Virology Journal - Latest Articles

Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge

Katie Rivers — 2013-06-19T00:00:00Z

Background: To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosolMethodTo evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled). Results: Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose. Conclusions: These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

Franziska Dag — 2013-06-17T00:00:00Z

Background: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood. Methods: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells. Results: MCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells. Conclusion: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

Subgroup J avian leukosis virus infection inhibits autophagy in DF-1 cells

Haixia Liu — 2013-06-17T00:00:00Z

Background: Subgroup J avian leukosis virus (ALV-J) infection can induce tumor-related diseases in chickens. Previous studies by our laboratory demonstrated that ALV-J infection of DF-1 cells resulted in altered activity and phosphorylation of AKT. However, little is known about the subsequent activation of host DF-1 cells. Results: In the current study, autophagy inhibition was observed for ALV-J infected DF-1 cells. Our data showed that the autophagosome protein, microtubule-associated protein 1 light chain 3-II (LC3-II), was reduced considerably in DF-1 cells infected with active ALV-J, while no change was observed for cells infected with inactivated ALV-J. Autophagy inhibition was also confirmed by fluorescence microscopy and transmission electron microscopy. Interestingly, when autophagy was promoted by rapamycin, the titers of ALV-J replication were decreased, and the replication level of ALV-J was significantly enhanced when atg5 (autophagy-related gene 5) was knocked out. Conclusions: These results suggested that ALV-J infection could down-regulate autophagy in DF-1 cells during viral replication. This study is the first to report on the relationship between ALV-J infection and autophagy in DF-1 cells.

A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment

Jan Varada — 2013-06-15T00:00:00Z

Background: Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting. Methods: Virus/antibody mixtures were incubated for one hour at 37[degree sign]C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies. Results: We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law). Conclusions: This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.

Gene delivery in a mouse xenograft of a retargeted retrovirus to a solid 143B osteosarcoma

Xia Zhang — 2013-06-14T00:00:00Z

Background: Osteosarcomas are the most common primary bone malignancies found in children and adolescents. An optimized system was developed for efficient retroviral gene delivery into solid 143B osteosarcoma tumors in mice using a retargeted Env. In these studies, the viral Env CP was isolated from an in vitro screen of a library of feline leukemia virus Env randomized in the receptor-binding domain and maintained high titer on human 143B osteosarcoma cell line.FindingsThe vector developed to express the random Env libraries encoded the drug selectable marker neo. To adapt this for studies in live animals, the murine based vector was modified to express the luciferase gene. The bicistronic vector developed expressed both the CP Env and luciferase in the presence of either the MPMV CTE or a WPRE element. Virus bearing the CP FeLV Env variant maintained high titers after concentration allowing for direct visualization of delivery of the luciferase gene in subcutaneous 143B osteosarcoma tumors. Conclusion: This system serves as a proof-of-concept for the use of novel FeLV Env pseudotyped MLV particles for in vivo gene delivery. Gene delivery and expression of lucerifase from viral particles bearing the CP Env was readily detected in live mice after a single round of intratumor injection.

Molecular characterization of human adenovirus infection in Thailand, 2009--2012

Punsinee Sriwanna — 2013-06-13T00:00:00Z

Background: Human adenovirus (HAdV) can cause a wide spectrum of human diseases worldwide. Methods: Using PCR and sequence analysis, we investigated HAdV infection prevalence in the Thai population for four years from January 2009 to December 2012. We collected Nasopharyngeal swab/aspirate (NP) specimens from patients in Bangkok, Khon Kaen, and Nakhon Si Thammarat province and fecal specimens only from Bangkok and Khon Kaen province. Results: We observed HAdV infection in 1.04% (82/7,921) of NP samples and in 5.84% (76/1,301) of fecal specimens. HAdV-B3 (32%) and HAdV-C1 (31%) were the genotypes most commonly associated with NP specimens followed by HAdV-C2 (13%) and HAdV-C5 (12%). In fecal specimens, we found that 25% harbored HAdV-F41 followed by HAdV-C1 (18%), HAdV-C2 (16%), and HAdV-B3 (13%). Out of all population subsets, children below the age of 3 years were the most likely to be HAdV positive (63.29%). In addition, HAdV infection occurred throughout the year without a seasonal distribution pattern, although HAdV infection of NP samples peaked from January-April while HAdV infection peaked from January to March and then again from May to July in fecal samples. Conclusions: This study has for the first time reported the HAdV infection rate in Thai NP and fecal specimens from 2009--2012. We observed that HAdV-B3 and HAdV-C1 were commonly found in NP specimens, and that HAdV-F41 was the most prevalence in fecal specimens in Thailand during the study period.

An antibody response to human polyomavirus 15-mer peptides is highly abundant in healthy human subjects

Lieven Stuyver — 2013-06-12T00:00:00Z

Background: Human polyomaviruses (HPyV) infections cause mostly unapparent or mild primary infections, followed by lifelong nonpathogenic persistence. HPyV, and specifically JCPyV, are known to co-diverge with their host, implying a slow rate of viral evolution and a large timescale of virus/host co-existence. Recent bio-informatic reports showed a large level of peptide homology between JCPyV and the human proteome. In this study, the antibody response to PyV peptides is evaluated Methods: The in-silico analysis of the HPyV proteome was followed by peptide microarray serology. A HPyV-peptide microarray containing 4,284 peptides was designed and covered 10 polyomavirus proteomes. Plasma samples from 49 healthy subjects were tested against these peptides Results: In-silico analysis of all possible HPyV 5-mer amino acid sequences were compared to the human proteome, and 1,609 unique motifs are presented. Assuming a linear epitope being as small as a pentapeptide, on average 9.3% of the polyomavirus proteome is unique and could be recognized by the host as non-self. Small t Ag (stAg) contains a significantly higher percentage of unique pentapeptides. Experimental evidence for the presence of antibodies against HPyV 15-mer peptides in healthy subjects resulted in the following observations: i) antibody responses against stAg were significantly elevated, and against viral protein 2 (VP2) significantly reduced; and ii) there was a significant correlation between the increasing number of embedded unique HPyV penta-peptides and the increase in microarray fluorescent signal. Conclusion: The anti-peptide HPyV-antibodies in healthy subjects are preferably directed against the penta-peptide derived unique fraction of the viral proteome.

Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma

Anuj Tyagi — 2013-06-11T00:00:00Z

Background: Quantification of titers of ubiquitous viruses such as Torque teno virus (TTV) that do not cause clinical symptoms might be helpful in assessing the immune status of an individual. We hereby describe the validation of a SYBR Green-based TTV quantification method for plasma samples. Methods: Plasmids with TTV specific inserts were used for preparing standards and absolute quantification of TTV was performed using SYBR Green methodology. The method was assessed for its accuracy and precision (intra and inter-day) on four non-consecutive days. TTV was also quantified from plasma samples of 20 healthy volunteers and from 30 hematopoietic stem cell transplant (HSCT) recipients. Results: The assay was specific and showed satisfactory efficiency (82.2 %, R2=0.99) with the limit of quantification defined as 100 copies per reaction. The assay had good precision (inter and intra-day coefficient of variation in cycle threshold (CT) < 4%) and accuracy (100 +/- 10 %) in the range of 100 to 1010 copies/reaction. We found TTV loads ranging from 2.5 -- 4.07 log copies/mL of plasma with CT (mean +/- SD) of 33.8 +/- 1.77 in healthy individuals and 2.06 -- 8.49 log copies/mL of plasma with CT (mean +/- SD) of 24.3 +/- 1.04 in HSCT recipients. Conclusion: SYBR Green-based q-PCR assay combines simplicity with satisfactory sensitivity and may be suitable for monitoring the immune status of transplant recipients, where TTV loads over time may serve as a marker for immune reconstitution in human plasma samples.

Monitoring of adenovirus serotypes in environmental samples by combined PCR and melting point analyses

Nils Hartmann — 2013-06-10T00:00:00Z

Background: Human adenoviruses are promising candidates for addressing health risks associated with enteric viruses in environmental waters. Relatively harmless but common, these DNA viruses persist within the population and are generally considered extremely stable, remaining infectious in water for long periods of time. Group-specific or single species detection of human adenoviruses in environmental samples is usually based on polymerase chain reaction assays. Simultaneous identification of specific species or serotypes needs additional processing. Here we present a simple molecular approach for the monitoring of serotypic diversity in the human adenovirus populations in contaminated water sites. Methods: Diversity patterns of human adenoviruses in environmental samples, collected in an outdoor artificial stream and pond simulation system, were analyzed using a closed tube polymerase chain reaction method with subsequent melting point analysis. Results: Human adenovirus serotype 41 was the most prominent adenovirus serotype detected in environmental water samples, but melting point analyses indicated the presence of additional adenovirus serotypes. Conclusions: Based on investigations with spiked and environmental samples, a combination of qPCR and melting point analysis was shown to identify adenovirus serotypes in sewage contaminated water.

Three new emerging subgroups of torque teno sus viruses (TTSuVs) and co-infection of TTSuVs with porcine circovirus type 2 in China

Jianbo Liu — 2013-06-10T00:00:00Z

Background: Torque teno sus viruses (TTSuVs) are non-enveloped viruses and have single-stranded, negative sense circular DNA genomes and are widely distributed in pigs. But till now, the prevalence of TTSuVs with porcine circovirus type 2 (PCV2) in pig herds of China is not very clear; and the genetic variation among different TTSuVs isolate is very large and need to divide the subgroups. In this study, the co-infection with TTSuVs and porcine circovrius (PCV) in the pig population of China was investigated and the subgroups of all TTSuVs genomes in Genbank were divided. Results: Results showed that the rate of co-infection with TTSuV1 and TTSuV2 reached 75% in PCV2-positive samples. Also Two TTSuV1 and four TTSuV2 isolates genome sequences were obtained, and the similarity of all TTSuV1 and TTSuV2 genomic sequences in GenBank were compared. Phylogenetic trees indicated that both the TTSuV1 and TTSuV2 sequences could be divided into four genotypes. Interestingly, the sub-genotypes TTSuV1d, TTSuV2c and TTSuV2d exist only in the pig population of China. Conclusions: This study demonstrates that co-infection with TTSuVs and PCVs is very common in the pig population of China, in which the viruses maybe contribute to clinical diseases cooperatively. In addition, three new subgroups of TTSuVs emerged in China for the first time and a high level of variation among different isolates of TTSuV1 and TTSuV2 was indicated by their genetic diversity.