bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of Hepatitis B Virus Genotypic Resistance Mutations by Coamplification at Lower Denaturation Temperature-PCR Coupled with Sanger Sequencing
Authors: Liu Can , Lin Jinpiao , Chen Huijuan , Shang Hongyan , Jiang Ling , Chen Jing , Ye Yang , Yang Bin , Ou Qishui 
Published: June 2014

ABSTRACT

Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (

T
c

) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (

T
m

)-reducing mutation occurred (e.g., C-G→T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in

T
m

(e.g., C-G→G-C) or raised

T
m

(e.g., T-A→C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases.
Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Blood Isolates of Acinetobacter Species
Authors: Hsueh Po-Ren , Kuo Lu-Cheng , Chang Tsung-Chain , Lee Tai-Fen , Teng Shih-Hua , Chuang Yu-Chung , Teng Lee-Jene , Sheng Wang-Huei 
Published: June 2014

ABSTRACT

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of
Acinetobacter baumannii
isolates, 72.4% (63/87) of
A. nosocomialis
isolates, and 97.6% (41/42) of
A. pittii
isolates. All
Acinetobacter junii
,
A. ursingii
,
A. johnsonii
, and
A. radioresistens
isolates (
n
= 28) could also be identified correctly by Bruker Biotyper.
Limited Dissemination and Shedding of the UL128 Complex-Intact, UL/b′-Defective Rhesus Cytomegalovirus Strain 180.92
Authors: Assaf Basel T. , Mansfield Keith G. , Strelow Lisa , Westmoreland Susan V. , Barry Peter A. , Kaur Amitinder 
Published: June 2014

ABSTRACTThe UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b′ region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b′ region predicts acute endothelial tropism and long-term sheddingin vivoin the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b′ region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropismin vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b′ region, rapidly emerged duringin vivoreplication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuatedin vivoand suggest that additional UL/b′ genes, besides the UL128 complex, are required for optimalin vivoCMV replication and dissemination.IMPORTANCEAn effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate thein vivofunction of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance ofin vivostudies in evaluating human CMV gene function and suggest that additional UL/b′ genes are required for optimal CMV dissemination and transmission.
Asthma Increases Susceptibility to Heterologous but Not Homologous Secondary Influenza
Authors: Furuya Yoichi , Roberts Sean , Hurteau Gregory J. , Sanfilippo Alan M. , Racine Rachael , Metzger Dennis W. 
Published: June 2014

ABSTRACTAsthma was the most common comorbidity observed among patients hospitalized with influenza A virus during the 2009 pandemic. However, little remains known about how the asthmatic phenotype influences protective immune responses against respiratory viral pathogens. Using the ovalbumin-induced allergic lung inflammation model, we found that asthmatic mice, unlike nonasthmatic mice, were highly susceptible to secondary heterologous virus challenge. While primary virus infection generated protective memory immune responses against homologous secondary virus challenge in both asthmatic and nonasthmatic mice, full protection against heterologous A/California/04/2009 (CA04) viral infection was observed only in nonasthmatic mice. Significant reductions in CA04-specific IgA, IgG, and IgM levels and in CA04-neutralizing activity of bronchoalveolar lavage fluid (BALF) was observed following secondary CA04 challenge of PR8-immunized asthmatic mice. Furthermore, transfer of immune BALF obtained from nonasthmatic, but not asthmatic, donors following secondary viral infection generated protection against CA04 in naive recipients. Nonspecific B-cell activation by CpG inoculation restored protection in PR8-immunized, CA04-challenged asthmatic mice. These results demonstrate a causal link between defective mucosal antibody responses and the heightened susceptibility of asthmatic mice to influenza infection and provide a mechanistic explanation for the observation that asthma was a major risk factor during the 2009 influenza pandemic.IMPORTANCEThe prevalence of asthma worldwide is increasing each year. Unfortunately, there is no cure for asthma. Asthmatic individuals not only suffer from consistent wheezing and coughing but are also believed to be more prone to serious lung infections that result in bronchitis and pneumonia. However, little is known about the influence of asthma on host mucosal immunity. Here we show that antibody responses during secondary heterologous influenza infections are suboptimal and that this is responsible for the increased mortality in asthmatic mice from viral infections. Understanding the mechanism of increased susceptibility will aid in developing new antiviral therapies for asthmatic patients.
Crucial Roles of Interleukin-7 in the Development of T Follicular Helper Cells and in the Induction of Humoral Immunity
Authors: Seo Yong Bok , Im Se Jin , Namkoong Hong , Kim Sae Won , Choi Young Woo , Kang Moon Cheol , Lim Hye Seong , Jin Hyun Tak , Yang Se Hwan , Cho Mi La , Kim You-Me , Lee Seung-Woo , Choi Young Ki , Surh Charles D. , Sung Young Chul 
Published: June 2014

ABSTRACT

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation
in vivo
, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses.

IMPORTANCE
Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.
Structure of Influenza Virus N7: the Last Piece of the Neuraminidase “Jigsaw” Puzzle
Authors: Sun Xiaoman , Li Qing , Wu Yan , Wang Mingyang , Liu Yue , Qi Jianxun , Vavricka Christopher J. , Gao George F. 
Published: June 2014

ABSTRACT

There are nine subtypes of influenza A virus neuraminidase (NA), N1 to N9. In addition, influenza B virus also contains NA, and there are two influenza virus NA-like molecules, N10 and N11, which were recently identified from bats. Crystal structures for all of these proteins have been solved, with the exception of N7, and there is no published report of N6, although a structure has been deposited in the Protein Data Bank. Here, we present the N7 and N6 structures at 2.1 Å and 1.8 Å, respectively. Structural comparison of all NA subtypes shows that both N7 and N6 highly resemble typical group 2 NA structures with some special characteristics, including an additional cavity adjacent to their active sites formed by novel 340-loop conformations. Comparative analysis also revealed new structural insights into the
N
-glycosylation, calcium binding, and second sialic acid binding site of influenza virus NA. This comprehensive study is critical for understanding the complexity of the most successful influenza drug target and for the structure-based design of novel influenza inhibitors.

IMPORTANCE
Influenza viruses impose a great burden on society, by the human-adapted seasonal types as well as by variants that occasionally jump from the avian reservoir to infect humans. The surface glycoprotein neuraminidase (NA) is essential for the propagation of the virus and currently the most successfully drug-targeted molecule. Therefore, the structural and functional analysis of NA is critical for the prevention and control of influenza infections. There are nine subtypes of influenza A virus NA (N1 to N9). In addition, influenza B virus also contains NA, and there are two influenza NA-like molecules, N10 and N11, which were recently identified in bats. Crystal structures for all of these proteins have been solved and reported with the exception of N7 and N6. The structural analysis of influenza virus N7 and N6 presented in this study therefore completes the puzzle and adds to a comprehensive understanding of influenza virus NA.
The Gammaherpesviruses Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Modulate the Toll-Like Receptor-Induced Proinflammatory Cytokine Response
Authors: Bussey Kendra A. , Reimer Elisa , Todt Helene , Denker Brigitte , Gallo Antonio , Konrad Andreas , Ottinger Matthias , Adler Heiko , Stürzl Michael , Brune Wolfram , Brinkmann Melanie M. 
Published: June 2014

ABSTRACT
The human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, establishes lifelong latency upon infection. Murine gammaherpesvirus 68 (MHV68) is a well-established model for KSHV. Toll-like receptors (TLRs) play a crucial role for the innate immune response to pathogens. Although KSHV and MHV68 are detected by TLRs, studies suggest they modulate TLR4 and TLR9 signaling, respectively. In this study, we show that in bone marrow-derived macrophages (BMDMs), MHV68 did not induce a detectable proinflammatory cytokine response. Furthermore, MHV68 abrogated the response to TLR2, -4, -7, and -9 agonists in BMDMs. Similarly to observations with MHV68, infection with KSHV efficiently inhibited TLR2 signaling in THP-1 monocytes. Using a KSHV open reading frame (ORF) library, we found that K4.2, ORF21, ORF31, and the replication and transcription activator protein (RTA)/ORF50 inhibited TLR2-dependent nuclear factor kappa B (NF-κB) activation in HEK293 TLR2-yellow fluorescent protein (YFP)- and Flag-TLR2-transfected HEK293T cells. Of the identified ORFs, RTA/ORF50 strongly downregulated TLR2 and TLR4 signaling by reducing TLR2 and TLR4 protein expression. Confocal microscopy revealed that TLR2 and TLR4 were no longer localized to the plasma membrane in cells expressing RTA/ORF50. In this study, we have shown that the gammaherpesviruses MHV68 and KSHV efficiently downmodulate TLR signaling in macrophages and have identified a novel function of RTA/ORF50 in modulation of the innate immune response.

IMPORTANCE
The Toll-like receptors (TLRs) are an important class of pattern recognition receptors of the innate immune system. They induce a potent proinflammatory cytokine response upon detection of a variety of pathogens. In this study, we found that the gammaherpesviruses murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently inhibit the TLR-mediated innate immune response. We further identified the KSHV-encoded replication and transcription activator protein (RTA) as a novel modulator of TLR signaling. Our data suggest that the gammaherpesviruses MHV68 and KSHV prevent activation of the innate immune response by targeting TLR signaling.
Infection and Pathogenesis of Canine, Equine, and Human Influenza Viruses in Canine Tracheas
Authors: Gonzalez Gaelle , Marshall John F. , Morrell Joanna , Robb David , McCauley John W. , Perez Daniel R. , Parrish Colin R. , Murcia Pablo R. 
Published: June 2014

ABSTRACT
Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity.

IMPORTANCE
Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as “mixing vessels” in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.
Disease Severity Is Associated with Differential Gene Expression at the Early and Late Phases of Infection in Nonhuman Primates Infected with Different H5N1 Highly Pathogenic Avian Influenza Viruses
Authors: Muramoto Yukiko , Shoemaker Jason E. , Le Mai Quynh , Itoh Yasushi , Tamura Daisuke , Sakai-Tagawa Yuko , Imai Hirotaka , Uraki Ryuta , Takano Ryo , Kawakami Eiryo , Ito Mutsumi , Okamoto Kiyoko , Ishigaki Hirohito , Mimuro Hitomi , Sasakawa Chihiro , Matsuoka Yukiko , Noda Takeshi , Fukuyama Satoshi , Ogasawara Kazumasa , Kitano Hiroaki , Kawaoka Yoshihiro 
Published: June 2014

ABSTRACTOccasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome.IMPORTANCEHighly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.
Modification of the Hepatitis B Virus Envelope Protein Glycosylation Pattern Interferes with Secretion of Viral Particles, Infectivity, and Susceptibility to Neutralizing Antibodies
Authors: Julithe Romain , Abou-Jaoudé Georges , Sureau Camille 
Published: June 2014

ABSTRACTThe envelope proteins of hepatitis B virus (HBV) bear an N-linked glycosylation site at N146 within the immunodominant a-determinant in the antigenic loop (AGL) region. This glycosylation site is never fully functional, leading to a nearly 1/1 ratio of glycosylated/nonglycosylated isoforms in the viral envelope. Here we investigated the requirement for a precise positioning of N-linked glycan at amino acid 146 and the functions associated with the glycosylated and nonglycosylated isoforms. We observed that the removal of the N146 glycosylation site by mutagenesis was permissive to envelope protein synthesis and stability and to secretion of subviral particles (SVPs) and hepatitis delta virus (HDV) virions, but it was detrimental to HBV virion production. Several positions in the AGL could substitute for position 146 as the glycosylation acceptor site. At position 146, neither a glycan chain nor asparagine was absolutely required for infectivity, but there was a preference for a polar residue. Envelope proteins bearing 5 AGL glycosylation sites became hyperglycosylated, leading to an increased capacity for SVP secretion at the expense of HBV and HDV virion secretion. Infectivity-compatible N-glycosylation sites could be inserted at 3 positions (positions 115, 129, and 136), but when all three positions were glycosylated, the hyperglycosylated mutant was substantially attenuated at viral entry, while it acquired resistance to neutralizing antibodies. Taken together, these findings suggest that the nonglycosylated N146 is essential for infectivity, while the glycosylated form, in addition to its importance for HBV virion secretion, is instrumental in shielding the a-determinant from neutralizing antibodies.IMPORTANCEAt the surface of HBV particles, the immunodominant a-determinant is the main target of neutralizing antibodies and an essential determinant of infectivity. It contains an N-glycosylation site at position 146, which is functional on only half of the envelope proteins. Our data suggest that the coexistence of nonglycosylated and glycosylated N146 at the surface of HBV reflects the dual function of this determinant in infectivity and immune escape. Hence, a modification of the HBV glycosylation pattern affects not only virion assembly and infectivity but also immune escape.