bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnostic Utility of a Multiplex Herpesvirus PCR Assay Performed with Cerebrospinal Fluid from Human Immunodeficiency Virus-Infected Patients with Neurological Disorders
Authors: Quereda C. , Corral I. , Laguna F. , Valencia M. E. , Tenorio A. , Echeverria J. E. , Navas E. , Martín-Dávila P. , Moreno A. , Moreno V. , Gonzalez-Lahoz J. M. , Arribas J. R. , Guerrero A.
Published: December 2019

We used a multiplex nested-PCR assay for the simultaneous detection in cerebrospinal fluid (CSF) of five human herpesviruses (HVs) (cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], herpes simplex virus [HSV], and human herpesvirus 6 [HHV-6]) in a clinical evaluation of human immunodeficiency virus (HIV)-infected patients with neurological disorders. This method, which has the advantages of being rapid and economical, would be of particular interest for the diagnosis of neurological syndromes caused by more than one HV. We studied 251 CSF samples from 219 patients. HV DNA was demonstrated in 93 (37%) of the CSF samples (34% of the patients). CMV was the HV most frequently detected in our patients (25%), while EBV, VZV, HSV, and HHV-6 DNAs were present in significantly fewer cases (7, 4, 3, and 1%, respectively). When results were compared with the final etiological diagnoses of the patients, the multiplex HV PCR showed high specificity for the diagnosis of CMV and VZV neurological diseases and for cerebral lymphoma (0.95, 0.97, and 0.99, respectively). The sensitivity of the assay was high for CMV disease (0.87), was low for cerebral lymphoma (0.33), and was not evaluable for VZV disease due to the small number of patients with this diagnosis. Nevertheless, detection of VZV DNA had possible diagnostic value in four of the nine cases, and EBV DNA amplification always predicted the diagnosis of cerebral lymphoma in patients with cerebral masses. Detection of HSV DNA was frequently associated with CMV amplification and fatal encephalitis. HHV-6 was not considered to have a pathogenetic role in the three cases in which it was detected. This multiplex HV PCR assay is a specific and clinically useful method for the evaluation of HIV-infected patients with neurological disorders related to HV.
Rapid Detection of
Campylobacter jejuni
in Stool Specimens by an Enzyme Immunoassay and Surveillance for
Campylobacter upsaliensis
in the Greater Salt Lake City Area
Authors: Hindiyeh Musa , Jense Sandra , Hohmann Sheri , Benett Hilary , Edwards Cheryl , Aldeen William , Croft Ann , Daly Judy , Mottice Susan , Carroll Karen C.
Published: December 2019

The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT
Campylobacter
microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 μg/ml), amphotericin B (10 μg/ml), and teicoplanin (4 μg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate
Campylobacter upsaliensis
. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for
C. upsaliensis
in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.
Isolation of
Legionella oakridgensis
from Two Patients with Pleural Effusion Living in the Same Geographical Area
Authors: Lo Presti François , Riffard Serge , Jarraud Sophie , Le Gallou Florence , Richet Hervé , Vandenesch François , Etienne Jerome
Published: December 2019

Two cases of Legionnaire's disease caused by
Legionella oakridgensis
were diagnosed at the university hospital in Nantes, France. The two patients' isolates were identified by means of phenotyping and genotyping methods. Epidemiological investigations concluded that the first case was hospital acquired while the second case was considered community acquired.
Diagnosis of Varicella-Zoster Virus Infections in the Clinical Laboratory by LightCycler PCR
Authors: Espy Mark J. , Teo Rosaline , Ross Teri K. , Svien Kathleen A. , Wold Arlo D. , Uhl James R. , Smith Thomas F.
Published: December 2019

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.
Use of Real-Time PCR and Fluorimetry for Rapid Detection of Rifampin and Isoniazid Resistance-Associated Mutations in Mycobacterium tuberculosis
Authors: Torres Maria J. , Criado Antonio , Palomares Jose C. , Aznar Javier
Published: December 2019

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of therpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3′ labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5′ labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.
Molecular Characterization of Mycobacterium tuberculosis H37Rv/Ra Variants: Distinguishing the Mycobacterial Laboratory Strain
†
Authors: Bifani P. , Moghazeh S. , Shopsin B. , Driscoll J. , Ravikovitch A. , Kreiswirth B. N.
Published: December 2019

The Mycobacterium tuberculosis strains H37Rv and H37Ra are the most commonly used controls for M. tuberculosis identification in the clinical and research laboratory setting. To reduce the likelihood of misidentification and possible cross-contamination with this laboratory neotype, it is important to be able to distinguish H37 from clinical isolates. To provide a reference for identifying H37, we used multiple molecular techniques to characterize H37 strains, including 18 of the most frequently used variants available through the American Type Culture Collection. Isolates were genotyped using gene probes to IS6110 and IS1085. In addition, we performed polymorphic GC-rich sequence typing (PGRS), spoligotyping, determination of variable number of tandem repeats (VNTR), and PCR amplification of the mtp40, msx4, andmpp8 polymorphic regions. Southern hybridization with IS6110 provided the most discrimination, differentiating the 18 H37 isolates into 10 discrete patterns made up of 9 H37Rv variants and 1 H37Ra variant. PGRS, IS1085,mpp8, and spoligotyping were not able to distinguish any H37 variants, while VNTR and msx4 discriminated two. Only IS6110 and spoligotyping could distinguish the H37 strain from clinical isolates. In summary, spoligotyping and IS6110 provide a rapid and accurate way to identify H37 contamination, though IS6110 can, in addition, classify many of the H37 variants that would otherwise require phenotypic segregation.
Cloning of the Rhesus Lymphocryptovirus Viral Capsid Antigen and Epstein-Barr Virus-Encoded Small RNA Homologues and Use in Diagnosis of Acute and Persistent Infections
Authors: Rao Pasupuleti , Jiang Hua , Wang Fred
Published: December 2019

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is associated with the development of several human malignancies. A closely related herpesvirus in the same lymphocryptovirus (LCV) genera as EBV naturally infects rhesus monkeys and provides an important animal model for studying EBV pathogenesis. We cloned the small viral capsid antigen (sVCA) homologue from the rhesus LCV and developed a peptide enzyme-linked immunosorbent assay (ELISA) to determine whether epitopes in the rhesus LCV sVCA are a reliable indicator of rhesus LCV infection. In order to define a “gold standard” for rhesus LCV infection, we also cloned the EBV-encoded small RNA 1 (EBER1) and EBER2 homologues from rhesus LCV and developed a reverse transcription (RT)-PCR assay to detect persistent LCV infection in rhesus monkey peripheral blood lymphocytes. Animals from a conventional and a hand-reared colony were studied to compare the prevalence of rhesus LCV infection in the two groups. There was a 100% correlation between the peptide ELISA and EBER RT-PCR results for rhesus LCV infection. In addition, specificity for LCV infection and exclusion of potential cross-reactivity to the rhesus rhadinovirus sVCA homologue could be demonstrated using sera from experimentally infected animals. These studies establish two novel assays for reliable diagnosis of acute and persistent rhesus LCV infections. The rhesus LCV sVCA peptide ELISA provides a sensitive and reliable assay for routine screening, and these studies of the hand-reared colony confirm the feasibility of raising rhesus LCV-naive animals.
Outbreak of Vancomycin-Resistant Enterococci in a Hospital in Gdańsk, Poland, due to Horizontal Transfer of Different Tn1546-Like Transposon Variants and Clonal Spread of Several Strains
Authors: Kawalec Magdalena , Gniadkowski Marek , Hryniewicz Waleria
Published: December 2019

Twenty-two vancomycin-resistant enterococcal (VRE) isolates of the VanA phenotype (21 Enterococcus faecium isolates and 1 E. faecalis isolate), representative of a large outbreak that occurred in a hospital in Gdańsk, Poland, were studied. All of the isolates demonstrated resistance to a wide variety of other antimicrobial agents in addition to glycopeptides. Several lines of evidence suggested that the outbreak most probably consisted of two epidemics that followed the independent introduction of VanA determinants into two separate hematological wards of the hospital. This hypothesis is supported by the fact that isolates recovered in these wards possessed two different polymorphs of the highly conserved DNA region encompassing the vanRSHAX genes and two distinct polymorph types of Tn1546-like transposons, which contain these genes. According to pulsed-field gel electrophoresis data, the outbreak in the adult hematology ward (HW) was highly polyclonal, which suggested a major role for the horizontal transmission of Tn1546-like elements among nonrelated strains of E. faecium and E. faecalis in this environment. On the other hand, the outbreak in the pediatric hematology ward (PHW) was most probably due to the clonal spread of two epidemic E. faecium strains, which had exchanged a plasmid carrying the Tn1546-like transposon. Restriction fragment length polymorphism studies of transposons and their insertionloci in plasmid DNA have suggested that numerous isolates from both HW and PHW contained two or more copies of Tn1546-like elements that underwent diversification due to various genetic modifications. The reported data demonstrated a very complex epidemiology of the first, and up to now the only, VanA VRE outbreak characterized in Poland.
Distribution of Neisseria meningitidisSerogroup B Serosubtypes and Serotypes Circulating in the United States
Authors: Tondella Maria Lucia C. , Popovic Tanja , Rosenstein Nancy E. , Lake D. B. , Carlone George M. , Mayer Leonard W. , Perkins Bradley A.
Published: December 2019

Because the Neisseria meningitidis serogroup B (NMSB) capsule is poorly immunogenic in humans, immunization strategies have focused on noncapsular antigens. Both PorA and to a lesser extent PorB are noncapsular protein antigens capable of inducing protective bactericidal antibodies, and vaccines based on the outer membrane protein (OMP) components of serogroup B meningococci have been shown to be effective in clinical trials. Multiple PorA antigens seem to be needed to prevent endemic meningococcal disease around the world, and a hexavalent PorA-based meningococcal vaccine has recently been developed in The Netherlands. To evaluate the distribution of NMSB PorA and PorB antigens in the United States, serosubtyping and serotyping were done on 444 NMSB strains isolated in the active surveillance areas of the United States (total population, 32 million) during the period 1992 to 1998. A total of 244 strains were isolated from sporadic cases of meningococcal disease, and 200 strains were isolated from an epidemic in Oregon. A panel of 16 mouse monoclonal antibodies reactive with PorA and 15 monoclonal antibodies reactive with PorB were used. Among the NMSB isolates obtained from sporadic cases, the most prevalent serosubtypes were P1.7,16 (14.3%), P1.19,15 (9.8%), P1.7,1 (8.6%), P1.5,2 (7.8%), P1.22a, 14 (7.8%), and P1.14 (5.3%) and the most prevalent serotypes were 4,7 (27.5%), 15 (16%), 14 (8.6%), 10 (6.1%), 1 (4.9%), and 2a (3.7%). A multivalent PorA-based OMP vaccine aimed at the six most prevalent serosubtypes could have targeted about half of the sporadic cases of NMSB disease that occurred between 1992 and 1998 in the surveillance areas. Twenty serosubtypes would have had to be included in a multivalent vaccine to achieve 80% coverage of strains causing sporadic disease. The relatively large number of isolates that did not react with murine monoclonal antibodies indicates that DNA sequence-based variable region typing of NMSB will be necessary to provide precise information on the distribution and diversity of PorA antigens and correlation with nonserosubtypeable isolates. The high degree of variability observed in the PorA and PorB proteins of NMSB in the United States suggests that vaccine strategies not based on OMPs should be further investigated.
Molecular Taxonomy of the Trichophyton rubrum Complex
Authors: Gräser Y. , Kuijpers A. F. A. , Presber W. , de Hoog G. S.
Published: December 2019

The validity of taxa around Trichophyton rubrum was evaluated by a combination of phenetic and molecular methods. Morphological and physiological features were compared to results of sequencing of the internal transcribed spacer region of the ribosomal operon, PCR fingerprinting, and amplified fragment length polymorphism analysis. The 15 species and varieties investigated (Trichophyton circonvolutum, Trichophyton fischeri,Trichophyton fluviomuniense, Trichophyton glabrum, Trichophyton gourvilii, Trichophyton kanei, Trichophyton kuryangei, Trichophyton megninii, Trichophyton pedis, Trichophyton raubitschekii, Trichophyton rodhaini,Trichophyton rubrum var. nigricans,Trichophyton soudanense, Trichophyton violaceumvar. indicum, and Trichophyton yaoundei) were reclassified or synonymized as T. rubrum or T. violaceum.
Rapid Extraction from and Direct Identification in Clinical Samples of Methicillin-Resistant Staphylococci Using the PCR
Authors: Jaffe Richard I. , Lane Janae D. , Albury Stephen V. , Niemeyer Debra M.
Published: December 2019

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecAgene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134), and coagulase-negativeStaphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.
Detection and Quantitation of Hepatitis C Virus RNA in Feces of Chronically Infected Individuals
Authors: Beld Marcel , Sentjens Roel , Rebers Sjoerd , Weel Jan , Wertheim-van Dillen Pauline , Sol Cees , Boom René
Published: December 2019

Hepatitis C virus (HCV) RNA was detected and quantified in human fecal specimens with the Roche COBAS AMPLICOR system adapted by us for fecal specimens. HCV RNA could be detected in the feces of four of six (67%) patients chronically infected with HCV, with loads up to about 2.8 × 105 copies/ml of feces. The same HCV genotypes were observed in feces and plasma as determined by direct sequencing of the 5′ untranslated region.
Cattle Can Be a Reservoir of Sorbitol-Fermenting Shiga Toxin-Producing Escherichia coli O157:H−Strains and a Source of Human Diseases
Authors: Bielaszewska Martina , Schmidt Herbert , Liesegang Almut , Prager Rita , Rabsch Wolfgang , Tschäpe Helmut , Cízek Alois , Janda Jan , Bláhová Kveta , Karch Helge
Published: December 2019

Using the immunomagnetic separation procedure, we isolated sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H− strains from two patients, one with hemolytic-uremic syndrome and the other with diarrhea, and from a dairy cow epidemiologically associated with the patients. The phenotypic and genotypic characteristics of all isolates were identical or closely related. Moreover, the bovine isolate showed a clonal relatedness to SF STEC O157:H− strains isolated from patients in Germany and the Czech Republic from 1988 to 1998. This is the first evidence that cattle can be a reservoir of SF STEC O157:H− and a source of human diseases.
Comparison of a Ligase Chain Reaction-Based Assay and Cell Culture for Detection of Pharyngeal Carriage ofChlamydia trachomatis
Authors: Winter Andrew J. , Gilleran Gerry , Eastick Kirstine , Ross Jonathan D. C.
Published: December 2019

In 264 genitourinary medicine clinic attenders reporting recent fellatio, the prevalence of pharyngeal Chlamydia trachomatis determined by an expanded standard including cell culture and two in-house PCR tests was 1.5% in 194 women and zero in 70 men. The ligase chain reaction (Abbott LCx) had a specificity of 99.2% and a positive predictive value of 60%.
Continuous Ambulatory Peritoneal Dialysis Peritonitis due to Enterococcus cecorum
Authors: De Baere Thierry , Claeys Geert , Verschraegen Gerda , Devriese Luc A. , Baele Margo , Van Vlem Bruno , Vanholder Raymond , Dequidt Clement , Vaneechoutte Mario
Published: December 2019

Enterococcus cecorum was isolated as the etiologic agent of a continuous ambulatory peritoneal dialysis peritonitis episode in an alcoholic patient. To date, this is only the third infection due to this bacterium, found in the intestinal tract of many domestic animals, that has been reported in humans.
Repression of the A8L Gene, Encoding the Early Transcription Factor 82-Kilodalton Subunit, Inhibits Morphogenesis of Vaccinia Virions
Authors: Hu Xiaolei , Wolffe Elizabeth J. , Weisberg Andrea S. , Carroll Lawrence J. , Moss Bernard
Published: December 2019

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and A8L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particles. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal recombinant vaccinia virus in which the A8L gene is stringently repressed by the
Escherichia coli lac
operator system. The amount of 82-kDa protein synthesized could be regulated by the amount of inducer: from undetectable to higher than normal levels. Virus replication, as determined by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa subunit was not required for viral early, intermediate, and late gene expression or DNA replication. Overexpression of the A8L gene alone, produced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluorescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated in the absence of inducer, indicating that the A8L protein has a role in morphogenesis of the core and subsequent events.
Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 in Immune Cells Develop a Severe AIDS-Like Disease
Authors: Hanna Zaher , Kay Denis G. , Cool Marc , Jothy Serge , Rebai Najet , Jolicoeur Paul
Published: December 2019

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (<1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.
Hepatitis B Virus X Protein Interferes with Cellular DNA Repair
Authors: Becker Sherry A. , Lee Teh-Hsiu , Butel Janet S. , Slagle Betty L.
Published: December 2019

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell’s capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.
Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain
Authors: Herrera Elizabeth , del Mar Lorenzo María , Blasco Rafael , Isaacs Stuart N.
Published: December 2019

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.
Defective Growth Correlates with Reduced Accumulation of a Viral DNA Replication Protein after Low-Multiplicity Infection by a Human Cytomegalovirus
ie1
Mutant
Authors: Greaves Richard F. , Mocarski Edward S.
Published: December 2019

To investigate the importance of the IE1 p72 regulatory protein during human cytomegalovirus replication, a recombinant virus unable to synthesize IE1 p72 was constructed. The Towne strain mutant CR208 lacked exon 4 of the major immediate-early gene and was isolated and complemented in an IE1-expressing immortalized human fibroblast line (ihfie1.3). Replication of CR208 in primary human fibroblasts was completed after an input multiplicity of 10 PFU/cell but was severely impaired at 0.1 PFU/cell. CR208 formed plaques with lower efficiency on primary fibroblasts than on ihfie1.3 cells, and the relationship between the CR208 inoculum size and the resulting number of undersized plaques was nonlinear, indicating that multiple particles of CR208 were required to initiate lytic replication in a single primary fibroblast. After infection of primary fibroblasts with CR208 at 5 PFU/cell, a normal pattern of viral antigens was detected, although IE1 p72 was absent. During lower-multiplicity infections, IE2 protein was consistently detected at similar levels in a similar proportion of CR208-infected cells relative to the case for a Towne infection, but many fewer CR208-infected cells contained the ppUL44 polymerase accessory protein when evaluated at 24 or 48 h after infection. Furthermore, fibroblasts infected with CR208 at a low multiplicity failed to form viral DNA replication compartments, despite having expressed IE2 p86. These low-multiplicity growth and expression defects were corrected in two rescued derivatives of CR208 able to synthesize IE1 p72. One rescued virus (CR249) carried a deletion removing the large intron between exons 1 and 2 of the
ie1-ie2
locus, revealing that this intron was dispensable for growth in cell culture.
Genetic Subtype-Independent Inhibition of Human Immunodeficiency Virus Type 1 Replication by CC and CXC Chemokines
Authors: Trkola Alexandra , Paxton William A. , Monard Simon P. , Hoxie James A. , Siani Michael A. , Thompson Darren A. , Wu Lijun , Mackay Charles R. , Horuk Richard , Moore John P.
Published: December 2019

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1α (MIP-1α), MIP-1β, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1α against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1β, MIP-1α. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1β, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1β, MIP-1α.
Sindbis Virus Induces Apoptosis through a Caspase-Dependent, CrmA-Sensitive Pathway
Authors: Nava Victor E. , Rosen Antony , Veliuona Michael A. , Clem Rollie J. , Levine Beth , Hardwick J. Marie
Published: December 2019

Sindbis virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis virus activates the cell suicide program is not known, we demonstrate here that Sindbis virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis virus-induced cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis virus-induced apoptosis.
Interaction of the Bovine Papillomavirus E6 Protein with the Clathrin Adaptor Complex AP-1
Authors: Tong Xiao , Boll Werner , Kirchhausen Tomas , Howley Peter M.
Published: December 2019

The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the γ subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.
Sequences within the Herpesvirus-Conserved
pac
1 and
pac
2 Motifs Are Required for Cleavage and Packaging of the Murine Cytomegalovirus Genome
Authors: McVoy Michael A. , Nixon Daniel E. , Adler Stuart P. , Mocarski Edward S.
Published: December 2019

The DNA sequence motifs
pac
1 [an A-rich region flanked by poly(C) runs] and
pac
2 (CGCGGCG near an A-rich region) are conserved near herpesvirus genomic termini and are believed to mediate cleavage of genomes from replicative concatemers. To determine their importance in the cleavage process, we constructed a number of recombinant murine cytomegaloviruses with a second cleavage site inserted at an ectopic location within the viral genome. Cleavage at a wild-type ectopic site occurred as frequently as at the natural cleavage site, whereas mutation of this ectopic site revealed that some of the conserved motifs of
pac
1 and
pac
2 were essential for cleavage whereas others were not. Within
pac
1, the left poly(C) region was very important for cleavage and packaging but the A-rich region was not. Within
pac
2, the A-rich region and adjacent sequences were essential for cleavage and packaging and the CGCGGCG region contributed to, but was not strictly essential for, efficient cleavage and packaging. A second A-rich region was not important at all. Furthermore, mutations that prevented cleavage also blocked duplication and deletion of the murine cytomegalovirus 30-bp terminal repeat at the ectopic site, suggesting that repeat duplication and deletion are consequences of cleavage. Given that the processes of genome cleavage and packaging appear to be highly conserved among herpesviruses, these findings should be relevant to other members of this family.
Disease Induction by Virus Derived from Molecular Clones of Equine Infectious Anemia Virus
Authors: Payne Susan L. , Qi Xiao-mei , Shao Hai , Dwyer Amy , Fuller Frederick J.
Published: December 2019

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and
rev
sequences.
Human Herpesvirus 8 Encodes an Interferon Regulatory Factor (IRF) Homolog That Represses IRF-1-Mediated Transcription
Authors: Zimring James C. , Goodbourn Stephen , Offermann Margaret K.
Published: December 2019

Human herpesvirus 8 (HHV-8) is the probable viral etiologic agent for Kaposi’s sarcoma. The HHV-8 genome encodes viral interferon regulatory factor (vIRF), a gene product that has homology to the IRF family of transcription factors. We demonstrate that vIRF inhibits responses to type I and type II interferons and blocks IRF-1-mediated transcription. vIRF does not compete with IRF-1 for binding to DNA or complex directly with IRF-1. The ability of vIRF to block IRF-1-mediated transcription is independent of the DNA binding domains of both vIRF and IRF-1. These data suggest that vIRF may contribute to viral pathogenesis and cellular transformation by interfering with interferon- and IRF-1-mediated gene expression through a novel mechanism.
Exchange of Viral Promoter/Enhancer Elements with Heterologous Regulatory Sequences Generates Targeted Hybrid Long Terminal Repeat Vectors for Gene Therapy of Melanoma
Authors: Diaz R. M. , Eisen T. , Hart I. R. , Vile R. G.
Published: December 2019

To generate transcriptionally targeted vectors, tissue-specific elements of the human tyrosinase promoter were exchanged with corresponding viral elements in the Moloney murine leukemia virus long terminal repeat (LTR). From these experiments, a vesicular stomatitis virus type G pseudotyped, hybrid LTR vector that contained three tyrosinase enhancer elements and gave high-level, tightly tissue-specific expression at high titers (3 × 10
7
CFU/ml) was constructed.
Intracellular Route of Canine Parvovirus Entry
Authors: Vihinen-Ranta Maija , Kalela Anne , Mäkinen Päivi , Kakkola Laura , Marjomäki Varpu , Vuento Matti
Published: December 2019

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.
Minimal Requirement for a Lentivirus Vector Based on Human Immunodeficiency Virus Type 1
Authors: Kim V. Narry , Mitrophanous Kyriacos , Kingsman Susan M. , Kingsman Alan J.
Published: December 2019

The use of human immunodeficiency virus vectors for gene therapy is hampered by concern over their safety. This concern might be ameliorated, in part, if the viral accessory genes and proteins could be eliminated from the vector genomes and particles. Here we describe a minimal vector system that is capable of transducing nondividing cells and which does not contain
tat
,
vif
,
vpr
,
vpu
, and
nef.
The Immediate-Early Gene Product Encoded by Open Reading Frame 57 of Herpesvirus Saimiri Modulates Gene Expression at a Posttranscriptional Level
Authors: Whitehouse Adrian , Cooper Matthew , Meredith David M.
Published: December 2019

The herpesvirus saimiri (HVS) immediate-early gene product encoded by open reading frame (ORF) 57 shares limited amino acid homology with HSV-1 ICP27 and Epstein-Barr virus BMLF1, both regulatory proteins. The ORF 57 gene has been proposed to be spliced based on the genome sequence, and here we confirm the intron-exon structure of the gene. We also demonstrate that a cDNA construct of the ORF 57 gene product represses the transactivating capability of the ORF 50a gene product (which is produced from a spliced transcript), but activates that of ORF 50b (an unspliced transcript). Further analyses with cotransfection experiments show that ORF 57 can either activate or repress expression from a range of both early and late HVS promoters, depending on the target gene. These results indicate that repression of gene expression mediated by the ORF 57 gene product is dependent on the presence of an intron within the target gene encoding region. Furthermore, Northern blot analysis demonstrates that the levels of mRNA transcribed from genes not containing an intron are not significantly affected in the presence of the ORF 57 gene product. This suggests that it regulates gene expression through a posttranscriptional mechanism.
The Role of Herpes Simplex Virus ICP27 in the Regulation of
UL24
Gene Expression by Differential Polyadenylation
Authors: Hann Louane E. , Cook W. James , Uprichard Susan L. , Knipe David M. , Coen Donald M.
Published: December 2019

Herpes simplex virus specifies two sets of transcripts from the
UL24
gene, short transcripts (e.g., 1.4 kb), processed at the
UL24
poly(A) site, and long transcripts (e.g., 5.6 kb), processed at the
UL26
poly(A) site. The 1.4- and 5.6-kb transcripts initiate from the same promoter but are expressed with early and late kinetics, respectively. Measurements of transcript levels following actinomycin D treatment of infected cells revealed that the 1.4- and 5.6-kb
UL24
transcripts have similar stabilities, consistent with
UL24
transcript kinetics being regulated by differential polyadenylation rather than by differential stabilities. Although the
UL24
poly(A) site, which gives rise to short transcripts, is encountered first during processing, long transcripts processed at the
UL26
site are equally or more abundant; thus, operationally, the
UL24
site is weak. Using a series of viral
ICP27
mutants, we investigated whether ICP27, which has been suggested to stimulate the usage of weak poly(A) sites, stimulates 1.4-kb transcript accumulation. We found that accumulation of 1.4-kb transcripts did not require ICP27 during viral infection. Rather, ICP27 was required for full expression of 5.6-kb transcripts, and the decrease in 5.6-kb transcripts relative to 1.4-kb transcripts was not due solely to reduced DNA synthesis. Our results indicate that temporal expression of
UL24
transcripts can be regulated by differential polyadenylation and that although ICP27 is not required for processing at the operationally weak
UL24
poly(A) site, it does modulate 5.6-kb transcript levels at a step subsequent to transcriptional initiation.
Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein
Authors: Plafker Scott M. , Gibson Wade
Published: December 2019

The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499–509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in
cis
to translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in
trans
to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed.
Membrane Fusion Promoted by Increasing Surface Densities of the Paramyxovirus F and HN Proteins: Comparison of Fusion Reactions Mediated by Simian Virus 5 F, Human Parainfluenza Virus Type 3 F, and Influenza Virus HA
Authors: Dutch Rebecca Ellis , Joshi Sangeeta Bagai , Lamb Robert A.
Published: December 2019

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.