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1.  Comparative efficacy of experimental inactivated and live-attenuated chimeric porcine circovirus (PCV) 1-2b vaccines derived from PCV1 and PCV2b isolates originated in China 
Virology Journal  2015;12:113.
Porcine circovirus type-2b (PCV2b) is recognized as the etiological agent of the various clinical manifestations of porcine circovirus-associated disease (PCVAD). Previous studies have demonstrated effectiveness of chimeric PCV1-2 vaccines against PCV2b challenge. In this study, the efficacy of inactivated and live-attenuated (2 × 103.5 or 2 × 104.0 50 % tissue culture infective dose [TCID50] dose) chimeric PCV1-2b vaccines was compared side-by-side in conventional pigs.
Twenty-seven non-PCV2 viremic pigs without PCV2-specific antibody were randomly divided into six groups, including four vaccinated and challenged groups, a nonvaccinated challenged group, and a mock group. All pigs except those in the mock group were challenged at 28 days post vaccination (DPV) using PCV2b.
Both inactivated and live-attenuated chimeric PCV1-2b vaccines induced a robust antibody responses, and significantly decreased microscopic lesion and lower viral loads in serum or superficial inguinal lymph nodes (SILN) compared with that in the nonvaccinated challenged group. PCV2 antibody titers decreased after 7 days post challenge (DPC) in pigs administered the inactivated PCV1-2b vaccine and they were lower than those in pigs inoculated with live-attenuated PCV1-2b on the day of necropsy. Moreover, no viremia was present in pigs inoculated with live-attenuated PCV1-2b vaccine at 21 DPC regardless of the dose difference.
The results demonstrated that both inactivated and live-attenuated chimeric PCV1-2b vaccines were effective to induce protective immunity against PCV2b infection.
PMCID: PMC4518523  PMID: 26220290
Porcine circovirus type 2; Inactivated and live-attenuated chimeric PCV1-2b vaccine; Inactivated PCV2b vaccine; Efficacy
2.  The Chinese herb-derived Sparstolonin B suppresses HIV-1 transcription 
Virology Journal  2015;12:108.
The Chines herb derived Sparstolonin B, (SsnB), is a recently identified natural compound that selectively blocks TLR2- and TLR4-mediated inflammatory signaling. But it is unknown whether this compound has any effect on HIV infection.
We found that SsnB treatment blocked HIV-1 transcription via a novel mechanism that requires the TAR region. Treatment of human T cell lines or peripheral blood mononuclear cells with SsnB at 1 μM significantly inhibited HIV production. Lastly, SsnB was able to inhibit HIV in synergy with AZT.
These data suggest that SsnB is a novel natural compound that inhibits HIV-1 transcription and may be a new drug in the treatment of HIV infection.
PMCID: PMC4513614  PMID: 26206295
Sparstolonin B; HIV transcription; TAR region
3.  Hepatitis C virus genotype and subtype distribution in Chinese chronic hepatitis C patients: nationwide spread of HCV genotypes 3 and 6 
Virology Journal  2015;12:109.
Hepatitis C virus (HCV) genotype and subtype are related to disease progression and response to antiviral therapy. Current HCV genotype and subtype distribution data, especially for genotypes 3 and 6, are limited in China. Our purpose was to investigate the current HCV genotype and subtype distributions in chronic hepatitis C patients in China.
Chronic hepatitis C patients (n = 1012) were enrolled, and demographic information and possible transmission risk factors were collected. Serum samples were subjected to reverse-transcription polymerase chain reaction, followed by direct DNA sequencing and phylogenetic analysis of the NS5B and core/E1 regions to determine HCV genotypes/subtypes. The geographical distributions of HCV genotypes/subtypes were analyzed. Demographic information and transmission risk factors were compared between different HCV genotypes/subtypes.
Four genotypes and seven subtypes of HCV were detected in 970 patients. Subtypes 1b, 2a, 3a, 6a, 3b, 6n, and 1a were detected at frequencies of 71.96 %, 19.90 %, 3.20 %, 2.16 %, 1.96 %, 0.41 %, and 0.41 %, respectively. Genotypes 3 and 6 showed an increasingly wide geographic distribution over time. Patients with subtypes 1b and 2a were older than those with 3a, 3b, 6a, and 6n subtypes (p < 0.05 in all subtypes). More genotype 1 and 2 patients underwent blood transfusion than those with genotype 3 (all p < 0.05). More genotype 3 and 6 patients had a history of intravenous drug use than those with genotypes 1 and 2 (all p < 0.05).
Though subtypes 1b and 2a are still the most prevalent HCV subtypes in China, genotype 3 and 6 HCV infections have already spread nationwide from southern and western China.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0341-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4513753  PMID: 26206422
Hepatitis C virus (HCV); Genotype; Phylogenetic analysis
4.  Phylogenetic and recombination analysis of Tobacco bushy top virus in China 
Virology Journal  2015;12:111.
During the past decade, tobacco bushy top disease, which is mainly caused by a combination of Tobacco bushy top virus (TBTV) and Tobacco vein-distorting virus (TVDV), underwent a sudden appearance, extreme virulence and degeneration of the epidemic in the Yunnan province of China. In addition to integrative control of its aphid vector, it is of interest to examine diversity and evolution among different TBTV isolates.
5’ and 3’ RACE, combined with one step full-length RT-PCR, were used to clone the full-length genome of three new isolates of TBTV that exhibited mild pathogenicity in Chinese fields. Nucleotide and amino acid sequences for the TBTV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates.
The genomes of three isolates, termed TBTV-JC, TBTV-MD-I and TBTV-MD-II, were 4152 nt in length and included one distinctive difference from previously reported TBTV isolates: the first nucleotide of the genome was a guanylate instead of an adenylate. Diversity and phylogenetic analyses among these three new TBTV isolates and five other available isolates suggest that ORFs and 3’UTRs of TBTV may have evolved separately. Moreover, the RdRp-coding region was the most variable. Recombination analysis detected a total of 29 recombination events in the 8 TBTV isolates, in which 24 events are highly likely and 5 events have low-level likelihood based on their correlation with the phylogenetic trees. The three new TBTV isolates have individual recombination patterns with subtle divergences in parents and locations.
The genome sizes of TBTV isolates were constant while different ORF-coding regions and 3’UTRs may have evolved separately. The RdRp-coding region was the most variable. Frequent recombination occurred among TBTV isolates. Three new TBTV isolates have individual recombination patterns and may have different progenitors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0340-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4514990  PMID: 26209518
TBTV; RACE; Phylogenetic analysis; Recombination
5.  Human papillomavirus (HPV) screening and cervical cancer burden. A Brazilian perspective 
Virology Journal  2015;12:112.
This review tackles the issues related to disease burden caused by cervical cancer (CC) and its precursor (CIN) lesions in Brazil. A special focus is given to new technologies with potential to interfere with the development of CC by reducing the high-risk human papillomavirus (hr-HPV)-induced lesions that remain a major public health burden in all developing countries where organized screening programs do not exist. Globally, 85 % of all incident CC and 50 % of CC deaths occur in the developing countries. Unfortunately, most regions of Brazil still demonstrate high mortality rates, ranking CC as the second most common cancer among Brazilian women. Recently, CC screening programs have been tailored in the country to enable early detection of CC precursor lesions and thereby reduce cancer mortality. A combination of HPV testing with liquid-based cytology (LBC) seems to be a promising new approach in CC screening, with high expectation to offer an adequate control of CC burden in this country.
PMCID: PMC4514998  PMID: 26208521
Cervical cancer; HPV DNA test; Molecular diagnostic techniques; Cancer screening; Pap test
6.  Complete genome sequence of Ostreid herpesvirus-1 associated with mortalities of Scapharca broughtonii broodstocks 
Virology Journal  2015;12:110.
Ostreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported.
In this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants.
OsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5 % G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2 % and 97.3 % identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV.
The determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0334-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4514999  PMID: 26209441
Herpesvirus; OsHV-1; Bivalves; Mortality; Genome sequence
7.  Molecular evidence of Ebola Reston virus infection in Philippine bats 
Virology Journal  2015;12:107.
In 2008–09, evidence of Reston ebolavirus (RESTV) infection was found in domestic pigs and pig workers in the Philippines. With species of bats having been shown to be the cryptic reservoir of filoviruses elsewhere, the Philippine government, in conjunction with the Food and Agriculture Organization of the United Nations, assembled a multi-disciplinary and multi-institutional team to investigate Philippine bats as the possible reservoir of RESTV.
The team undertook surveillance of bat populations at multiple locations during 2010 using both serology and molecular assays.
A total of 464 bats from 21 species were sampled. We found both molecular and serologic evidence of RESTV infection in multiple bat species. RNA was detected with quantitative PCR (qPCR) in oropharyngeal swabs taken from Miniopterus schreibersii, with three samples yielding a product on conventional hemi-nested PCR whose sequences differed from a Philippine pig isolate by a single nucleotide. Uncorroborated qPCR detections may indicate RESTV nucleic acid in several additional bat species (M. australis, C. brachyotis and Ch. plicata). We also detected anti-RESTV antibodies in three bats (Acerodon jubatus) using both Western blot and ELISA.
The findings suggest that ebolavirus infection is taxonomically widespread in Philippine bats, but the evident low prevalence and low viral load warrants expanded surveillance to elaborate the findings, and more broadly, to determine the taxonomic and geographic occurrence of ebolaviruses in bats in the region.
PMCID: PMC4504098  PMID: 26184657
Reston; Ebolavirus; Filovirus; Philippine; Bat; Molecular; Serology
8.  In vitro characterisation of a pleconaril/pirodavir-like compound with potent activity against rhinoviruses 
Virology Journal  2015;12:106.
Rhinovirus infections do not only cause common colds, but may also trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Even though rhinoviruses have been the focus of extensive drug development efforts in the past, an anti-rhinoviral drug still has to make it to the market. In the past, the viral capsid protein VP1 has been shown to be an important target for the development of antiviral molecules. Furthermore, many different chemical scaffolds appear to possess the properties that are required to inhibit virus replication by this mechanism of action. I-6602, an analogue of the rhinovirus inhibitor pirodavir, was previously identified as a potent inhibitor of rhinovirus infection. Here, we describe the antiviral activity of its analogue ca603, a molecule with a modified linker structure, and corroborate its mechanism of action as a capsid binder.
The molecule ca603 shows antiviral activity against a panel of rhino-and enteroviruses. Cross-resistance is observed against viruses with mutations that render them resistant to the inhibitory effect of the capsid binder pleconaril and thermostability assays demonstrate that the compound binds and stabilizes the viral capsid. Binding of the molecule to the VP1 protein is corroborated by in silico modeling.
It is confirmed that ca603 inhibits rhinovirus replication by interaction with the VP1 protein and, by this, allows to further expand the chemical diversity of capsid-binding molecules.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0330-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4501209  PMID: 26169596
9.  Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels 
Virology Journal  2015;12:102.
Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. Three distinct viruses may cause them: camelpox virus (CMLV), camel parapox virus (CPPV) and camelus dromedary papilloma virus (CdPV). These diseases are often difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify these diseases, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost-and timesaving benefits.
Methods and results
In the present communication, we describe the development, optimization and validation of a multiplex PCR assay able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets and was validated with viral genomic DNA extracted from known virus strains (n = 14) and DNA extracted from homogenized clinical skin specimens (n = 86). The assay detects correctly the target pathogens by amplification of targeted genes, even in case of co-infection. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing.
This assay provide rapid, sensitive and specific method for identifying three important viruses in specimens collected from dromedary camels with varying clinical presentations.
PMCID: PMC4491865  PMID: 26148497
Development; Multiplex PCR; Diagnosis; Pox and pox-like diseases; Camels
10.  Identification of Hop stunt viroid infecting Citrus limon in China using small RNAs deep sequencing approach 
Virology Journal  2015;12:103.
The advent of next generation sequencing technology has allowed for significant advances in plant virus discovery, particularly for identification of covert viruses and previously undescribed viruses. The Citrus limon Burm. f. (C. limon) is a small evergreen tree native to Asia, and . China is the world’s top lemon-producing nation.
In this work, lemon samples were collected from southwestern of China, where an unknown disease outbreak had caused huge losses in the lemon production industry. Using high-throughput pyrosequencing and the assembly of small RNAs, we showed that the Hop stunt viroid (HSVd) was present in C. limon leaf sample. The majority of it is a main lemon producing agricultural cultivarHop stunt viroid derived siRNAs (HSVd-siRNAs) in C. limon were 21 nucleotides in length, and nearly equal amount of HSVd-siRNAs originated from the plus-genomic RNA strand as from the complementary strand. A bias of HSVd-siRNAs toward sequences beginning with a 5′-Guanine was observed. Furthermore, hotspot analysis showed that a large amount of HSVd-siRNAs derived from the central and variant domains of the HSVd genome.
Our results suggest that C. limon could set up a small RNA-mediated gene silencing response to Hop stunt viroid, Interestingly, based on bioinformatics analysis, our results also suggest that the large amounts of HSVd-siRNAs from central and variant domains might be involved in interference with host gene expression and affect symptom development.
PMCID: PMC4492010  PMID: 26148502
Citrus limon; Viroid; Small RNA; RNA interference
11.  DAXX modulates human papillomavirus early gene expression and genome replication in U2OS cells 
Virology Journal  2015;12:104.
The human papillomavirus (HPV) genomes can replicate, and are maintained as autonomously replicating extrachromosomal plasmids in human U2OS cells. Previous studies have shown that HPV genomes are transcriptionally active in U2OS cells and can express the viral early proteins required for initiation and establishment of HPV replication. In the present work, we have examined the involvement of cellular DAXX protein in HPV replication in U2OS cells.
We have used indirect immunofluorescence and FISH analysis in order to study HPV replication compartments in U2OS cells. In addition, we have used siRNA knock-down for examining the effect of the DAXX protein on HPV replication and transcription in U2OS cells.
We show that a portion of HPV replication foci are partially co-localized with components of ND10, cellular DAXX and PML proteins. In addition, we demonstrate that the knock-down of the cellular DAXX protein modulates the HPV genome replication and transcription in U2OS cells – papillomavirus replication is reduced in the absence of this component of ND10.
The DAXX protein modulates the early gene expression and the transient replication of HPV genomes in U2OS cells.
PMCID: PMC4492069  PMID: 26148509
Papillomavirus; Transcription; Replication; Virus-host interaction; DAXX
12.  Mucosal Alpha-Papillomavirus (HPV89) in a rare skin lesion 
Virology Journal  2015;12:105.
Apocrine acrosyringeal keratosis is a rare skin lesion showing a unique benign keratotic lesion associated with syringocystoadenoma papilliferum. It is characterized by an exophytic proliferation of the epidermis with two distinct keratinocytic structures: a) columns of hyperkeratotic mass surrounded by acanthotic epidermis and b) papillated and/or cystic invaginations typical of syringocystoadenoma papilliferum. No causative agents were reported.
The present report describes a typical case of apocrine acrosyringeal keratosis localized in the right retro-auricular area of 57-year-old man in which the presence of HPV was evaluated. PCR analysis and direct sequencing revealed the presence of HPV 89. The presence of this low risk mucosal HPV in a skin localization was never reported as well as in association with this rare tumor. Furthermore rolling circle amplification, RT-PCR and in situ hybridization confirmed the presence of a transcriptionally active HPV 89.
Taken together our results suggest that HPV89 plays a role in apocrine acrosyringeal keratosis with syringocystoadenoma papilliferum, in consideration of the documented biological activity of the virus. The association of low risk mucosal HPV infection with this skin lesion opens new perspectives in its clinical management. Further studies on samples from other patients are needed to confirm this association.
PMCID: PMC4492091  PMID: 26148544
HPV 89; Syringocystoadenoma papilliferum; Apocrine acrosyringeal keratosis
13.  Standardization of the methods and reference materials used to assess virus content in varicella vaccines 
Virology Journal  2015;12:101.
In Korea, every vaccine lot is tested by the National Center for Lot Release (NCLR) in accordance with the national lot release procedures to ensure the safety and efficacy of vaccines. These quality tests examine the virus content in varicella vaccines via plaque assays (either the agar overlay method [AOM] or plaque staining method [PSM]), according to the procedures suggested by the Korean Reference Material for the Varicella Vaccine (KRMVV) or the manufacturer’s standard in-house protocol.
To standardize the virus content tests, viral titers in the KRMVV were measured using the PSM at four participating laboratories in a collaborative study. With the aim of developing a standardized method using the KRMVV as a positive control, we compared the ability of the two test methods, AOM and PSM, to accurately and reproducibly determine the virus content of two commercial varicella vaccines.
The results showed that the standardized method (PSM) was more suitable for quality control analysis of the varicella vaccine.
Use of a standardized method (PSM) according to the Korean reference material will improve the reliability and objectivity of lot release testing.
PMCID: PMC4491273  PMID: 26141063
Varicella; Vaccine; Virus content; National Center for Lot Release; Korean reference material
14.  Abnormal CD4 + T helper (Th) 1 cells and activated memory B cells are associated with type III asymptomatic mixed cryoglobulinemia in HCV infection 
Virology Journal  2015;12:100.
Mixed cryoglobulinemia (MC) in hepatitis C virus (HCV) infection is associated with abnormal immune responses mediated by T cells and B cells, while the relationships of different subsets of CD4 + T helper (Th) cells, B cells and associated cytokines with type III asymptomatic MC in HCV infection are poorly understood.
Fifty-four chronic hepatitis C (CHC) patients and 23 healthy controls (HCs) were enrolled in the study. Serum cryoglobulins were detected by cryoprecipitation. The types of cryoglobulin were determined by western blot. The phenotypes and frequencies of Th cell and B cell subsets were detected by flow cytometric analysis. The cytokines IFN-γ, IL-4, IL-17, IL-21, IL-22, and TGF-β were measured by enzyme-linked immunosorbent assay.
Twenty-six CHC patients were detected with type III asymptomatic MC. The frequencies of Th2, Th17, follicular helper T (Tfh cells), Th22, and tissue-like B cells were significantly higher in CHC patients compared to HCs, while these cell subsets were not significantly different between CHC patients and HCV-related MC patients. The frequencies of Th1 and activated memory B cells increased in HCV-related MC patients compared to HCs, although the difference between the two cell subsets in CHC patients and HCs was not significant. The frequency of regulatory T cells (Treg cells) was higher in CHC patients than in HCV-related MC patients and HCs. Higher expressions of serum IFN-γ, IL-17, IL-21, and IL-22 were observed in CHC patients than in HCs, but the differences were not significantly different in CHC patients and HCV-related MC patients. The frequency of Th1 cells was associated with activated memory B cells in HCV-related MC patients, and the frequency of Th1 cells and activated memory B cells was closely related to HCV RNA in HCV-related MC patients.
The increased frequencies of Th17 cells, Tfh cells, Th22 cells, Treg cells, cytokines IL-17, IL-21, IL-22, and tissue-like B cells, were related to HCV infection but not type III asymptomatic MC. Higher frequencies of Th1 cells and activated memory B cells were associated with type III asymptomatic MC in HCV infection.
PMCID: PMC4501109  PMID: 26129991
Hepatitis C; Type III asymptomatic cryoglobulinemia; CD4+ T helper cells; Activated memory B cells
15.  iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation 
Virology Journal  2015;12:99.
Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear.
We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes.
As a whole, 681 (65.8 % downregulated, 34.2 % upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results.
The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0328-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4489111  PMID: 26113023
Rice; Proteome; iTRAQ; Magnesium chelatase; Peptidase; Plant defense
16.  Incidence of human adenoviruses and Hepatitis A virus in the final effluent of selected wastewater treatment plants in Eastern Cape Province, South Africa 
Virology Journal  2015;12:98.
Municipal effluent constitutes a large reservoir of human enteric viruses and bacteria. Contemporary monitoring practices rely on indicator bacteria, and do not test for viruses. Different viruses, including Norwalk-like viruses, Hepatitis A virus (HAV), adenoviruses, and rotaviruses, are important agents of illnesses in humans. The burden of disease caused by adenoviruses manifests as pneumonia, bronchiolitis, otitis media, conjunctivitis, and tonsillitis, whereas HAV infection can manifest as acute inflammatory diseases of the liver, fever, anorexia, malaise, nausea, and abdominal discomfort, followed by jaundice and dark urine. The public health implications of these viruses depend upon the physiological status of the wastewater microbial community.
The occurrence of human adenovirus (HAdV) and HAV was determined in the final effluents of five wastewater treatment plants (WWTPs) in the Eastern Cape, South Africa, over 12 months (September 2012–August 2013). The viruses were detected with real-time PCR, and conventional PCR was used for serotyping.
Adenovirus was detected in effluent samples from all five WWTPs and in 64 % of the total samples, whereas HAV was not detected in any effluent sample. At WWPT-A, samples were collected from the final effluent tank (adenoviral concentrations ranged from 1.05 × 101 to 1.10 × 104 genome/L, with a 41.7 % detection rate) and the discharge point (adenoviral concentrations ranged between 1.2 × 101 and 2.8 × 104 genome/L, with a 54.5 % detection rate). At WWPT-B, HAdV was detected in 91.7 % of samples, with viral concentrations of 7.92 × 101–2.37 × 105 genome/L. The HAdV concentrations at WWPT-C were 5.32 × 101–2.20 × 105 genome/L, and the detection rate was 75 %. The adenoviral concentrations at WWPT-D were 1.23 × 103–1.05 × 104 genome/L, and the detection rate was 66.7 %. At WWPT-E, the viral concentrations were 1.08 × 101–5.16 × 104 genome/L, and the detection rate was 54.5 %. Characterization of the adenoviruses revealed HAdV serotypes 2 (1.4 %) and 41 (7.1 %), in species C and F, respectively.
This study is the first to report the prevalence of HAdV in the final effluents of WWTPs in the Eastern Cape, South Africa. The adenoviral detection rates indicate the potential contamination of the environment, with adverse effects on public health.
PMCID: PMC4481066  PMID: 26104284
Adenovirus; Hepatitis A virus; Wastewater; Eastern Cape; Effluent; Public health
17.  Characteristics of oseltamivir-resistant influenza A (H1N1) pdm09 virus during the 2013–2014 influenza season in Mainland China 
Virology Journal  2015;12:96.
In this study, we analyzed the characteristics of oseltamivir-resistant influenza A (H1N1) pdm09 virus isolated from patients in mainland China during the influenza season from September 2013 through March 2014, and provide guidance on which antiviral to be used for clinical treatment.
The all viruses collected from September 1, 2013 through March 31, 2014 were obtained from the Chinese National Influenza Surveillance Network. A fluorescence-based assay was used to detect virus sensitivity to neuraminidase inhibitors (NAIs). The hemagglutinin (HA) and neuraminidase (NA) gene of the oseltamivir-resistant viruses were sequenced.
A total of 24 (2.14 %) influenza A (H1N1) pdm09 viruses that were resistant to oseltamivir were identified. These 24 viruses were isolated from 23 patients and no epidemiological link among them could be identified. Except for one virus with the H275H/Y mixture substitution, all the other 23 viruses had H275Y substitution in the NA protein. Sequence analysis revealed that the amino acid substitutions in the HA protein of influenza A (H1N1) pdm09 viruses with H275Y substitution isolated from mainland China were similar to the viruses from clustered cases reported in the United States, and the amino acid substitutions in the NA protein were similar to the viruses reported in Sapporo, Japan in 2013–2014. All of the oseltamivir-resistant viruses in mainland China and Japan possessed additional substitutions N386K, V241I and N369K in the NA protein, while most (>89 %) resistant-viruses from the United States during the same period possess V241I and N369K and did not have the N386K substitution. The N386K substitution was also exist in most sensitive viruses during the same period in mainland China. The amino acid substitutions in both HA and NA protein differed from the clustered cases from Australia reported in 2011 with additional substitutions. The drug-resistant influenza A(H1N1) pdm09 viruses were from patients without any known NAIs medication history prior to sampling.
During the influenza season from September 2013 through March 2014 in Mainland China, oseltamivir-resistant influenza A(H1N1)pdm09 viruses were much more frequently detected than ever since the appearance of the virus in 2009.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0317-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4484626  PMID: 26103966
Influenza A (H1N1) pdm09; 50 % Inhibitory concentration; Oseltamivir; Antiviral-resistant
18.  Initial combination anti-viral therapy with lamivudine and adefovir dipivoxil decreases short-term fatality rate of hepatitis-B-virus-related acute-on-chronic liver failure 
Virology Journal  2015;12:97.
Acute-on-chronic liver failure (ACLF) is a common serious hepatitis B virus (HBV)-related disease and has a poor prognosis. Until recently, initial combination antiviral treatment in ACLF patients was rarely reported. This study evaluated the effect of initial combination treatment with lamivudine and adefovir dipivoxil on the prognosis of HBV-related ACLF.
In this retrospective study, 131 eligible ACLF patients, including 61 treated with 100 mg lamivudine and 10 mg adefovir dipivoxil daily and 70 not treated with any nucleoside analogs (NAs), were selected and assigned into the NA and non-NA groups. All the patients received standard medicinal therapy. At weeks 0–4 and 12, serum markers for hepatic and renal functions were measured in all patients and accumulated fatality rates were calculated. Statistical analyses, including Student’s t test, χ2 test and unconditional logistic regression analysis, were performed using SPSS version 17.0 software.
Clinical data indicated that improvement of hepatic function was better in the NA than in the non-NA group. The accumulated fatality rate in the NA group was lower than in the non-NA group at weeks 2–4 and 12, and these differences were significant. Univariate analysis showed that age, prothrombin activity, model of end-stage liver disease (MELD) score, and treatment without NAs were risk factors for short-term survival of ACLF. Further research by unconditional logistic regression analysis identified that older age, high MELD score and treatment without NAs were independent risk factors for short-term survival of ACLF.
Initial combination antiviral treatment is effective in decreasing short-term fatality of HBV-related ACLF.
PMCID: PMC4501091  PMID: 26104153
Hepatitis B virus; Acute-on-chronic liver failure; Antiviral treatment; Lamivudine; Adefovir dipivoxil
19.  Efficient generation of influenza virus with a mouse RNA polymerase I-driven all-in-one plasmid 
Virology Journal  2015;12:95.
The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse PolI-driven plasmid for efficient production of influenza virus.
A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 105–106 50 % tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin–Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method.
An all-in-one plasmid and a 3-plasmid murine PolI-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust “1 + 2” approach to generate influenza vaccine seed virus.
PMCID: PMC4495709  PMID: 26093583
Influenza virus; Reverse genetics; Mouse RNA polymerase I promoter
20.  Patient-derived hepatitis C virus inhibits CD4+ but not CD8+ T lymphocyte proliferation in primary T cells 
Virology Journal  2015;12:93.
Hepatitis C virus (HCV) can replicate in cells of the immune system and productively propagate in primary T lymphocytes in vitro. We aimed to determine whether exposure to authentic, patient-derived HCV can modify the proliferation capacity, susceptibility to apoptosis and phenotype of T cells.
Primary total T cells from a healthy donor were used as targets and plasma-derived HCV from patients with chronic hepatitis C served as inocula. T cell phenotype was determined prior to and at different time points after exposure to HCV. T cell proliferation and apoptosis were measured by flow cytometry-based assays.
The HCV inocula that induced the highest intracellular expression of HCV also caused a greatest shift in the T cell phenotype from predominantly CD4-positive to CD8-positive. This shift was associated with inhibition of CD4+ but not CD8+ T cell proliferation and did not coincide with altered apoptotic death of either cell subset.
The data obtained imply that exposure to native HCV can have an impact on the relative frequencies of CD4+ and CD8+ T cells by selectively suppressing CD4+ T lymphocyte proliferation and this may occur in both the presence and the absence of measurable HCV replication in these cells. If the virus exerts a similar effect in vivo, it may contribute to the impairment of virus-specific T cell response by altering cooperation between immune cell subsets.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0322-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4474354  PMID: 26084511
HCV; HCV lymphotropism; HCV infection of T cells; T cell proliferation; T cell apoptosis; T cell cytokine expression
21.  Detection of human parvovirus 4 viremia in the follow-up blood samples from seropositive individuals suggests the existence of persistent viral replication or reactivation of latent viral infection 
Virology Journal  2015;12:94.
The transmission routes for human parvovirus 4 (PARV4) infections in areas with high seroprevalence are not known. In the work described here, persistent PARV4 viral replication was investigated by conducting a longitudinal study.
Ten healthcare workers each provided a blood sample at the beginning of the study (first sample) and 12 months later (second sample). The paired samples were tested for PARV4-positivity by immunoblotting analysis and nested polymerase chain reactions.
IgG antibodies against PARV4 were detected in six participants, three of whom also had IgM antibodies against PARV4. The immunoblotting results did not vary over time. PARV4 DNA was detected in the first blood sample from one participant who had IgG antibodies against PARV4 and in the second blood samples from 2 participants who had IgG and IgM antibodies against PARV4.
Detection of PARV4 DNA in the second blood samples from two seropositive participants suggests the existence of persistent PARV4 replication or reactivation of inactive virus in the tissues. The finding of persistent or intermittent PARV4 replication in individuals with past infections provides an important clue toward unraveling the non-parenteral transmission routes of PARV4 infection in areas where the virus is endemic.
PMCID: PMC4480887  PMID: 26088443
Parvovirus 4; Persistent PARV4 infection; Latent PARV4 infection; Anti-PARV4 IgM
22.  Outbreak of febrile illness caused by coxsackievirus A4 in a nursery school in Beijing, China 
Virology Journal  2015;12:92.
Coxsackievirus A4 (CV-A4) is classified as human enterovirus A according to its serotype. CV-A4, an etiological agent of hand, foot, and mouth disease, affects children worldwide and can circulate in closed environments such as schools and hospitals for long periods.
An outbreak of febrile illness at a nursery school in Beijing, China, was confirmed to be caused by CV-A4. Phylogenetic analysis of the complete genome of the isolated strain showed that the virus belongs to the same cluster as the predominant CV-A4 strain in China. This outbreak was controlled by effective measures.
The early identification of the pathogen and timely intervention may be the most critical factors in controlling an outbreak caused by CV-A4 in a preschool.
PMCID: PMC4495935  PMID: 26084565
Coxsackievirus A4; Outbreak; Preschool; Prevention; Control
23.  Successful rescue of disseminated varicella infection with multiple organ failure in a pediatric living donor liver transplant recipient: a case report and literature review 
Virology Journal  2015;12:91.
A 12-year-old female patient with biliary atresia underwent living donor liver transplantation (LDLT). Twelve months after the LDLT, she developed acute hepatitis (alanine aminotransferase 584 IU/L) and was diagnosed with disseminated varicella-zoster virus (VZV) infection with high level of serum VZV-DNA (1.5 × 105 copies/mL) and generalized vesicular rash. She had received the VZV vaccination when she was 5-years-old and had not been exposed to chicken pox before the LDLT, and her serum was positive for VZV immunoglobulin G at the time of the LDLT. Although she underwent treatment with intravenous acyclovir, intravenous immunoglobulin, and withdrawal of immunosuppressants, her symptoms worsened and were accompanied by disseminated intravascular coagulation, pneumonia, and encephalitis. These complications required treatment in the intensive care unit for 16 days. Five weeks later, her clinical findings improved, although her VZV-DNA levels remained high (8.5 × 103copies/mL). Oral acyclovir was added for 2 weeks, and she was eventually discharged from our hospital on day 86 after admission; she has not experienced a recurrence. In conclusion, although disseminated VZV infection with multiple organ failure after pediatric LDLT is a life-threatening disease, it can be cured via an early diagnosis and intensive treatment.
PMCID: PMC4480512  PMID: 26081644
Disseminated varicella-zoster infection; Living donor liver transplantation; Multiple organ failure; VZV-DNA PCR
24.  Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg 
Virology Journal  2015;12:89.
Approaches to simplify and accelerate the construction of full-length infectious cDNA clones for plant potyviruses have been described, based on cloning strategies involving in vitro ligation or homologous recombination in yeast. In the present study, we developed a faster and more efficient in vitro recombination system using Gibson assembly (GA), to engineer a Lettuce mosaic virus (LMV) infectious clone expressing an ectopic mcherry-tagged VPg (Viral protein genome-linked) for in planta subcellular localization of the viral protein in an infection context.
Three overlapping long distance PCR fragments were amplified and assembled in a single-step process based on in vitro recombination (Gibson assembly). The resulting 17.5 kbp recombinant plasmids (LMVmchVPg_Ec) were inoculated by biolistic on lettuce plants and then propagated mechanically on Nicotiana benthamiana. Confocal microscopy was used to analyze the subcellular localization of the ectopically expressed mcherry-VPg fusion protein.
The Gibson assembly allowed the cloning of the expected plasmids without any deletion. All the inoculated plants displayed symptoms characteristic of LMV infection. The majority of the mcherry fluorescent signal observed using confocal microscopy was located in the nucleus and nucleolus as expected for a potyviral VPg.
This is the first report of the use of the Gibson assembly method to construct full-length infectious cDNA clones of a potyvirus genome. This is also the first description of the ectopic expression of a tagged version of a potyviral VPg without affecting the viability of the recombinant potyvirus.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0315-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4475333  PMID: 26070311
Full-length infectious clone; Gibson assembly; Potyvirus; VPg; Improved cloning
25.  Macaque models of enhanced susceptibility to HIV 
Virology Journal  2015;12:90.
There are few nonhuman primate models of enhanced HIV susceptibility. Such models can improve comprehension of HIV acquisition risk factors and provide rigorous testing platforms for preclinical prevention strategies. This paper reviews past, current, and proposed research on macaque HIV acquisition risk models and identifies areas where modeling is significantly lacking. We compare different experimental approaches and provide practical considerations for designing macaque susceptibility studies. Modifiable (mucosal and systemic coinfections, hormonal contraception, and rectal lubricants) and non-modifiable (hormonal fluctuations) risk factors are highlighted. Risk acquisition models via vaginal, rectal, and penile challenge routes are discussed. There is no consensus on the best statistical model for evaluating increased susceptibility, and additional research is required. The use of enhanced susceptibility macaque models would benefit multiple facets of the HIV research field, including basic acquisition and pathogenesis studies as well as the vaccine and other biomedical preventions pipeline.
PMCID: PMC4479314  PMID: 26070461
SIV; SHIV; HIV; Susceptibility; Risk; Nonhuman primate model; Coinfection model

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