bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of the Alere i Influenza A&B Nucleic Acid Amplification Test by Use of Respiratory Specimens Collected in Viral Transport Medium
Authors: Bell J. Jeremiah , Selvarangan Rangaraj 
Published: September 2014

ABSTRACT

The Alere i Influenza A&B assay is a newly developed rapid molecular assay which has the potential to generate results within 15 min from sample collection. In this study, we evaluated the Alere i Influenza A&B assay by using salvaged frozen respiratory specimens that were collected in viral transport medium from children ages 10 months to 19 years. Alere i Influenza A&B assay test results were compared with viral culture and ProFlu
+
real-time reverse transcription-PCR (RT-PCR) assay results. We found that the overall sensitivity and specificity of the Alere i Influenza A&B assay were 93.3% and 94.5% for the detection of influenza A virus and 100% and 100% for the detection of influenza B virus, respectively, compared to viral culture. In comparison to ProFlu
+
real-time RT-PCR, overall sensitivity and specificity of the Alere i Influenza A&B assay for the detection of influenza A virus were 88.8% and 98.3% and 100% and 100% for detecting influenza B virus. Overall, the Alere i Influenza A&B assay performed well compared to either virus cell culture or RT-PCR.
PCR-Based Detection of Toxoplasma gondii DNA in Blood and Ocular Samples for Diagnosis of Ocular Toxoplasmosis
Authors: Bourdin C. , Busse A. , Kouamou E. , Touafek F. , Bodaghi B. , Le Hoang P. , Mazier D. , Paris L. , Fekkar A. 
Published: September 2014

ABSTRACT

PCR detection of
Toxoplasma gondii
in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis (OT) and furthermore for genotyping the strain involved in the disease. To assess this hypothesis, we performed PCR with 121 peripheral blood samples from 104 patients showing clinical and/or biological evidence of ocular toxoplasmosis and from 284 (258 patients) controls. We tested 2 different extraction protocols, using either 200 μl (small volume) or 2 ml (large volume) of whole blood. Sensitivity was poor, i.e., 4.1% and 25% for the small- and large-volume extractions, respectively. In comparison, PCR with ocular samples yielded 35.9% sensitivity, while immunoblotting and calculation of the Goldmann-Witmer coefficient yielded 47.6% and 72.3% sensitivities, respectively. Performing these three methods together provided 89.4% sensitivity. Whatever the origin of the sample (ocular or blood), PCR provided higher sensitivity for immunocompromised patients than for their immunocompetent counterparts. Consequently, PCR detection of
Toxoplasma gondii
in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT, and ocular sampling remains necessary for the biological diagnosis of OT.
Evaluation of Five Real-Time PCR Assays for Detection of Mycoplasma pneumoniae
Authors: Dumke Roger , Jacobs Enno 
Published: September 2014

ABSTRACT

Four commercial real-time PCR assays to detect
Mycoplasma pneumoniae
were tested, and the results were compared with the results for an in-house approach. Despite differences of crossing threshold values of up to 4, assays were able to detect at least 20 CFU/5 μl (52 fg DNA/5 μl) of sample with the Diagenode kit showing the best clinical sensitivity.
Interaction of Adenovirus Type 5 E4orf4 with the Nuclear Pore Subunit Nup205 Is Required for Proper Viral Gene Expression
Authors: Lu YiQing , Kucharski Thomas J. , Gamache Isabelle , Blanchette Paola , Branton Philip E. , Teodoro Jose G. 
Published: September 2014

ABSTRACT

Adenovirus type 5 E4orf4 is a multifunctional protein that regulates viral gene expression. The activities of E4orf4 are mainly mediated through binding to protein phosphatase 2A (PP2A). E4orf4 recruits target phosphoproteins into complexes with PP2A, resulting in dephosphorylation of host factors, such as SR splicing factors. In the current study, we utilized immunoprecipitation followed by mass spectrometry to identify novel E4orf4-interacting proteins. In this manner we identified Nup205, a component of the nuclear pore complex (NPC) as an E4orf4 interacting partner. The arginine-rich motif (ARM) of E4orf4 was required for interaction with Nup205 and for nuclear localization of E4orf4. ARMs are commonly found on viral nuclear proteins, and we observed that Nup205 interacts with three different nuclear viral proteins containing ARMs. E4orf4 formed a trimolecular complex containing both Nup205 and PP2A. Furthermore, Nup205 complexed with E4orf4 was hypophosphorylated, suggesting that the protein is specifically targeted for dephosphorylation. An adenovirus mutant that does not express E4orf4 (Orf4
−
) displayed elevated early and reduced late gene expression relative to that of the wild type. We observed that knockdown of Nup205 resulted in the same phenotype as that of the Orf4
−
virus, suggesting that the proteins function as a complex to regulate viral gene expression. Furthermore, knockdown of Nup205 resulted in a more than a 4-fold reduction in the replication of wild-type adenovirus. Our data show for first time that Ad5 E4orf4 interacts with and modifies the NPC and that Nup205-E4orf4 binding is required for normal regulation of viral gene expression and viral replication.

IMPORTANCE
Nuclear pore complexes (NPCs) are highly regulated conduits in the nuclear membrane that control transport of macromolecules between the nucleus and cytoplasm. Viruses that replicate in the nucleus must negotiate the NPC during nuclear entry, and viral DNA, mRNA, and proteins must then be exported from the nucleus. Several types of viruses restructure the NPC to facilitate replication, and the current study shows that adenovirus type 5 (Ad5) utilizes a novel mechanism to modify NPC function. We demonstrate that a subunit of the NPC, Nup205, is a phosphoprotein that is actively dephosphorylated by the Ad5-encoded protein E4orf4. Moreover, Nup205 is required by Ad5 to regulate viral gene expression and efficient viral replication. Nup205 is a nonstructural subunit that is responsible for the gating functions of the NPC, and this study suggests for the first time that the NPC is regulated by phosphorylation both during normal physiology and viral infection.
Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells
Authors: Danzy Shamika , Studdard Lydia R. , Manicassamy Balaji , Solorzano Alicia , Marshall Nicolle , García-Sastre Adolfo , Steel John , Lowen Anice C. 
Published: September 2014

ABSTRACT
Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate.

IMPORTANCE
Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells.
Viruses and RNA Interference: Issues and Controversies
Authors: Cullen Bryan R. 
Published: September 2014

ABSTRACT
The question of whether any mammalian cells are able to mount an effective RNA interference-mediated antiviral innate immune response has remained highly controversial. In this Gem, I review recent data addressing this important issue and propose a testable hypothesis that can explain many of the apparently contradictory results published in this area of research.
A Bivalent Vaccine Based on a Replication-Incompetent Influenza Virus Protects against Streptococcus pneumoniae and Influenza Virus Infection
Authors: Katsura Hiroaki , Piao Zhenyu , Iwatsuki-Horimoto Kiyoko , Akeda Yukihiro , Watanabe Shinji , Horimoto Taisuke , Oishi Kazunori , Kawaoka Yoshihiro 
Published: September 2014

ABSTRACT

Streptococcus pneumoniae
is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both
S. pneumoniae
and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either
S. pneumoniae
or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both
S. pneumoniae
and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases.

IMPORTANCE
Streptococcus pneumoniae
and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or
Streptococcus pneumoniae
. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.
Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom
Authors: Elderfield Ruth A. , Watson Simon J. , Godlee Alexandra , Adamson Walt E. , Thompson Catherine I. , Dunning Jake , Fernandez-Alonso Mirian , Blumenkrantz Deena , Hussell Tracy ,   The MOSAIC Investigators, Zambon Maria , Openshaw Peter , Kellam Paul , Barclay Wendy S. 
Published: September 2014

ABSTRACT
The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves.

IMPORTANCE
Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.
In Vivo
Cellular Tropism of Gorilla Simian Foamy Virus in Blood of Infected Humans
Authors: Rua Rejane , Betsem Edouard , Montange Thomas , Buseyne Florence , Gessain Antoine 
Published: September 2014

ABSTRACT

Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a
Gorilla gorilla
SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8
+
, CD4
+
, and CD19
+
lymphocyte samples and rarely in CD14
+
monocyte and CD56
+
NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10
5
cells in CD8
+
T lymphocytes, CD4
+
T lymphocytes, and CD19
+
B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4
+
T lymphocytes. SFV DNA levels in CD4
+
T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8
+
, CD4
+
, and B lymphocytes are major cellular targets of SFV in the blood of infected humans.

IMPORTANCE
Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV
in vivo
. Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4
+
T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.
Identification of Molecular Markers Associated with Alteration of Receptor-Binding Specificity in a Novel Genotype of Highly Pathogenic Avian Influenza A(H5N1) Viruses Detected in Cambodia in 2013
Authors: Rith Sareth , Davis C. Todd , Duong Veasna , Sar Borann , Horm Srey Viseth , Chin Savuth , Ly Sovann , Laurent Denis , Richner Beat , Oboho Ikwo , Jang Yunho , Davis William , Thor Sharmi , Balish Amanda , Iuliano A. Danielle , Sorn San , Holl Davun , Sok Touch , Seng Heng , Tarantola Arnaud , Tsuyuoka Reiko , Parry Amy , Chea Nora , Allal Lotfi , Kitsutani Paul , Warren Dora , Prouty Michael , Horwood Paul , Widdowson Marc-Alain , Lindstrom Stephen , Villanueva Julie , Donis Ruben , Cox Nancy , Buchy Philippe 
Published: September 2014

ABSTRACT
Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.
Prime-Boost Immunization Strategies against Chikungunya Virus
Authors: Hallengärd David , Lum Fok-Moon , Kümmerer Beate M. , Lulla Aleksei , Lulla Valeria , García-Arriaza Juan , Fazakerley John K. , Roques Pierre , Le Grand Roger , Merits Andres , Ng Lisa F. P. , Esteban Mariano , Liljeström Peter 
Published: September 2014

ABSTRACTChikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that causes debilitating arthralgia in humans. Here we describe the development and testing of novel DNA replicon and protein CHIKV vaccine candidates and evaluate their abilities to induce antigen-specific immune responses against CHIKV. We also describe homologous and heterologous prime-boost immunization strategies using novel and previously developed CHIKV vaccine candidates. Immunogenicity and efficacy were studied in a mouse model of CHIKV infection and showed that the DNA replicon and protein antigen were potent vaccine candidates, particularly when used for priming and boosting, respectively. Several prime-boost immunization strategies eliciting unmatched humoral and cellular immune responses were identified. Further characterization by antibody epitope mapping revealed differences in the qualitative immune responses induced by the different vaccine candidates and immunization strategies. Most vaccine modalities resulted in complete protection against wild-type CHIKV infection; however, we did identify circumstances under which certain immunization regimens may lead to enhancement of inflammation upon challenge. These results should help guide the design of CHIKV vaccine studies and will form the basis for further preclinical and clinical evaluation of these vaccine candidates.IMPORTANCEAs of today, there is no licensed vaccine to prevent CHIKV infection. In considering potential new vaccine candidates, a vaccine that could raise long-term protective immunity after a single immunization would be preferable. While humoral immunity seems to be central for protection against CHIKV infection, we do not yet fully understand the correlates of protection. Therefore, in the absence of a functional vaccine, there is a need to evaluate a number of different candidates, assessing their merits when they are used either in a single immunization or in a homologous or heterologous prime-boost modality. Here we show that while single immunization with various vaccine candidates results in potent responses, combined approaches significantly enhance responses, suggesting that such approaches need to be considered in the further development of an efficacious CHIKV vaccine.
A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway
Authors: Schuster Susan , Zirkel Florian , Kurth Andreas , van Cleef Koen W. R. , Drosten Christian , van Rij Ronald P. , Junglen Sandra 
Published: September 2014

ABSTRACT

Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Côte d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nucleotides (nt) (RNA 1) and 1,801 nt (RNA 2). Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative, that of the Pariacoto virus (family
Nodaviridae
). Unexpectedly, for the putative capsid protein, maximal pairwise identity of 16% to Lake Sinai virus 2, an unclassified virus with a nonsegmented RNA genome, was found. Moreover, MoNV virions are nonenveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of ∼56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNA interference (RNAi) that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long double-stranded RNA (dsRNA) and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into small interfering RNAs (siRNAs). Phylogenetic analyses indicate that MoNV is a novel member of the family
Nodaviridae
that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level.

IMPORTANCE
The identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus with a distant relationship to nodaviruses. Another remarkable feature of MoNV is that, unlike other nodaviruses, it expresses two subgenomic RNAs (sgRNAs). One of the sgRNAs expresses a protein that counteracts antiviral defense of its mosquito host, whereas the function of the other sgRNA remains unknown. Our results show that complete genome segments can be exchanged beyond the species level and suggest that insects harbor a large repertoire of exceptional viruses.