bluuebunny/crossref_metadata_2025 huggingface dataset:
Recent High Incidence of Fulminant Hepatitis in Samara, Russia: Molecular Analysis of Prevailing Hepatitis B and D Virus Strains
Authors: Flodgren Erik , Bengtsson Susanne , Knutsson Mikael , Strebkova Elena A. , Kidd Alistair H. , Alexeyev Oleg A. , Kidd-Ljunggren Karin
Published: December 2019

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the δ-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.
Molecular Characterization of Rotavirus in Ireland: Detection of Novel Strains Circulating in the Population
Authors: O'Halloran F. , Lynch M. , Cryan B. , O'Shea H. , Fanning S.
Published: December 2019

A collection of three hundred thirty rotavirus-positive stool samples from children with diarrhea in the southern and eastern regions of Ireland between 1997 and 1999 were submitted to the Molecular Diagnostics Unit of the Cork Institute of Technology, Cork, Ireland, for investigation. These strains were characterized by several methods, including polyacrylamide gel electropherotyping and G and P genotyping. A subset of the G types was confirmed by nucleic acid sequencing. The most prevalent types found in this collection included G1P[8] (n = 106; 32.1%), G2P[4] (n = 94; 28.5%), and G4P[8] (n = 37; 11.2%). Novel strains were also detected, including G1P[4] (n = 19; 5.8%), and G4P[4] (n = 2; 0.6%). Interestingly, mixed infections accounted for 18.8% (n = 62) of the total collection, with only 3% (n = 10) which were not G and/or P typeable. Significantly, six G8 and five G9 strains were identified as part of mixed infections. These strains have not previously been identified in Irish children, suggesting a greater diversity in rotavirus strains currently circulating in Ireland.
Cutaneous Infection Caused by Cylindrocarpon lichenicola in a Patient with Acute Myelogenous Leukemia
Authors: Iwen Peter C. , Tarantolo Stefano R. , Sutton Deanna A. , Rinaldi Michael G. , Hinrichs Steven H.
Published: December 2019

Cylindrocarpon lichenicola is a saprophytic soil fungus which has rarely been associated with human disease. We report the first case of localized invasive cutaneous infection caused by this fungus in a 53-year-old male from the rural midwestern United States with relapsed acute myelogenous leukemia. On admission for induction chemotherapy, the patient was noted to have an abrasive laceration between the fourth and fifth metacarpophalangeal joints and on the dorsum of the right hand, which progressed to frank ulceration following chemotherapy. A biopsy provided an initial diagnosis of an invasive fungal infection consistent with aspergillosis based on the histopathological appearance of the mold in tissue. Multiple positive fungal cultures which were obtained from the biopsied tissue were subsequently identified by microscopic and macroscopic characteristics to be C. lichenicola. The infection resolved following marrow regeneration, aggressive debridement of the affected tissue, and treatment with amphotericin B. This case extends the conditions associated with invasive disease caused by C. lichenicola.
Detection of Human Papillomavirus Type 16 DNA in Consecutive Genital Samples Does Not Always Represent Persistent Infection as Determined by Molecular Variant Analysis
Authors: Mayrand Marie-Hélène , Coutlée François , Hankins Catherine , Lapointe Normand , Forest Pierre , de Ladurantaye Manon , Roger Michel
Published: December 2019

Persistent human papillomavirus (HPV) infection of the uterine cervix is a risk factor for progression to high-grade squamous intraepithelial lesions. Detection in consecutive genital samples of HPV-16 DNA, a frequently encountered HPV type, may represent persistent infection or reinfection. We undertook a study using PCR–single-strand conformation polymorphism (SSCP) analysis and sequencing of PCR products (PCR-sequencing) to determine if consecutive HPV-16-positive samples contained the same HPV-16 variant. Fifty women (36 human immunodeficiency virus [HIV] seropositive, 14 HIV seronegative) had at least two consecutive genital specimens obtained at 6-month intervals that contained HPV-16 DNA as determined by a consensus L1 PCR assay. A total of 144 samples were amplified with two primer pairs for SSCP analysis of the entire long control region. Fifteen different SSCP patterns were identified in our population, while 22 variants were identified by PCR-sequencing. The most frequent SSCP pattern was found in 75 (53%) samples from 27 (54%) women. The SSCP patterns obtained from consecutive specimens were identical for 46 (92%) of 50 women, suggesting persistent infection. Four women exhibited in consecutive specimens different HPV-16 SSCP patterns that were all confirmed by PCR-sequencing. The additional information on the nature of persistent infection provided by molecular variant analysis was useful for 6% of women, since three of the four women who did not have identical consecutive specimens would have been misclassified as having persistent HPV-16 infection on the basis of HPV typing.
Characterization of Actinomyces Isolates from Infected Root Canals of Teeth: Description of Actinomyces radicidentis sp. nov.
Authors: Collins Matthew D. , Hoyles Lesley , Kalfas Sotos , Sundquist Goran , Monsen Tor , Nikolaitchouk Natalia , Falsen Enevold
Published: December 2019

Two strains of a previously undescribedActinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces andArcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain ofActinomyces radicidentis is CCUG 36733.
Distribution of Rotavirus VP7 Serotypes and VP4 Genotypes Circulating in Sousse, Tunisia, from 1995 to 1999: Emergence of Natural Human Reassortants
Authors: Trabelsi Abdelhalim , Peenze Ina , Pager Cara , Jeddi M. , Steele Duncan
Published: December 2019

Rotavirus strains circulating in Sousse, Tunisia, between 1995 and 1999 were characterized antigenically by monoclonal antibodies to the VP6 subgroup and the VP7 serotype. The VP4 genotype was determined by reverse transcription-PCR, as were the strains with untyped VP7. Only 17% of 375 children were shedding rotavirus as determined by latex agglutination assay. Most rotavirus strains were G1P[8] (50%), followed by G4P[8] and G4P[6]. Reassortant G1P[4] strains emerged in Sousse during the 1998–1999 season.
Hospital-Based Evaluation of Two Rapid Human Immunodeficiency Virus Antibody Screening Tests
Authors: Kannangai Rajesh , Ramalingam Sandeep , Pradeepkumar Selvaraj , Damodharan Kannan , Sridharan Gopalan
Published: December 2019

Two rapid human immunodeficiency virus (HIV) screening assays, HIV TRI-DOT and HIV-SPOT were compared with standard enzyme-linked immunosorbent assays according to a testing algorithm. Sensitivities and specificities in the real-time evaluation were 99.5 and 99.9% for TRI-DOT and 98.2 and 99.7% for HIV-SPOT, respectively. These two tests are suitable for use where facilities and laboratory expertise are limited.
Brevibacterium casei Sepsis in an 18-Year-Old Female with AIDS
Authors: Brazzola P. , Zbinden R. , Rudin C. , Schaad U. B. , Heininger U.
Published: December 2019

Brevibacterium sp. was isolated from the blood of an acutely ill 18-year-old female with AIDS. The isolate was identified asBrevibacterium casei by use of carbohydrate assimilation tests. Treatment was successful with intravenously administered ciprofloxacin. To our knowledge, this is the first report of sepsis caused by B. casei in a human immunodeficiency virus-infected patient.
A Lymph Node-Derived Cytopathic Simian Immunodeficiency Virus Mne Variant Replicates in Nonstimulated Peripheral Blood Mononuclear Cells
Authors: Kimata Jason T. , Mozaffarian Afsaneh , Overbaugh Julie
Published: December 2019

Lymph nodes (LNs) are sites of active human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) replication and disease at both early and late stages of infection. Consequently, variant viruses that replicate efficiently and subsequently cause immune dysfunction may be harbored in this tissue. To determine whether LN-associated SIVs have an increased capacity to replicate and induce cytopathology, a molecular clone of SIV was isolated directly from DNA extracted from unpassaged LN tissue of a pig-tailed macaque (Macaca nemestrina) infected with SIVMne. The animal had declining CD4+T-lymphocyte counts at the time of the LN biopsy. In human CD4+T-cell lines, the LN-derived virus, SIVMne027, replicated with relatively slow kinetics and was minimally cytopathic and non-syncytium inducing compared to other SIVMne clones. However, in phytohemagglutinin-stimulated pig-tailed macaque peripheral blood mononuclear cells (PBMCs), SIVMne027 replicated efficiently and was highly cytopathic for the CD4+T-cell population. Interestingly, unlike other SIVMne clones, SIVMne027 also replicated to a high level in nonstimulated macaque PBMCs. High-level replication depended on the presence of both the T-cell and monocyte/macrophage populations and could be enhanced by interleukin-2 (IL-2). Finally, the primary determinant governing the ability of SIVMne027 to replicate in nonstimulated and IL-2-stimulated PBMCs mapped togag-pol-vif. Together, these data demonstrate that LNs may harbor non-syncytium-inducing, cytopathic viruses that replicate efficiently and are highly responsive to the effects of cytokines such as IL-2.
Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding
Authors: Dudas Kathleen C. , Ruyechan William T.
Published: December 2019

We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation of 1 mol of FM per mol of ICP8 have been established. The binding properties of the modified protein were analyzed by polyacrylamide gel shift analysis with model oligonucleotides. This analysis indicates that while intrinsic binding is similar to that observed with unmodified protein, the cooperative binding of the modified protein to single-stranded DNA is significantly altered. Helix-destabilizing assays, whose results are a reflection of cooperative binding, also indicate that this property of ICP8 is decreased upon modification with FM. Mapping of the site of modification by cyanogen bromide cleavage and peptide sequencing has shown that the major site of modification is cysteine 254. This position in the primary structure of ICP8 is distinct from the regions previously shown to be involved in the interaction of this protein with single-stranded DNA.
Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2
Authors: Rutledge Elizabeth A. , Halbert Christine L. , Russell David W.
Published: December 2019

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.
Characterization of Replication-Competent Hepatitis A Virus Constructs Containing Insertions at the N Terminus of the Polyprotein
Authors: Zhang Yuan , Kaplan Gerardo G.
Published: December 2019

To determine whether hepatitis A virus (HAV) could tolerate the insertion of exogenous sequences, we constructed full-length HAV cDNAs containing in-frame insertions at the N terminus of the polyprotein and transfected the derived T7 RNA polymerase in vitro transcripts into FRhK-4 cells. Replication of HAVvec1, a construct containing an insertion of 60 nucleotides coding for a polylinker, a 2B/2C cleavage site for HAV protease 3C
pro
, and two initiation codons that restored the sequence of the N terminus of the polyprotein, was detected 2 weeks after transfection by indirect immunofluorescence analysis using anti-HAV monoclonal antibodies. Western blot analysis of HAVvec1-infected cells using anti-VP2 and anti-VP4 antibodies failed to detect the expression of the inserted sequences. Insertion of a 24-mer oligonucleotide coding for a FLAG epitope into HAVvec1 resulted in its HAV-mediated expression which was retained upon deletion of a Gln residue from the inserted 2B/2C cleavage site. Western blot analysis using anti-FLAG and anti-VP2 antibodies showed that the FLAG epitope accumulated in infected cells fused to VP0. Replacement of the FLAG epitope with an epitope of the circumsporozoite protein (CSP) of
Plasmodium falciparum
resulted in its stable HAV-mediated expression for at least six serial passages in FRhK-4 cells. Sedimentation analysis in sucrose density gradients showed that the CSP epitope accumulated in infected cells fused to VP0, forming 80S empty capsids which also contained native VP0. Our data suggest that the HAV internal ribosome entry site can efficiently direct dual initiation of translation of the polyprotein from AUG codons separated by 66 to 78 nucleotides and show that HAV can tolerate insertions at the N terminus of the polyprotein.
Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus
Authors: Meulenberg J. J. M. , Bos-de Ruijter J. N. A. , van de Graaf R. , Wensvoort G. , Moormann R. J. M.
Published: December 2019

The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique
Pac
I restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the
Pac
I site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.
Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis
Authors: Jerome Keith R. , Tait Jonathan F. , Koelle David M. , Corey Lawrence
Published: December 2019

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4+CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.
71-Kilodalton Heat Shock Cognate Protein Acts as a Cellular Receptor for Syncytium Formation Induced by Human T-Cell Lymphotropic Virus Type 1
Authors: Sagara Yasuko , Ishida Chuzo , Inoue Yukiko , Shiraki Hiroshi , Maeda Yoshiaki
Published: December 2019

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and β-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-β-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.
Mutagenesis of the NS3 Protease of Dengue Virus Type 2
Authors: Valle Rosaura P. C. , Falgout Barry
Published: December 2019

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.
Human Rhinovirus Type 14:Human Immunodeficiency Virus Type 1 (HIV-1) V3 Loop Chimeras from a Combinatorial Library Induce Potent Neutralizing Antibody Responses against HIV-1
Authors: Smith Allen D. , Geisler Sheila C. , Chen Anne A. , Resnick Dawn A. , Roy Birgit M. , Lewi Paul J. , Arnold Edward , Arnold Gail Ferstandig
Published: December 2019

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.
Extent of Antigenic Diversity in the V3 Region of the Surface Glycoprotein, gp120, of Human Immunodeficiency Virus Type 1 Group M and Consequences for Serotyping
Authors: Plantier Jean-Christophe , Le Pogam Sophie , Poisson Francis , Buzelay Laurence , Lejeune Bernard , Barin Francis
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) may be studied by molecular or immunological approaches. Most analyses have been performed by genetic comparison of isolates and have led to the definition of clades or subtypes within the major (M) group of HIV-1. Five subtypes (A to E) were initially identified by comparison of genomic sequences. Four new subtypes (F to I) were identified more recently. Amino acid differences in the immunogenic V3 loop between isolates have also been studied, leading to a phenetic classification of at least 14 clusters (1 to 14) of sequences (B. T. M. Korber, K. McInnes, R. F. Smith, and G. Myers, J. Virol. 68:6730–6744, 1994). In this study, we compared the antigenicity of the V3 consensus sequences defined by phylogenetic analysis to the antigenicity of those defined by phenetic analysis. We used a recently developed subtype-specific enzyme immunoassay (SSEIA) that uses the principle of blocking with an excess of peptide in the liquid phase. Two SSEIAs were performed, the first with five V3 sequences defined by phylogenetic analysis and the second with 14 V3 sequences defined by phenetic analysis. A total of 168 HIV-1 sera taken from seropositive individuals from seven different countries or regions were studied. Experimental and statistical data, including correlation matrix and cluster analyses, demonstrated associations between the genetic subtypes and phenetically associated groups. Most of these were predicted by Korber et al. (J. Virol. 68:6730–6744, 1994) by theoretical analysis. We also found that V3 sequences can be grouped into between three and five antigenically unrelated categories. Residues that may be responsible for major antigenic differences were identified at the apex of the V3 loop, within the octapeptide xIGPGxxx, where x represents the critical positions. Our study provides evidence that there is a limited number of V3 serotypes which could be easily monitored by serological assays to study the diversity and dynamics of HIV-1 strains.
Prophylactic Administration of a Complementarity-Determining Region Derived from a Neutralizing Monoclonal Antibody Is Effective against Respiratory Syncytial Virus Infection in BALB/c Mice
Authors: Bourgeois C. , Bour J. B. , Aho L. S. , Pothier P.
Published: December 2019

Immunotherapy with antibodies against respiratory syncytial virus (RSV) is a treatment option given the absence of any vaccine or other available satisfactory treatment. We selected one of our monoclonal antibodies, RS-348, that is highly neutralizing. We showed that a single peptide (PEP3H) derived from complementarity-determining region 3 (CDR3) of its heavy chain was capable of neutralizing the virus
in vitro
. When intranasally administered 24 h before challenge, this peptide protected BALB/c mice against RSV lung infection. These results indicate that a single CDR can be effective against RSV infection.
A Moloney Murine Leukemia Virus-Based Retroviral Vector Pseudotyped by the Insect Retroviral
gypsy
Envelope Can Infect
Drosophila
Cells
Authors: Teysset Laure , Burns Jane C. , Shike Hiroko , Sullivan Barbara L. , Bucheton Alain , Terzian Christophe
Published: December 2019

The
gypsy
element of
Drosophila melanogaster
is the first retrovirus identified so far in invertebrates. Previous data suggest that
gypsy
ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp70lucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the
gypsy
ENV-like protein. We have shown by immunostaining that the
gypsy
envelope protein is produced in 293GP/LNhsp70lucL.3 cells and that vector particles collected from these cells can infect
Drosophila
cells. Our results provide direct evidence that the infectious property of
gypsy
is due to its ORF3 gene product.
Glycoproteins gB, gD, and gHgL of Herpes Simplex Virus Type 1 Are Necessary and Sufficient To Mediate Membrane Fusion in a Cos Cell Transfection System
Authors: Turner Angelina , Bruun Birgitte , Minson Tony , Browne Helena
Published: December 2019

Herpes simplex virus type 1 glycoproteins gB, gD, and gHgL were expressed by transient transfection of Cos cells. Polykaryocyte formation above the background level seen in untransfected controls was observed only if all three components were expressed. Thus, gB, gD, and gHgL are necessary and sufficient to induce membrane fusion.
The Secondary Structure of the R Region of a Murine Leukemia Virus Is Important for Stimulation of Long Terminal Repeat-Driven Gene Expression
Authors: Cupelli Lisa , Okenquist Sharon A. , Trubetskoy Alla , Lenz Jack
Published: December 2019

In addition to their role in reverse transcription, the R-region sequences of some retroviruses affect viral transcription. The first 28 nucleotides of the R region within the long terminal repeat (LTR) of the murine type C retrovirus SL3 were predicted to form a stem-loop structure. We tested whether this structure affected the transcriptional activity of the viral LTR. Mutations that altered either side of the stem and thus disrupted base pairing were generated. These decreased the level of expression of a reporter gene under the control of viral LTR sequences about 5-fold in transient expression assays and 10-fold in cells stably transformed with the LTR-reporter plasmids. We also generated a compensatory mutant in which both the ascending and descending sides of the stem were mutated such that the nucleotide sequence was different but the predicted secondary structure was maintained. Most of the activity of the wild-type SL3 element was restored in this mutant. Thus, the stem-loop structure was important for the maximum activity of the SL3 LTR. Primer extension analysis indicated that the stem-loop structure affected the levels of cytoplasmic RNA. Nuclear run-on assays indicated that deletion of the R region had a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Thus, the main effect of the R-region element was on one or more steps that occurred after the template was transcribed by RNA polymerase. This finding implied that the main function of the R-region element involved RNA processing. R-region sequences of human immunodeficiency virus type 1 or mouse mammary tumor virus could not replace the SL3 element. R-region sequences from an avian reticuloendotheliosis virus partially substituted for the SL3 sequences. R-region sequences from Moloney murine leukemia virus or feline leukemia virus did function in place of the SL3 element. Thus, the R region element appears to be a general feature of the mammalian type C genus of retroviruses.
Dendritic Cells Route Human Immunodeficiency Virus to Lymph Nodes after Vaginal or Intravenous Administration to Mice
Authors: Masurier Carole , Salomon Benoît , Guettari Nadia , Pioche Catherine , Lachapelle François , Guigon Martine , Klatzmann David
Published: December 2019

We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.
Serp2, an Inhibitor of the Interleukin-1β-Converting Enzyme, Is Critical in the Pathobiology of Myxoma Virus
Authors: Messud-Petit Frederique , Gelfi Jacqueline , Delverdier Maxence , Amardeilh Marie-France , Py Robert , Sutter Gerd , Bertagnoli Stephane
Published: December 2019

Recently, myxoma virus was shown to encode an additional member of the serpin superfamily. The viral gene, calledserp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1β (IL-1β)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1β by the protease (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860–5866, 1996). Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit. A Serp2 mutant myxoma virus was constructed by disruption of the single-copyserp2gene and insertion of theEscherichia coli gptgene serving as the selectable marker. A revertant virus was obtained by replacing theE. coli gptgene by the intactserp2open reading frame. The Serp2−mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4+T-cell line (RL5). Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2−mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo. After the infection of European rabbits, the Serp2−mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals. Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level. In contrast, rapid inflammatory reactions occurred upon infection with the Serp2−mutant virus. Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis. We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes. The weakening of host defense is directly linked toserp2gene function and is likely to involve the inhibition of IL-1β-converting-enzyme-dependent pathways.
Importance of the Positive-Strand RNA Secondary Structure of a Murine Coronavirus Defective Interfering RNA Internal Replication Signal in Positive-Strand RNA Synthesis
Authors: Repass John F. , Makino Shinji
Published: December 2019

The RNA elements that are required for replication of defective interfering (DI) RNA of the JHM strain of mouse hepatitis virus (MHV) consist of three discontinuous genomic regions: about 0.46 to 0.47 kb from both terminal sequences and an internal 58-nucleotide (nt)-long sequence (58-nt region) present at about 0.9 kb from the 5′ end of the DI genome. The internal region is important for positive-strand DI RNA synthesis (Y. N. Kim and S. Makino, J. Virol. 69:4963–4971, 1995). We further characterized the 58-nt region in the present study and obtained the following results. (i) The positive-strand RNA structure in solution was comparable with that predicted by computer modeling. (ii) Positive-strand RNA secondary structure, but not negative-strand RNA structure, was important for the biological function of the region. (iii) The biological function had a sequence-specific requirement. We discuss possible mechanisms by which the internal
cis
-acting signal drives MHV positive-strand DI RNA synthesis.
Establishment and Characterization of Japanese Encephalitis Virus-Specific, Human CD4
+
T-Cell Clones: Flavivirus Cross-Reactivity, Protein Recognition, and Cytotoxic Activity
Authors: Aihara Hirokuni , Takasaki Tomohiko , Matsutani Takaji , Suzuki Ryuji , Kurane Ichiro
Published: December 2019

We analyzed the CD4
+
T-lymphocyte responses of two donors who had received Japanese encephalitis virus (JEV) vaccine 6 or 12 months earlier. Bulk culture proliferation assays showed that peripheral blood mononuclear cells (PBMC) responded to JEV antigens (Ag) but also responded at lower levels to West Nile virus (WNV) and dengue virus type 1, 2, and 4 (D1V, D2V, and D4V, respectively) Ag. Five JEV-specific CD4
+
human T-cell clones and one subclone were established from PBMC of these two donors. Two clones responded to WNV Ag as well as to JEV Ag, whereas the others responded only to JEV Ag. Three of five CD4
+
T-cell clones had JEV-specific cytotoxic activity and recognized E protein. The HLA restriction of the JEV-specific T-cell clones was examined. Three clones were HLA-DR4 restricted, one was HLA-DQ3 restricted, and the HLA restriction of one clone was not determined. T-cell receptor analysis showed that these clones expressed different T-cell receptors, suggesting that they originated from different T lymphocytes. These results indicate that JEV vaccine induces JEV-specific and flavivirus-cross-reactive CD4
+
T lymphocytes and that these T lymphocytes recognize E protein. The functions and HLA restriction patterns of these T lymphocytes are, however, heterogeneous.
Molecular and Cellular Analysis of Human Immunodeficiency Virus-Induced Apoptosis in Lymphoblastoid T-Cell-Line-Expressing Wild-Type and Mutated CD4 Receptors
Authors: Moutouh Laure , Estaquier Jérôme , Richman Douglas D. , Corbeil Jacques
Published: December 2019

We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39–48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56lck(mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56lckor NF-κB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56lcksignaling, and may involve a critical region for CD4 dimerization.
In Vivo Selection of Rous Sarcoma Virus Mutants with Randomized Sequences in the Packaging Signal
Authors: Doria-Rose Nicole A. , Vogt Volker M.
Published: December 2019

Retrovirus genomes contain a sequence at the 5′ end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Ψ, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Ψ and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Ψ were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.
Avian Hepatitis B Virus Infection Is Initiated by the Interaction of a Distinct Pre-S Subdomain with the Cellular Receptor gp180
Authors: Urban Stephan , Breiner Klaus M. , Fehler Frank , Klingmüller Ursula , Schaller Heinz
Published: December 2019

Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay. Recombinant DHBV pre-S polypeptides, produced in
Escherichia coli
, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction. Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding. An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition. Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes. These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.
Cleavage Susceptibility of Reovirus Attachment Protein ς1 during Proteolytic Disassembly of Virions Is Determined by a Sequence Polymorphism in the ς1 Neck
Authors: Chappell James D. , Barton Erik S. , Smith Trent H. , Baer Geoffrey S. , Duong David T. , Nibert Max L. , Dermody Terence S.
Published: December 2019

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ς1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ς1 cleavage upon conversion of virions to ISVPs. We examined the ς1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ς1 cleavage. The ς1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ς1 proteins of the remaining strains. Thr
249
occupies the
d
position of a heptad repeat motif predicted to stabilize ς1 oligomers through α-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ς1 known as the neck. Substitution of Thr
249
with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ς1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ς1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ς1 after Arg
245
. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ς1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ς1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.