2002

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVΔE3L) resulted in increased phosphorylation levels of both PKR and eIF2α. IFN-pretreated cells infected with VV in which theE3Llocus was replaced with theNS5Agene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2α in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVΔE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2α and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVΔE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.
Cytomegalovirus Basic Phosphoprotein (pUL32) Binds to Capsids In Vitro through Its Amino One-Third
Authors: Baxter Michael K. , Gibson Wade
Published: July 2002

The cytomegalovirus (CMV) basic phosphoprotein (BPP) is a component of the tegument. It remains with the nucleocapsid fraction under conditions that remove most other tegument proteins from the virion, suggesting a direct and perhaps tight interaction with the capsid. As a step toward localizing this protein within the molecular structure of the virion and understanding its function during infection, we have investigated the BPP-capsid interaction. In this report we present evidence that the BPP interacts selectively, through its amino one-third, with CMV capsids. Radiolabeled simian CMV (SCMV) BPP, synthesized in vitro, bound to SCMV B-capsids, and C-capsids to a lesser extent, following incubation with either isolated capsids or lysates of infected cells. Human CMV (HCMV) BPP (pUL32) also bound to SCMV capsids, and SCMV BPP likewise bound to HCMV capsids, indicating that the sequence(s) involved is conserved between the two proteins. Analysis of SCMV BPP truncation mutants localized the capsid-binding region to the amino one-third of the molecule—the portion of BPP showing the greatest sequence conservation between the SCMV and HCMV homologs. This general approach may have utility in studying the interactions of other proteins with conformation-dependent binding sites.
Downstream Sequences Influence the Choice between a Naturally Occurring Noncanonical and Closely Positioned Upstream Canonical Heptameric Fusion Motif during Bovine Coronavirus Subgenomic mRNA Synthesis
Authors: Ozdarendeli Aykut , Ku Seulah , Rochat Sylvie , Williams Gwyn D. , Senanayake Savithra D. , Brian David A.
Published: July 2002

Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5′ and 3′ coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (
GG
UA
G
AC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (−17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5′ end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.
Expression and Immunogenicity of Human Immunodeficiency Virus Type 1 Gag Expressed by a Replication-Competent Rhabdovirus-Based Vaccine Vector
Authors: McGettigan James P. , Sarma Satyam , Orenstein Jan M. , Pomerantz Roger J. , Schnell Matthias J.
Published: July 2002

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544–3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430–4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8
+
cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8
+
T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.
Assembly and Catalysis of Concerted Two-End Integration Events by Moloney Murine Leukemia Virus Integrase
Authors: Yang Fan , Roth Monica J.
Published: July 2002

Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.
Interleukin-12- and Gamma Interferon-Dependent Innate Immunity Are Essential and Sufficient for Long-Term Survival of Passively Immunized Mice Infected with Herpes Simplex Virus Type 1
Authors: Vollstedt Sabine , Franchini Marco , Alber Gottfried , Ackermann Mathias , Suter Mark
Published: July 2002

Interferon (IFN) type I (alpha/beta IFN [IFN-α/β]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-γ) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to104PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-γ-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.
Recombinant Norwalk Virus-Like Particles Administered Intranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal) Immune Responses
Authors: Guerrero Roberto A. , Ball Judith M. , Krater Sharon S. , Pacheco Susan E. , Clements John D. , Estes Mary K.
Published: July 2002

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345–1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant
Escherichia coli
heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 μg) of rNV VLPs and the majority of mice that received two high doses orally (200 μg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.
Differential Expression of Interleukin-2 and Gamma Interferon in Human Immunodeficiency Virus Disease
Authors: Sieg Scott F. , Bazdar Douglas A. , Harding Clifford V. , Lederman Michael M.
Published: July 2002

Subnormal T-cell production of interleukin-2 (IL-2) in human immunodeficiency virus (HIV) disease has been described; however, it is not clear whether failure to synthesize IL-2 represents a selective or global defect in T-cell cytokine production. We evaluated the intracellular production of gamma interferon (IFN-γ) and IL-2 in CD4+cells that were stimulated with staphylococcal enterotoxin B or cytomegalovirus antigen. Strikingly, IFN-γ and IL-2 are differentially regulated in T cells of HIV-infected patients such that the numbers of CD69+cells or IFN-γ-positive cells that make IL-2 are proportionally decreased in CD4+T cells from HIV-infected patients. These findings demonstrate a selective defect in IL-2 production and suggest that enumeration of IFN-γ-producing cells in response to T-cell receptor stimulation, while providing some estimate of antigen-reactive cell frequency, may not reflect or predict “normal” T-cell function in HIV-infected patients.
Ten-Kilodalton Domain in Ty3 Gag3-Pol3p between PR and RT Is Dispensable for Ty3 Transposition
Authors: Claypool Jonathan A. , Malik Harmit S. , Eickbush Thomas H. , Sandmeyer Suzanne B.
Published: July 2002

Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast
Saccharomyces cerevisiae
. In cells overexpressing Ty3 under the
GAL1
upstream activation sequence, Ty3 RNA, proteins, and DNA are made. Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein. A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains. Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA. These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.
Evolution and Characterization of Tetraonine Endogenous Retrovirus: a New Virus Related to Avian Sarcoma and Leukosis Viruses
Authors: Dimcheff Derek E. , Krishnan Mallika , Mindell David P.
Published: July 2002

In a previous study, we found avian sarcoma and leukosis virus (ASLV)
gag
genes in 19 species of birds in the order Galliformes including all grouse and ptarmigan (Tetraoninae) surveyed. Our data suggested that retroviruses had been transmitted horizontally among some host species. To further investigate these elements, we sequenced a replication-defective retrovirus, here named tetraonine endogenous retrovirus (TERV), from
Bonasa umbellus
(ruffed grouse). This is the first report of a complete, replication-defective ASLV provirus sequence from any bird other than the domestic chicken. We found a replication-defective proviral sequence consisting of putative Gag and Env proteins flanked by long terminal repeats. Reverse transcription-PCR analysis showed that retroviral
gag
sequences closely related to TERV are transcribed, supporting the hypothesis that TERV is an active endogenous retrovirus. Phylogenetic analyses suggest that TERV may have arisen via recombination between different retroviral lineages infecting birds. Southern blotting using
gag
probes showed that TERV occurs in tetraonines but not in chickens or ducks, suggesting that integration occurred after the earliest phasianid divergences but prior to the radiation of tetraonine birds.
A GP64-Null Baculovirus Pseudotyped with Vesicular Stomatitis Virus G Protein
Authors: Mangor J. T. , Monsma S. A. , Johnson M. C. , Blissard G. W.
Published: July 2002

The
Autographa californica
multiple nucleopolyhedrovirus (Ac
M
NPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc
64−
, could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9
VSV-G
) that inducibly expresses the VSV-G protein upon infection with Ac
M
NPV Sf9
VSV-G
and Sf9 cells were infected with vAc
64−
, and cells were monitored for infection and for movement of infection from cell to cell. vAc
64−
formed plaques on Sf9
VSV-G
cells but not on Sf9 cells, and plaques formed on Sf9
VSV-G
cells were observed only after prolonged intervals. Passage and amplification of vAc
64−
on Sf9
VSV-G
cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc
64−
in the G-expressing cells was delayed in comparison to wild-type (wt) Ac
M
NPV, and growth curves showed that pseudotyped vAc
64−
was generated at titers of approximately 10
6
to 10
7
infectious units (IU)/ml, compared with titers of approximately 10
8
IU/ml for wt Ac
M
NPV. Propagation and amplification of pseudotyped vAc
64−
virions in Sf9
VSV-G
cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc
64−
-derived virus that had acquired the G gene through recombination with Sf9
VSV-G
cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.
Antiviral Response in Cells Containing Stat1 with Heterologous Transactivation Domains
Authors: Shen Yuhong , Darnell James E.
Published: July 2002

The STATs (signal transducers and activators of transcription), latent cytoplasmic transcription factors, are activated by binding of extracellular polypeptides to cell surface receptors. Dimerization, accumulation in the nucleus, and transcriptional inductions of specific genes then occur. The COOH terminus of the STATs acts as a transcriptional activation domain (TAD). Stat1, one of seven mammalian STAT genes, forms a homodimer after activation by gamma interferon and induces transcription of a number of genes. These induced genes in turn produce the antiviral state. In the present experiments we used a Stat1-deficient cell line complemented with Stat1 or various fusion constructs in which the wild-type Stat1 TAD was replaced by other TADs to test the possibility that a specific activating domain was necessary for the induction of the antiviral response. We found that a wide variety of TADs with different activation potential appended to the Stat1 COOH terminus could substitute for the wild-type protein in inducing the antiviral state.
Evolutionary Relationships among Parvoviruses: Virus-Host Coevolution among Autonomous Primate Parvoviruses and Links between Adeno-Associated and Avian Parvoviruses
Authors: Lukashov Vladimir V. , Goudsmit Jaap
Published: July 2002

The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family
Parvoviridae
. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary groups of parvoviruses from vertebrates: (i) the human helper-dependent adeno-associated virus (AAV) serotypes 1 to 6 and the autonomous avian parvoviruses; (ii) the bovine, chipmunk, and autonomous primate parvoviruses, including human viruses B19 and V9; and (iii) the parvoviruses from rodents (except for chipmunks), carnivores, and pigs. Each of these three evolutionary groups could be further subdivided, reflecting both virus-host coevolution and multiple cross-species transmissions in the evolutionary history of parvoviruses. No parvoviruses from invertebrates clustered with vertebrate parvoviruses. Our analysis provided evidence for negative selection among parvoviruses, the independent evolution of their genes, and recombination among parvoviruses from rodents. The topology of the phylogenetic tree of autonomous human and simian parvoviruses matched exactly the topology of the primate family tree, as based on the analysis of primate mitochondrial DNA. Viruses belonging to the AAV group were not evolutionarily linked to other primate parvoviruses but were linked to the parvoviruses of birds. The two lineages of human parvoviruses may have resulted from independent ancient zoonotic infections. Our results provide an argument for reclassification of
Parvovirinae
based on evolutionary relationships among viruses.
Linkage between STAT Regulation and Epstein-Barr Virus Gene Expression in Tumors
Authors: Chen Honglin , Lee Jae Myun , Zong Yongsheng , Borowitz Michael , Ng Mun Hon , Ambinder Richard F. , Hayward S. Diane
Published: July 2002

Epstein-Barr virus (EBV) latency gene expression in lymphoblastoid cell lines is regulated by EBNA2. However, the factors regulating viral expression in EBV-associated tumors that do not express EBNA2 are poorly understood. In EBV-associated tumors, EBNA1 and frequently LMP1 are synthesized. We found that an alternative latent membrane protein 1 (LMP1) promoter, L1-TR, located within the terminal repeats is active in both nasopharyngeal carcinoma and Hodgkin's disease tissues. Examination of the L1-TR and the standard ED-L1 LMP1 promoters in electrophoretic mobility shift assays revealed that both promoters contain functional STAT binding sites. Further, both LMP1 promoters responded in reporter assays to activation of JAK-STAT signaling. Cotransfection of JAK1 or v-Src or treatment of cells with the cytokine interleukin-6 upregulated expression from ED-L1 and L1-TR reporter plasmids. Cotransfection of a dominant negative STAT3β revealed that STAT3 is likely to be the biologically relevant STAT for EBNA1 Qp and LMP1 L1-TR promoter regulation. In contrast, LMP1 expression from ED-L1 was not abrogated by STAT3β, indicating that the two LMP1 promoters are regulated by different STAT family members. Taken together with the previous demonstration of JAK-STAT activation of Qp driven EBNA1 expression, this places two of the EBV genes most commonly expressed in tumors under the control of the same signal transduction pathway. Immunohistochemical analyses of nasopharyngeal carcinoma tumors revealed that STAT3, STAT5, and STAT1 are constitutively activated in these tumors while STAT3 is constitutively activated in the malignant cells of Hodgkin's disease. We hypothesize that chronic or aberrant STAT activation may be both a necessary and predisposing event for EBV-driven tumorigenesis in immunocompetent individuals.
Roles of Macrophages in Measles Virus Infection of Genetically Modified Mice
Authors: Roscic-Mrkic Branka , Schwendener Reto A. , Odermatt Bernhard , Zuniga Armando , Pavlovic Jovan , Billeter Martin A. , Cattaneo Roberto
Published: July 2002

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.
RGD Tripeptide of Bluetongue Virus VP7 Protein Is Responsible for Core Attachment to
Culicoides
Cells
Authors: Tan Boon-Huan , Nason Emma , Staeuber Norbert , Jiang Wenrong , Monastryrskaya Katherine , Roy Polly
Published: July 2002

Bluetongue virus (BTV) is an arthropod-borne virus transmitted by
Culicoides
species to vertebrate hosts. The double-capsid virion is infectious for
Culicoides
vector and mammalian cells, while the inner core is infectious for only
Culicoides
-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to
Culicoides
cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the
Culicoides
cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for
Culicoides
cell binding activity.
Binding of LFA-1 (CD11a) to Intercellular Adhesion Molecule 3 (ICAM-3; CD50) and ICAM-2 (CD102) Triggers Transmigration of Human Immunodeficiency Virus Type 1-Infected Monocytes through Mucosal Epithelial Cells
Authors: Carreno Marie-Paule , Chomont Nicolas , Kazatchkine Michel D. , Irinopoulou Theano , Krief Corrine , Mohamed Ali-Si , Andreoletti Laurent , Matta Mathieu , Belec Laurent
Published: July 2002

Transmigration of human immunodeficiency virus (HIV)-infected mononuclear cells through the genital mucosa is one of the possible mechanisms of sexual transmission of HIV. Here, we investigated the transmigration of cell-associated R5-tropic HIV type 1 (HIV-1) through a tight monolayer of human epithelial cells in vitro. We show that this process is dependent on an initial interaction between αLβ2 integrin CD11a/CD18 on infected monocytic cells and intercellular adhesion molecule 2 (ICAM-2; CD102) and ICAM-3 (CD50) on the apical membrane of epithelial cells. The CD50 and CD102 ligands were overexpressed on epithelial cells when the cells were activated by proinflammatory cytokines in the cellular microenvironment. An accumulation of proviral DNA was found in the transmigrated cells, clearly reflecting the preferential transepithelial migration of HIV-1-infected cells under proinflammatory conditions. Our observations provide new insights supporting the hypothesis that HIV-infected mononuclear cells contained in genital secretions from infected individuals may cross the epithelial genital mucosa of an exposed receptive sexual partner, particularly under inflammatory conditions of damaged genital tissue. Understanding the fundamental aspects of the initial HIV entry process during sexual transmission remains a critical step for preventing human infection and developing further vaccinal strategies and virucidal agents.
Molecular Epidemiology of Human Immunodeficiency Virus Type 1 Transmission in a Heterosexual Cohort of Discordant Couples in Zambia
Authors: Trask Stanley A. , Derdeyn Cynthia A. , Fideli Ulgen , Chen Yalu , Meleth Sreelatha , Kasolo Francis , Musonda Rosemary , Hunter Eric , Gao Feng , Allen Susan , Hahn Beatrice H.
Published: July 2002

Most human immunodeficiency virus type 1 (HIV-1) transmissions in sub-Saharan Africa are believed to occur between married adults who are discordant for their HIV-1 infection status; however, no studies to date have investigated the molecular epidemiology of such transmission events. Here we report the genetic characterization of HIV-1 strains from 149 transmission pairs that were identified prospectively in a cohort of discordant couples in Lusaka, Zambia. Subgenomic
gag
, gp120, gp41, and/or long terminal repeat regions were amplified by PCR analysis of uncultured blood samples from both partners and sequenced without interim cloning. Pairwise genetic distances were calculated for the regions analyzed and compared to those of subtype-specific reference sequences as well as local controls. Sequence relationships were also examined by phylogenetic tree analysis. By these approaches, epidemiological linkage was established for the majority of transmission pairs. Viruses from 129 of the 149 couples (87%) were very closely related and clustered together in phylogenetic trees in a statistically highly significant manner. In contrast, viruses from 20 of the 149 couples (13%) were only distantly related in two independent genomic regions, thus ruling out transmission between the two partners. The great majority (95%) of transmitted viruses were of subtype C origin, although representatives of subtypes A, D, G, and J were also identified. There was no evidence for extensive transmission networks within the cohort, although two phylogenetic subclusters of viruses infecting two couples each were identified. Taken together, these data indicate that molecular epidemiological analyses of presumed transmission pairs are both feasible and required to determine behavioral, virological, and immunological correlates of heterosexual transmission in sub-Saharan Africa with a high level of accuracy.
Retrograde Transport of Transmissible Mink Encephalopathy within Descending Motor Tracts
Authors: Bartz Jason C. , Kincaid Anthony E. , Bessen Richard A.
Published: July 2002

The spread of the abnormal conformation of the prion protein, PrPSc, within the spinal cord is central to the pathogenesis of transmissible prion diseases, but the mechanism of transport has not been determined. For this report, the route of transport of the HY strain of transmissible mink encephalopathy (TME), a prion disease of mink, in the central nervous system following unilateral inoculation into the sciatic nerves of Syrian hamsters was investigated. PrPScwas detected at 3 weeks postinfection in the lumbar spinal cord and ascended to the brain at a rate of approximately 3.3 mm per day. At 6 weeks postinfection, PrPScwas detected in the lateral vestibular nucleus and the interposed nucleus of the cerebellum ipsilateral to the site of sciatic nerve inoculation and in the red nucleus contralateral to HY TME inoculation. At 9 weeks postinfection, PrPScwas detected in the contralateral hind limb motor cortex and reticular thalamic nucleus. These patterns of PrPScbrain deposition at various times postinfection were consistent with that of HY TME spread from the sciatic nerve to the lumbar spinal cord followed by transsynaptic spread and retrograde transport to the brain and brain stem along descending spinal tracts (i.e., lateral vestibulospinal, rubrospinal, and corticospinal). The absence of PrPScfrom the spleen suggested that the lymphoreticular system does not play a role in neuroinvasion following sciatic nerve infection. The rapid disease onset following sciatic nerve infection demonstrated that HY TME can spread by retrograde transport along specific descending motor pathways of the spinal cord and, as a result, can initially target brain regions that control vestibular and motor functions. The early clinical symptoms of HY TME infection such as head tremor and ataxia were consistent with neuronal damage to these brain areas.
Intracutaneous DNA Vaccination with the E8 Gene of Cottontail Rabbit Papillomavirus Induces Protective Immunity against Virus Challenge in Rabbits
Authors: Hu Jiafen , Han Ricai , Cladel Nancy M. , Pickel Martin D. , Christensen Neil D.
Published: July 2002

The cottontail rabbit papillomavirus (CRPV)-rabbit model has been used in several studies for testing prophylactic and therapeutic papillomavirus vaccines. Earlier observations had shown that the CRPV nonstructural genes E1, E2, and E6 induced strong to partial protective immunity against CRPV infection. In this study, we found that CRPV E8 immunization eliminated virus-induced papillomas in EIII/JC inbred rabbits (100%) and provided partial protection (55%) against virus challenge in outbred New Zealand White rabbits. CRPV-E8 is a small open reading frame, coding for a 50-amino-acid protein, that is colinear with the CRPV E6 gene and has features similar to those of the bovine papillomavirus and human papillomavirus E5 genes. Papillomas that grew on E8-vaccinated outbred rabbits were significantly smaller than those on vector-vaccinated rabbits (
P
< 0.01;
t
test). Delayed-type hypersensitivity skin tests showed that some of the E8-vaccinated rabbits had positive responses to E8-specific peptides.
A Mutation in the Latency-Related Gene of Bovine Herpesvirus 1 Disrupts the Latency Reactivation Cycle in Calves
Authors: Inman Melissa , Lovato Luciane , Doster Alan , Jones Clinton
Published: July 2002

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated via the ocular or nasal cavity. Following acute infection, the primary site for BHV-1 latency is the sensory neuron. Reactivation from latency occurs sporadically, resulting in virus shedding and transmission to uninfected cattle. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, suggesting that it mediates some aspect of latency. An LR mutant was constructed by inserting three stop codons near the beginning of the LR-RNA, suggesting that expression of LR proteins would be altered. The LR mutant grew with wild-type (wt) efficiency in bovine kidney cells (MDBK). When calves were infected with the LR mutant, a dramatic decrease (3 to 4 logs) in ocular, but not nasal, viral shedding occurred during acute infection relative to the wt or the LR-rescued virus (M. Inman, L. Lovato, A. Doster, and C. Jones, J. Virol.
75:
8507-8515, 2001). In this study, we examined the latency reactivation cycle in calves infected with the LR mutant and compared these results to those from calves infected with wt BHV-1 or the LR-rescued virus. During acute infection, lower levels of infectious virus were detected in trigeminal ganglion homogenates from calves infected with the LR mutant. As judged by in situ hybridization, BHV-1-positive neurons were detected in trigeminal ganglia of calves infected with the wt but not the LR mutant. Although LR-RNA was detected by reverse transcription-PCR in calves latently infected with the LR mutant, a semiquantitative PCR analysis revealed that lower levels of viral DNA were present in trigeminal ganglia of calves infected with the LR mutant. Dexamethasone treatment of calves latently infected with wt BHV-1 or the LR-rescued virus, but not the LR mutant, consistently induced reactivation from latency, as judged by shedding of infectious virus from the nose or eyes and increases in BHV-1-specific antibodies. In summary, this study demonstrates that wt expression of LR gene products plays an important role in the latency reactivation cycle of BHV-1 in cattle.