bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity and Mechanism of Immunoglobulin M (IgM)-and IgG-Dependent Protective Immunity to LarvalStrongyloides stercoralisinMice
Authors: Ligas Jessica A. , Kerepesi Laura A. , Galioto Ann Marie , Lustigman Sara , Nolan Thomas J. , Schad Gerhard A. , Abraham David
Published: November 2003

Protective immunity in mice to the infective third-stage larvae (L3) ofStrongyloides stercoraliswas shown to be dependent on immunoglobulin M (IgM), complement activation, and granulocytes. The objectives of the present study were to determine whether IgG was also a protective antibody isotype and to define the specificity and the mechanism by which IgG functions. Purified IgG recovered from mice 3 weeks after a booster immunization with live L3 was shown to transfer high levels of protective immunity to naïve mice. IgG transferred into mice treated to block complement activation or to eliminate granulocytes failed to kill the challenge larvae. Transfer of immune IgG into IL-5 knockout (KO) mice, which are deficient in eosinophils, resulted in larval attrition, while transfer into FcRγ KO mice did not result in larval killing. These findings suggest that IgG from mice immunized with live L3 requires complement activation and neutrophils for killing of L3 through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This is in contrast to the results of investigations using IgM from mice immunized with live L3 and IgG from mice immunized with larval antigens soluble in deoxycholate in which protective immunity was shown to be ADCC independent. Western blot analyses with immune IgM and IgG identified few antigens recognized by all protective antibody isotypes. Results from immunoelectron microscopy demonstrated that the protective antibodies bound to different regions in the L3. It was therefore concluded that while IgM and IgG antibodies are both protective against larvalS. stercoralis, they recognize different antigens and utilize different killing mechanisms.
Intestinal Antilectin Immunoglobulin A AntibodyResponse and Immunity to
Entamoeba dispar
Infection followingCure of Amebic LiverAbscess
Authors: Ravdin Jonathan I. , Abd-Alla Mohamed D. , Welles Seth L. , Reddy Selvan , Jackson Terry F. H. G.
Published: November 2003

We
followed 93 subjects with amebic liver abscess (ALA) and 963 close
associate controls at 3-month intervals for 36 months to characterize
intestinal and humoral antibody responses to the amebic
galactose-inhibitable lectin and to determine whether immunity
developed to
Entamoeba histolytica
or
Entamoeba
dispar
infection following cure of ALA. We found that ALA subjects
had a higher prevalence and level of intestinal antilectin
immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant
lectin protein) IgA and IgG antibodies,
P
< 0.01 and
P
< 0.05, respectively, compared to controls. The
intestinal antilectin IgA antibody response was sustained over a longer
time period in ALA subjects (71.8% remained positive at 18
months and 52.6% at 36 months,
P
< 0.001
compared to 17.6% and 10.3% of controls, respectively).
ALA subjects were highly immune to
E. dispar
infection
throughout the study (0% infected at 6 and 36 months, compared
to 6.5% and 4.9% of control subjects, respectively,
P
< 0.05). Upon entry into the study, 6.3% of
ALA subjects were infected with
E. histolytica;
the incidence
of new
E. histolytica
infections in controls (as determined by
culture) was too low (1.4%) to determine whether ALA subjects
exhibited immunity to new infections. We found that stool cultures
every 3 months markedly underestimated the occurrence of new
E.
histolytica
infections, as 15.3% of controls seroconverted
after 12 months of follow-up. Unfortunately, under the field conditions
present in Durban, South Africa, enzyme-linked immunosorbent assay for
detection of lectin antigen in stool yielded unreliable results. In
summary, subjects cured of ALA exhibited sustained mucosal IgA antibody
responses to the amebic galactose-inhibitable lectin and a high level
of immunity to
E. dispar
infection. Determination of immunity
to
E. histolytica
following cure of ALA will require the use
of more sensitive and reliable diagnostic
methods.
Cloning and Expression of the Major SecretedCathepsin B-Like Protein from Juvenile
Fasciola hepatica
andAnalysis of Immunogenicity following Liver FlukeInfection
Authors: Law Ruby H. P. , Smooker Peter M. , Irving James A. , Piedrafita David , Ponting Rebecca , Kennedy Nicholas J. , Whisstock James C. , Pike Robert N. , Spithill Terry W.
Published: November 2003

The
functions of the cathepsin B-like proteases in liver flukes are unknown
and analysis has been hindered by a lack of protein for study, since
the protein is produced in small amounts by juvenile flukes. To
circumvent this, we isolated and characterized a cDNA encoding the
major secreted cathepsin B from
Fasciola hepatica
. The
predicted preproprotein is 339 amino acids in length, with the mature
protease predicted to be 254 amino acids long, and shows significant
similarity to parasite and mammalian cathepsin B. Only one of the two
conserved histidine residues required for cathepsin B exopeptidase
activity is predicted to be present. Recombinant preproprotein was
produced in yeast, and it was shown that the recombinant proprotein can
undergo a degree of self-processing in vitro to the mature form, which
is active against gelatin and synthetic peptide substrates. The
recombinant protein is antigenic in vaccinated rats, and antibodies to
the protein are detected early after infection of rats and sheep with
F. hepatica
. The kinetics of the response to cathepsin B and
cathepsin L after infection of sheep and rats confirm the temporal
expression of these proteins during the life cycle of the
parasite.
Temporal Analysis of
Borrelia burgdorferi
Erp Protein Expression throughout the Mammal-Tick InfectiousCycle
Authors: Miller Jennifer C. , von Lackum Kate , Babb Kelly , McAlister Jason D. , Stevenson Brian
Published: November 2003

Previous
immunological studies indicated that the Lyme disease spirochete,
Borrelia burgdorferi
, expresses Erp outer surface proteins
during mammalian infection. We conducted analyses of Erp expression
throughout the entire tick-mammal infectious cycle, which revealed that
the bacteria regulate Erp production in vivo. Bacteria within unfed
nymphal ticks expressed little to no Erp proteins. However, as infected
ticks fed on mice,
B. burgdorferi
increased production of Erp
proteins, with essentially all transmitted bacteria expressing these
proteins. Mice infected with
B. burgdorferi
mounted rapid IgM
responses to all tested Erp proteins, followed by strong immunoglobulin
G responses that generally increased in intensity throughout 11 months
of infection, suggesting continued exposure of Erp proteins to the host
immune system throughout chronic infection. As naive tick larvae
acquired
B. burgdorferi
by feeding on infected mice,
essentially all transmitted bacteria produced Erp proteins, also
suggestive of continual Erp expression during mammalian infection.
Shortly after the larvae acquired bacteria, Erp production was
drastically downregulated. The expression of Erp proteins on
B.
burgdorferi
throughout mammalian infection is consistent with
their hypothesized function as factor H-binding proteins that protect
the bacteria from host innate immune
responses.
Role of Receptor Proteins for Enterobactin and 2,3-Dihydroxybenzoylserine in Virulence of
Salmonella enterica
Authors: Rabsch W. , Methner U. , Voigt W. , Tschäpe H. , Reissbrodt R. , Williams P. H.
Published: November 2003

Single, double, and triple mutants of an enterobactin-deficient mutant strain of
Salmonella enterica
serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The
fepA
,
iroN
, and
cir
alleles were transduced to enterobactin-proficient strains of
S. enterica
serovar Typhimurium and
S. enterica
serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the
S. enterica
serovar Typhimurium strains indicated that the parental strain and the
fepA iroN
double mutant were similarly virulent but that the
fepA iroN cir
triple mutant was significantly attenuated. Moreover, in mixed infections, the
fepA iroN
mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with
S. enterica
serovar Enteritidis strains also indicated that mutation of the
fepA iroN
genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of
S. enterica
serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the
S. enterica
serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of
S. enterica
.
A Humoral Immune Response Confers Protection againstHaemophilus ducreyiInfection
Authors: Cole Leah E. , Toffer Kristen L. , Fulcher Robert A. , San Mateo Lani R. , Orndorff Paul E. , Kawula Thomas H.
Published: November 2003

Haemophilus ducreyiis the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Neither naturally occurring chancroid nor experimental infection withH. ducreyiresults in protective immunity. Likewise, a single inoculation ofH. ducreyidoes not protect pigs against subsequent infection. Accordingly, we used the swine model of chancroid infection to examine the impact of multiple inoculations on a host's immune response. After three successive inoculations withH. ducreyi, pigs developed a modestly protective immune response evidenced by the decreased recovery of viable bacteria from lesions. All lesions biopsied 2 days after the first and second inoculations contained viableH. ducreyicells, yet only 55% of the lesions biopsied 2 days after the third inoculation did. Nearly 90% of the lesions biopsied 7 days after the first inoculation contained viableH. ducreyicells, but this percentage dropped to only 16% after the third inoculation. Between the first and third inoculations, the average recovery of CFU from lesions decreased approximately 100-fold. The reduced recovery of bacteria corresponded directly with a fivefold increase inH. ducreyi-specific antibody titers and the emergence of bactericidal activity. These immune sera were protective when administered to naïve pigs prior to challenge withH. ducreyi. These data suggest that pigs mount an effective humoral immune response toH. ducreyiafter multiple exposures to the organism.
Monoclonal Antibody MG96 Completely Blocks
Plasmodium yoelii
Developmentin
Anophelesstephensi
Authors: Dinglasan Rhoel R. , Fields Iesha , Shahabuddin Mohammed , Azad Abdu F. , Sacci John B.
Published: November 2003

In
spite of research efforts to develop vaccines against the causative
agent of human malaria,
Plasmodium falciparum
, effective
control remains elusive. The predominant vaccine strategy focuses on
targeting parasite blood stages in the vertebrate host. An alternative
approach has been the development of transmission-blocking vaccines
(TBVs). TBVs target antigens on parasite sexual stages that persist
within the insect vector, anopheline mosquitoes, or target mosquito
midgut proteins that are presumed to mediate parasite development. By
blocking parasite development within the insect vector, TBVs
effectively disrupt transmission and the resultant cascade of secondary
infections. Using a mosquito midgut-specific mouse monoclonal antibody
(MG96), we have partially characterized membrane-bound midgut
glycoproteins in
Anopheles gambiae
and
Anopheles
stephensi
. These proteins are present on the microvilli of midgut
epithelial cells in both blood-fed and unfed mosquitoes, suggesting
that the expression of the protein is not induced as a result of blood
feeding. MG96 exhibits a dose-dependent blocking effect against
Plasmodium yoelii
development in
An. stephensi.
We
achieved 100% blocking of parasite development in the mosquito
midgut. Preliminary deglycosylation assays indicate that the epitope
recognized by MG96 is a complex oligosaccharide. Future investigation
of the carbohydrate epitope as well as gene identification should
provide valuable insight into the possible mechanisms of ookinete
attachment and invasion of mosquito midgut epithelial
cells.
Analysis of Virulence and Inflammatory Potential of
Shigella flexneri
Purine Biosynthesis Mutants
Authors: Cersini Antonella , Martino Maria Celeste , Martini Irene , Rossi Giacomo , Bernardini Maria Lina
Published: November 2003

Several
Shigella flexneri
mutants with defects in aromatic amino acid and/or purine biosynthesis have been evaluated as vaccines in humans or in animal models. To be suitable as a vaccine, a mutant has to show virulence attenuation, minimal reactogenicity, and a good immunogenic potential in animal models. With this aim, we have constructed five
S. flexneri
5 (wild-type strain M90T) mutants with inactivation of one or two of the loci
purEK
,
purHD
, and
guaBA
, governing early or late steps of purine biosynthesis. The mutants have been analyzed in vitro in cell cultures and in vivo in the Sereny test and in the murine pulmonary model of shigellosis. M90T
guaBA
, M90T
guaBA purEK
, M90T
guaBA purHD
, and M90T
purHD purEK
gave a negative result in the Sereny test. In contrast, in the murine pulmonary model all of the strains had the same 50% lethal dose as the wild type, except M90T
guaBA purHD
, which did not result in death of the animals. Nevertheless, bacterial counts in infected lungs, immunohistochemistry, and reverse transcription-PCR analysis of mRNAs for tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), interleukin-1β (IL-1β), IL-6, IL-12, and inducible nitric oxide synthase (iNOS) revealed significant differences among the strains. At 72 h postinfection, M90T
guaBA purHD
still induced proinflammatory cytokines and factors such as IL-1β, IL-6, TNF-α, and iNOS, along with cytokines such as IL-12 and IFN-γ. Moreover, in the absence of evident lesions in murine tissues, this mutant highly stimulated major histocompatibility complex class II expression, showing a significant ability to activate the innate immunity of the host.
Critical Roles for
stx
2
,
eae
, and
tir
in Enterohemorrhagic
Escherichia coli
-Induced Diarrhea and Intestinal Inflammation in Infant Rabbits
Authors: Ritchie Jennifer M. , Thorpe Cheleste M. , Rogers Arlin B. , Waldor Matthew K.
Published: November 2003

Enterohemorrhagic
Escherichia coli
(EHEC) is a group of food-borne pathogens
that can cause diarrhea, colitis, and the hemolytic uremic syndrome
(HUS). The importance of several of the proposed EHEC virulence factors
lacks experimental verification in animal models. The limitations of
current animal models led us to reexamine the infant rabbit model for
the study of EHEC pathogenicity. Here, we report that intragastric
inoculation of a Shiga toxin 2 (Stx2)-producing
E. coli
O157:H7 clinical isolate into infant rabbits led to severe diarrhea and
intestinal inflammation but no signs of HUS. We constructed a set of
isogenic derivatives of this isolate with deletions in several putative
virulence genes, including
stx
2
,
eae
,
tir
, and
ehxA
, to investigate the contribution of
individual virulence factors to EHEC pathogenicity.
stx
2
increased the severity and duration of
EHEC-induced diarrhea. Furthermore, although
stx
2
had no role in EHEC intestinal colonization nor was it required for
EHEC-induced inflammation,
stx
2
altered how the
host responded to EHEC infection by promoting heterophilic infiltration
of the colonic epithelium and lamina propria. Intragastric inoculation
of purified Stx2 also induced inflammation and diarrhea in this model.
Diarrhea and intestinal inflammation were also dependent on EHEC
colonization, as EHEC derivatives with deletions in
eae
and
tir
did not colonize, form attaching and effacing lesions, or
develop clinical signs of disease. Our studies indicate that infant
rabbits are a useful model for investigation of the intestinal stage of
EHEC pathogenesis and suggest that Shiga toxin may play a critical role
in causing diarrhea and inflammation in patients infected with
EHEC.
PneumococcalSurface Protein A Is Expressed In Vivo, and Antibodies to PspA AreEffective for Therapy in a Murine Model of PneumococcalSepsis
Authors: Swiatlo E. , King J. , Nabors G. S. , Mathews B. , Briles D. E.
Published: November 2003

Pneumococcal
surface protein A (PspA) is an immunogenic protein expressed on the
surface of all strains of
Streptococcus pneumoniae
(pneumococcus) and induces antibodies which protect against invasive
infection in mice. Pneumococci used for infectious challenge in
protection studies are typically collected from cultures grown in
semisynthetic medium in vitro. The purpose of these studies is to
confirm that PspA is expressed by pneumococci during growth in vivo at
a level sufficient for antibodies to PspA to be protective. Mice were
actively immunized with purified PspA or by passive transfer of
monoclonal antibody (MAb) and challenged with a capsular type 3 strain
in diluted whole blood from bacteremic mice. All were protected against
challenge with 10 times the 50% lethal dose (LD
50
),
and mice challenged with 1,000 times the LD
50
had increased
survival compared with controls. Additionally, nonimmune mice treated
with MAbs to PspA or PspA immune serum at 6 and 12 h after
infection with 10 times the LD
50
also showed increased
survival. Northern blot analysis of RNA from pneumococci grown either
in vitro or in vivo showed similar levels of PspA mRNA. These results
demonstrate that PspA is expressed in vivo in a mouse model and that
immunization with PspA induces antibodies to an antigen which is
expressed during the course of invasive infection.
Immunotherapy with antibodies to PspA may have some utility in treating
pneumococcal infections in
humans.
CD8+-T-CellResponses of Mycobacterium-Infected Mice to a Newly Identified MajorHistocompatibility Complex Class I-Restricted Epitope Shared byProteins of the ESAT-6Family
Authors: Majlessi Laleh , Rojas Marie-Jésus , Brodin Priscille , Leclerc Claude
Published: November 2003

Here we describe the identification of a new CD8+-T-cell epitope, the GYAGTLQSL nonamer, shared by the TB10.3 and TB10.4 proteins of theMycobacterium tuberculosisESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently recognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were able to recognizeMycobacterium bovisBCG-infected macrophages, were generated and now allow investigation of mycobacterial-antigen processing through the major histocompatibility complex class I pathway.
Inducible Nitric Oxide Synthase Regulates Production of Isoprostanes In Vivo during Chlamydial Genital Infection in Mice
Authors: Ramsey K. H. , Sigar I. M. , Rana S. V. , Gupta J. , Holland S. M. , Byrne G. I. , Morrow J. D.
Published: November 2003

Urinary nitrite and F2-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F2-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F2-isoprostane production and excretion during murine chlamydial genital infection.
High Levels of Susceptibility and T Helper 2 Response in MyD88-Deficient Mice Infected withLeishmania majorAre Interleukin-4 Dependent
Authors: Debus Andrea , Gläsner Joachim , Röllinghoff Martin , Gessner André
Published: November 2003

Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected withLeishmania majorparasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.
Targeting ofAdenovirus via Genetic Modification of the Viral Capsid Combined with aProteinBridge
Authors: Korokhov Nikolay , Mikheeva Galina , Krendelshchikov Alexander , Belousova Natalya , Simonenko Vera , Krendelshchikova Valentina , Pereboev Alexander , Kotov Alexander , Kotova Olga , Triozzi Pierre L. , Aldrich Wayne A. , Douglas Joanne T. , Lo Kin-Ming , Banerjee Papia T. , Gillies Stephen D. , Curiel David T. , Krasnykh Victor
Published: November 2003

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain ofStaphylococcus aureusprotein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.
Role of the Cytoplasmic Domain of the NewcastleDisease Virus Fusion Protein in Association with LipidRafts
Authors: Dolganiuc V. , McGinnes L. , Luna E. J. , Morrison T. G.
Published: November 2003

To
explore the association of the Newcastle disease virus (NDV) fusion (F)
protein with cholesterol-rich membrane domains, its localization in
detergent-resistant membranes (DRMs) in transfected cells was
characterized. After solubilization of cells expressing the F protein
with 1% Triton X-100 at 4°C, ca. 40% of total,
cell-associated F protein fractionated with classical DRMs with
densities of 1.07 to l.14 as defined by flotation into sucrose density
gradients. Association of the F protein with this cell fraction was
unaffected by the cleavage of F
0
to F
1
and
F
2
or by coexpression of the NDV attachment protein, the
hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by
mutation, of potential palmitate addition sites in and near the
F-protein transmembrane domain had no effect on F-protein association
with DRMs. Rather, specific deletions of the cytoplasmic domain of the
F protein eliminated association with classical DRMs. Comparisons of
deletions that affected fusion activity of the protein and deletions
that affected DRM association suggested that there is no direct link
between the cell-cell fusion activity of the F protein and DRM
association. Furthermore, depletion of cholesterol from cells
expressing F and HN protein, while eliminating DRM association, had no
effect on the ability of these cells to fuse with avian red blood
cells. These results suggest that specific localization of the F
protein in cholesterol-rich membrane domains is not required for
cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have
been implicated in virus assembly. The results presented here raise the
possibility that the cytoplasmic domain is important in virus assembly
at least in part because it directs the protein to cholesterol-rich
membrane
domains.
Macrophage-Tropic Simian/Human Immunodeficiency VirusChimeras Use CXCR4, Not CCR5, for Infections of Rhesus MacaquePeripheral Blood Mononuclear Cells and AlveolarMacrophages
Authors: Igarashi Tatsuhiko , Donau Olivia K. , Imamichi Hiromi , Dumaurier Marie-Jeanne , Sadjadpour Reza , Plishka Ronald J. , Buckler-White Alicia , Buckler Charles , Suffredini Anthony F. , Lane H. Clifford , Moore John P. , Martin Malcolm A.
Published: November 2003

After the nearly complete and irreversible depletion of CD4+T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>106viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.
High-Frequency Homologous Recombination betweenBaculoviruses Involves DNAReplication
Authors: Kamita Shizuo George , Maeda Susumu , Hammock Bruce D.
Published: November 2003

We
determined the frequency of DNA recombination between
Bombyx
mori
nucleopolyhedroviruses (BmNPVs) and
between BmNPV and the closely related
Autographa californica
NPV (Ac
M
NPV) in BmN cells, Sf-21 cells, and larvae of
Heliothis virescens
. The BmN cells were coinfected with two
BmNPVs, one with a mutation at the polyhedrin gene (
polh
)
locus and a second carrying a
lacZ
gene marker cassette.
Eleven different BmNPV mutants carrying the
lacZ
gene marker
at various distances (1.4 to 61.7 kb) from
polh
were used for
the coinfections. The Sf-21 cells and larvae of
H. virescens
were coinfected with wild-type Ac
M
NPV and 1 of the 11
lacZ
-marked BmNPV mutants. In BmN cells, high-frequency
recombination was detected as early as 15 h postcoinfection
but not at 12 h postcoinfection. At 18 h
postcoinfection, the mean frequency of recombination ranged between
20.0 and 35.4% when the
polh
and
lacZ
marker
genes were separated by at least 9.7 kb. When these marker genes were
separated by only 1.4 kb, the mean frequency of recombination was
2.7%. In BmN cells, the mean recombination frequency between two
BmNPVs increased only marginally when the multiplicity of infection of
each virus was increased 10-fold. In Sf-21 cells and the larvae of
H. virescens
, the recombination frequency between BmNPV and
Ac
M
NPV was ≤1.0%. Ac
M
NPV DNA
replication occurred normally after the coinfection of Sf-21 cells.
BmNPV DNA replication, however, was not detected, indicating that
normal DNA replication by both viruses is required for high-frequency
recombination.
Evolution of Human Calicivirus RNA In Vivo:Accumulation of Mutations in the Protruding P2 Domain of the CapsidLeads to Structural Changes and Possibly a NewPhenotype
Authors: Nilsson Mikael , Hedlund Kjell-Olof , Thorhagen Margareta , Larson Göran , Johansen Kari , Ekspong Anders , Svensson Lennart
Published: November 2003

In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype.FUT2gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor+, a finding in accordance with the hypothesis ofFUT2gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with theFUT2gene characterization, may lead to a better understanding of calicivirus pathogenesis.
Different Heparan Sulfate Proteoglycans Serve asCellular Receptors for HumanPapillomaviruses
Authors: Shafti-Keramat Saeed , Handisurya Alessandra , Kriehuber Ernst , Meneguzzi Guerrino , Slupetzky Katharina , Kirnbauer Reinhard
Published: November 2003

Papillomaviruses
replicate in stratified epithelia of skin and mucosa. Infection with
certain human papillomavirus (HPV) types is the main cause of
anogenital neoplasia, in particular cervical cancer. Early events of
papillomavirus infectivity are poorly understood. While heparan sulfate
proteoglycans (HSPGs) mediate initial binding to the cell surface, the
class of proteins carrying heparan sulfates has not been defined. Here
we examined two processes of papillomavirus infection, attachment of
virus-like particles (VLP) to cells and infection with authentic HPV
type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major
epithelial form and is strongly upregulated in wound edge
keratinocytes. We employed K562 cells, which lack HSPGs except minor
amounts of endogenous betaglycan, and stable clones that express cDNAs
of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated
with levels of heparan sulfate on the cell surface. Parental K562 bound
HPV16 VLP weakly, whereas all three K562 transfectants demonstrated
enhanced binding, with the highest binding capacity observed for
syndecan-1-transfected cells, which also expressed the most HSPG. For
HPV11 infectivity assays, a high virion inoculum was required to infect
K562 cells, whereas ectopic expression of syndecan-1 increased
permissiveness eightfold and expression of syndecan-4 or glypican-1
fourfold. Infection of keratinocytes was eliminated by treatment with
heparitinase, but not phospholipase C, further implicating the syndecan
family of integral membrane proteins as receptor proteins. Human
keratinocytes with a homozygous deletion of α6 integrin are
permissive for HPV11 infection. These results indicate that several
HSPGs can serve as HPV receptors and support a putative role for
syndecan-1, rather than α6 integrin, as a primary receptor
protein in natural HPV infection of
keratinocytes.
HerpesSimplex Virus 1 Mutant in Which the ICP0 HUL-1 E3 Ubiquitin Ligase SiteIs Disrupted Stabilizes cdc34 but Degrades D-Type Cyclins andExhibits DiminishedNeurotoxicity
Authors: Hagglund Ryan , Roizman Bernard
Published: November 2003

Herpes
simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a
multifunctional protein that functions as a promiscuous transactivator
and promotes the degradation of multiple cellular proteins. In vitro
studies indicated that it encodes two physically separated functional
E3 ubiquitin ligase domains. One, designated herpesvirus ubiquitin
ligase 1 (HUL-1), maps to a region encoded by exon 3 and is contained
between residues 543 and 680. Deletion of amino acids 621 to 625
abolishes this activity. The second, designated HUL-2, maps to the RING
finger domain present in ICP0 encoded by exon 2. Earlier studies have
shown that ICP0 stabilizes cyclins D1 and D3, and several lines of
investigation led to the hypothesis that this function of ICP0 is the
consequence of degradation of the E2 enzyme cdc34, known to be involved
in the proteasome-dependent degradation of D-type cyclins. Consistent
with this hypothesis, we have previously shown that cdc34 physically
interacts with ICP0 at or near aspartate 199 and at amino acids 621 to
625 and that the former site is required for effective ubiquitylation
and degradation of cdc34. Furthermore, the ICP0 HUL-1 domain promotes
the polyubiquitination of cdc34 in vitro. If the mechanism by which
D-type cyclins are salvaged in wild-type-infected cells is dependent on
polyubiquitination and consequent destruction of cdc34, than the mutant
virus R6701, which was constructed for these studies and lacks ICP0
residues 621 to 625, should destabilize the D cyclins and preclude the
degradation of cdc34. We report that ICP0 residues 621 to 625 are
essential for degradation of cdc34 in infected cells and for the
ICP0-mediated stabilization of D-type cyclins, that a mutation that
specifically disrupted the ring finger domain of the HUL-2 site had no
effect on the degradation of cdc34 in infected cells, and that deletion
of ICP0 residues 621 to 625 decreased the replicative capacity of the
virus in growth-arrested but not in dividing cells and resulted in
diminished pathogenicity on intracerebral inoculation of mice. We
conclude that the ICP0 HUL-1 domain acts in infected cells to degrade
cdc34 and that this function requires the interaction of cdc34 with
sequences in exons 2 and 3 but does not involve the HUL-2 RING finger
E3
domain.
Mechanisms Governing Maintenance of Cdk1/Cyclin B1Kinase Activity in Cells Infected with HumanCytomegalovirus
Authors: Sanchez Veronica , McElroy Anita K. , Spector Deborah H.
Published: November 2003

Previous
work has demonstrated dysregulation of key cell cycle components in
human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in
cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti,
E. A. Fortunato, C. L. Clark, J. Corbeil,
D. D. Richman, and D. H. Spector, J.
Virol. 69:6697-6704, 1995). The activation of the mitotic
kinase Cdk1/cyclin B, which was detected as early as 8 h
postinfection (p.i.) and maintained throughout the time course, was
particularly interesting. To understand the mechanisms underlying the
induction of this kinase activity, we have examined the pathways that
regulate the activation of Cdk1/cyclin B1 complexes. The accumulation
of the cyclin B1 subunit in HCMV-infected cells is the result of
increased synthesis and reduced degradation of the protein. In
addition, the catalytic subunit, Cdk1, accumulates in its active form
in virus-infected cells. The decreased level of the
Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due
in part to the down-regulation of the expression and activity of the
Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1
via the proteasome also accounts for its absence at 24 h p.i.
At late times, we observed accumulation of the Cdc25 phosphatases that
remove the inhibitory phosphates from Cdk1. Interestingly, biochemical
fractionation studies revealed that the active form of Cdk1, a fraction
of total cyclin B1, and the Cdc25 phosphatases reside predominantly in
the cytoplasm of infected cells. Collectively, these data suggest that
the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected
cells can be explained by three mechanisms: the accumulation of cyclin
B1, the inactivation of negative regulatory pathways for Cdk1, and the
accumulation of positive factors that promote Cdk1
activity.
Dendritic Cells Harbor Infectious Porcine CircovirusType 2 in the Absence of Apparent Cell Modulation or Replication of theVirus
Authors: Vincent I. E. , Carrasco C. P. , Herrmann B. , Meehan B. M. , Allan G. M. , Summerfield A. , McCullough K. C.
Published: November 2003

Dendritic cells (DCs) play crucial roles in innate and adaptive immune responses, rendering them critical targets for virus infections. Porcine circovirus type 2 (PCV2) is associated with the development of postweaning multisystemic wasting syndrome (PMWS) in piglets. We demonstrate here that 80 to 90% of monocyte-derived and bone marrow-derived DCs interact with PCV2 similar to the early stages of an infection. There was no evidence for virus replication, but the virus did persist in DCs without loss of infectivity nor the induction of cell death. This could reflect an abortive infection, but there was no evidence of virus uncoating—the infectivity remained intact for at least 5 days. Alternatively, the results may reflect DC endocytosis of antigenic material. However, there was no modulation of DC surface major histocompatibility complex class I and class II, CD80/86, CD25, CD16, or CD14. Furthermore, infected DC did not transmit virus to syngeneic T lymphocytes, even when the latter were activated. Such coculture did not induce PCV2 replication or death of the lymphocytes or DCs. These results demonstrate that PCV2 can persist in DCs in the absence of virus replication or degradation. Such a silent virus infection presents a novel mechanism of not only immune evasion but also escaping the DC degradation pathway. Because of their migratory capacity, infection of DCs thus provides a potent vehicle for transport of the virus throughout the host without the need for replication. In addition, the lymphopenia seen in PMWS is not a direct effect of the virus on lymphocytes but would require additional events, as proposed by others.
Complete Genomic Sequence and Comparative Analysis ofthe Tumorigenic Poxvirus Yaba Monkey TumorVirus
Authors: Brunetti Craig R. , Amano Hiroko , Ueda Yoshiaki , Qin Jing , Miyamura Tatsuo , Suzuki Tetsuro , Li Xing , Barrett John W. , McFadden Grant
Published: November 2003

TheYatapoxvirusgenus of poxviruses is comprised ofYaba monkey tumor virus(YMTV),Tanapox virus, andYaba-like disease virus(YLDV), which all have the ability to infect primates, including humans. Unlike other poxviruses, YMTV induces formation of focalized histiocytomas upon infection. To gain a greater understanding of theYatapoxvirusgenus and the unique tumor formation properties of YMTV, we sequenced the 134,721-bp genome of YMTV. The genome of YMTV encodes at least 140 open reading frames, all of which are also found as orthologs in the closely related YLDV. However, 13 open reading frames found in YLDV are completely absent from YMTV. Common to both YLDV and YMTV are the unusually large noncoding regions between many open reading frames. To determine whether any of these noncoding regions might be functionally significant, we carried out a comparative analysis between the putative noncoding regions of YMTV and similar noncoding regions from other poxviruses. This approach identified three new gene poxvirus families, defined as orthologs of YMTV23.5L, YMTV28.5L, and YMTV120.5L, which are highly conserved in virtually all poxvirus species. Furthermore, the comparative analysis also revealed a 40-bp nucleotide sequence at approximately 14,700 bases from the left terminus that was 100% identical in the comparable intergene site within members of theYatapoxvirus,Suipoxvirus, andCapripoxvirusgenera and 95% conserved in theLeporipoxvirusgenus. This conserved sequence was shown to function as a poxvirus late promoter element in transfected and infected cells, but other functions, such as an involvement in viral replication or packaging, cannot be excluded. Finally, we summarize the predicted immunomodulatory protein repertoire in theYatapoxvirusgenus as a whole.
Improved Coreceptor Usage Prediction and GenotypicMonitoring of R5-to-X4 Transition by Motif Analysis of HumanImmunodeficiency Virus Type 1
env
V3 LoopSequences
Authors: Jensen Mark A. , Li Fu-Sheng , van ’t Wout Angélique B. , Nickle David C. , Shriner Daniel , He Hong-Xia , McLaughlin Sherry , Shankarappa Raj , Margolick Joseph B. , Mullins James I.
Published: November 2003

Early
in infection, human immunodeficiency virus type 1 (HIV-1) generally
uses the CCR5 chemokine receptor (along with CD4) for cellular entry.
In many HIV-1-infected individuals, viral genotypic changes arise that
allow the virus to use CXCR4 (either in addition to CCR5 or alone) as
an entry coreceptor. This switch has been associated with an
acceleration of both CD3
+
T-cell decline and
progression to AIDS. While it is well known that the V3 loop of gp120
largely determines coreceptor usage and that positively charged
residues in V3 play an important role, the process of genetic change in
V3 leading to altered coreceptor usage is not well understood. Further,
the methods for biological phenotyping of virus for research or
clinical purposes are laborious, depend on sample availability, and
present biosafety concerns, so reliable methods for sequence-based“
virtual phenotyping” are desirable. We introduce a
simple bioinformatic method of scoring V3 amino acid sequences that
reliably predicts CXCR4 usage (sensitivity, 84%; specificity,
96%). This score (as determined on the basis of
position-specific scoring matrices [PSSM]) can be interpreted
as revealing a propensity to use CXCR4 as follows: known R5 viruses had
low scores, R5X4 viruses had intermediate scores, and X4 viruses had
high scores. Application of the PSSM scoring method to reconstructed
virus phylogenies of 11 longitudinally sampled individuals revealed
that the development of X4 viruses was generally gradual and involved
the accumulation of multiple amino acid changes in V3. We found that X4
viruses were lost in two ways: by the dying off of an established X4
lineage or by mutation back to low-scoring V3
loops.
SequenceAnalysis of Recent H7 Avian Influenza Viruses Associated with ThreeDifferent Outbreaks in Commercial Poultry in the UnitedStates
Authors: Spackman Erica , Senne Dennis A. , Davison Sherrill , Suarez David L.
Published: November 2003

The
hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza
virus (AIV) isolated between 1994 and 2002 from live-bird markets
(LBMs) in the northeastern United States and from three outbreaks in
commercial poultry have been characterized. Phylogenetic analysis of
the HA and NA genes demonstrates that the isolates from commercial
poultry were closely related to the viruses circulating in the LBMs.
Also, since 1994, two distinguishing genetic features have appeared in
this AIV lineage: a deletion of 17 amino acids in the NA protein stalk
region and a deletion of 8 amino acids in the HA1 protein which is
putatively in part of the receptor binding site. Furthermore, analysis
of the HA cleavage site amino acid sequence, a marker for pathogenicity
in chickens and turkeys, shows a progression toward a cleavage site
sequence that fulfills the molecular criteria for highly pathogenic
AIV.
Robust Adenoviral and Adeno-Associated Viral Gene Transfer to the In Vivo Murine Heart
Authors: Champion Hunter C. , Georgakopoulos Dimitrios , Haldar Saptarsi , Wang Lili , Wang Yibin , Kass David A.
Published: November 2003

Background—

Viral gene transfer to the whole heart in vivo has been achieved in several mammalian species but remained difficult to accomplish in murine hearts. We postulated that a key impediment derives from the use of proximal aortic occlusion during virus injection, because this eliminates coronary perfusion gradients in mice as aortic root and left ventricle pressures equalize.

Methods and Results—

Pressure-volume analysis confirmed these mechanics. In contrast, descending aortic occlusion with whole-body cooling (20°C) preserved transmyocardial perfusion gradients and allowed for sustained (>10-minute) dwell times in an upper-body perfusion circuit. This approach yielded robust cardiac transfection with adenovirus (AdV) and adeno-associated virus (AAV) injected into the left ventricle cavity or more simply via a central vein. Cardio-specific expression was achieved with a myocyte-specific promotor. Optimal AdV transfection required 9-minute aortic occlusion, versus 5-minute occlusion for AAV. Using this method, we examined the in vivo function of phospholamban (PLB) by stably transfecting PLB-null mice with AAV encoding PLB (AAV
PLB
). AAV
PLB
restored PLB protein to near control levels that colocalized with SERCA2A in cardiomyocytes. At baseline, PLB-null hearts exhibited enhanced systolic and diastolic function, but frequency-dependent reserve was blunted versus wild-type controls. These properties, particularly the frequency response, returned toward control 3 months after AAV
PLB
transfection.

Conclusions—

The new simplified approach for murine whole-heart viral transfection should assist molecular physiology studies.
Ouabain- and Marinobufagenin-Induced Proliferation of Human Umbilical Vein Smooth Muscle Cells and a Rat Vascular Smooth Muscle Cell Line, A7r5
Authors: Abramowitz Joel , Dai Cuiping , Hirschi Karen K. , Dmitrieva Renata I. , Doris Peter A. , Liu Lijun , Allen Julius C.
Published: November 2003

Background—

We studied the growth-promoting effects of 2 sodium pump–selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5.

Methods and Results—

Both ouabain and marinobufagenin activated proliferation of these cells in a concentration-dependent manner, reflecting the cardiotonic steroid sensitivity of the specific α
1
subunit contained within each cell source. The observed effective concentration ranges of both compounds was below that necessary to induce cytoplasmic ion alterations by sodium pump inhibition.

Conclusions—

These data indicate that the ouabain-activated proliferative effect previously observed in canine VSMCs occurs in other VSMC sources. This growth effect seems to be initiated by drug interaction with the sodium pump, reflected by the affinity of the steroid for the pump, and is independent of altered transmembrane ionic gradients.