bluuebunny/crossref_metadata_2025_split huggingface dataset:
Safety and Immunogenicity of an Intranasal Sendai Virus-Based Human Parainfluenza Virus Type 1 Vaccine in 3- to 6-Year-Old Children
Authors: Adderson Elisabeth , Branum Kristen , Sealy Robert E. , Jones Bart G. , Surman Sherri L. , Penkert Rhiannon , Freiden Pamela , Slobod Karen S. , Gaur Aditya H. , Hayden Randall T. , Allison Kim , Howlett Nanna , Utech Jill , Allay Jim , Knight James , Sleep Susan , Meagher Michael M. , Russell Charles J. , Portner Allen , Hurwitz Julia L. 
Published: January 2015

ABSTRACTHuman parainfluenza virus type 1 (hPIV-1) is the most common cause of laryngotracheobronchitis (croup), resulting in tens of thousands of hospitalizations each year in the United States alone. No licensed vaccine is yet available. We have developed murine PIV-1 (Sendai virus [SeV]) as a live Jennerian vaccine for hPIV-1. Here, we describe vaccine testing in healthy 3- to 6-year-old hPIV-1-seropositive children in a dose escalation study. One dose of the vaccine (5 × 105, 5 × 106, or 5 × 10750% egg infectious doses) was delivered by the intranasal route to each study participant. The vaccine was well tolerated by all the study participants. There was no sign of vaccine virus replication in the airway in any participant. Most children exhibited an increase in antibody binding and neutralizing responses toward hPIV-1 within 4 weeks from the time of vaccination. In several children, antibody responses remained above incoming levels for at least 6 months after vaccination. Data suggest that SeV may provide a benefit to 3- to 6-year-old children, even when vaccine recipients have preexisting cross-reactive antibodies due to previous exposures to hPIV-1. Results encourage the testing of SeV administration in young seronegative children to protect against the serious respiratory tract diseases caused by hPIV-1 infections.
Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR
Authors: Edouard Sophie , Prudent Elsa , Gautret Philippe , Memish Ziad A. , Raoult Didier 
Published: January 2015

ABSTRACT
We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.
Four Carbapenem-Resistant Gram-Negative Species Carrying Distinct Carbapenemases in a Single Patient
Authors: Ding Baixing , Hu Fupin , Yang Yang , Guo Qinglan , Huang Jinwei , Wang Minggui 
Published: January 2015

ABSTRACT

Carbapenem-resistant
Escherichia coli
,
Klebsiella pneumoniae
,
Enterobacter aerogenes
, and
Acinetobacter baumannii
were isolated from a single patient, each producing different carbapenemases (NDM-1, KPC-2, IMP, and OXA-23, respectively). The NDM-1-producing
E. coli
strain was preceded by a clonally related carbapenem-susceptible strain a month earlier, suggesting
in vivo
acquisition of
bla
NDM-1
.
The Herpes Simplex Virus 1 UL51 Protein Interacts with the UL7 Protein and Plays a Role in Its Recruitment into the Virion
Authors: Roller Richard J. , Fetters Rachel 
Published: January 2015

ABSTRACT
The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions.

IMPORTANCE
The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion.
Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein
Authors: Earley Lauriel F. , Kawano Yasuhiro , Adachi Kei , Sun Xiao-Xin , Dai Mu-Shui , Nakai Hiroyuki 
Published: January 2015

ABSTRACT

Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2
144–184
). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2
144–184
has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.

IMPORTANCE
Adeno-associated virus (AAV) has become a popular and successful vector for
in vivo
gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.
Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2
Authors: Utt Age , Das Pratyush Kumar , Varjak Margus , Lulla Valeria , Lulla Aleksei , Merits Andres 
Published: January 2015

ABSTRACT

Chikungunya virus (CHIKV) (genus
Alphavirus
) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels.

IMPORTANCE
CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.
Impact of Natural IgM Concentration on Gene Therapy with Adenovirus Type 5 Vectors
Authors: Qiu Qi , Xu Zhili , Tian Jie , Moitra Rituparna , Gunti Sreenivasulu , Notkins Abner L. , Byrnes Andrew P. 
Published: January 2015

ABSTRACTNatural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentrationin vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.
Differential Effects of the G118R, H51Y, and E138K Resistance Substitutions in Different Subtypes of HIV Integrase
Authors: Quashie Peter K. , Oliviera Maureen , Veres Tamar , Osman Nathan , Han Ying-Shan , Hassounah Said , Lie Yolanda , Huang Wei , Mesplède Thibault , Wainberg Mark A. 
Published: January 2015

ABSTRACT
Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients.

IMPORTANCE
Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how “minor” polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3′ processing indicates the importance of the 3′ processing step and highlights a therapeutic angle that needs to be better exploited.
Ebola Virus and Severe Acute Respiratory Syndrome Coronavirus Display Late Cell Entry Kinetics: Evidence that Transport to NPC1
+
Endolysosomes Is a Rate-Defining Step
Authors: Mingo Rebecca M. , Simmons James A. , Shoemaker Charles J. , Nelson Elizabeth A. , Schornberg Kathryn L. , D'Souza Ryan S. , Casanova James E. , White Judith M. 
Published: January 2015

ABSTRACT

Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1
+
LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity.

IMPORTANCE
Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1
+
LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1
+
LE/Lys, as a therapeutic target for SARS and EBOV.
Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment
Authors: Xie Maorong , Xuan Baoqin , Shan Jiaoyu , Pan Deng , Sun Yamei , Shan Zhao , Zhang Jinping , Yu Dong , Li Bin , Qian Zhikang 
Published: January 2015

ABSTRACT
Recently, interferon-induced transmembrane proteins (IFITMs) have been identified to be key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5 cells). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and knockdown of IFITMs by RNA interference reduced the virus titer by about 100-fold on day 8 postinfection, according to the findings of a virus yield assay at a low multiplicity of infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of the vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is closely related to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication.

IMPORTANCE
HCMV is known to repurpose the interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a proviral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we identified a new function of IFITMs during the very late stage of virus replication, i.e., virion assembly. Virus entry and assembly both involve vesicle transport and membrane fusion; thus, a common biochemical activity of IFITMs is likely to be involved. Therefore, our findings may provide a new platform for dissecting the molecular mechanism of action of IFITMs during the blocking or enhancement of virus infection, which are under intense investigation in this field.
Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus
Authors: Brennan Benjamin , Li Ping , Zhang Shuo , Li Aqian , Liang Mifang , Li Dexin , Elliott Richard M. 
Published: January 2015

ABSTRACT

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed that
SFTS virus
represents a new lineage within the
Phlebovirus
genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family
Bunyaviridae
. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M- and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen.

IMPORTANCE
SFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.