The zinc finger antiviral protein (ZAP) is a mammalian host restriction factor, and it could inhibit HBV RNA synthesis in vitro experiments. However, the role of ZAP against HBV in vivo environment is unclear. This study aimed to investigate whether ZAP could act against HBV transcription and replication in ZAP tansgenic mouse model.
HBV-replication-competent plasmid pHBV4.1 was transferred to ZAP transgenic ICR mouse via the tail vein using a hydrodynamic in vivo transfection procedure, and ICR mouse were used as controls. HBV RNA and HBV DNA replication intermediates in the liver were respectively analyzed by Northern blotting and Southern blotting. The expression of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in the liver tissue was detected by immunohistochemical staining.
As compared to ICR control mouse, the levels of 3.5 kb mRNA in ZAP transgenic mouse were decreased by 8.4%; while the level of HBV DNA replication intermediates was decreased by 82%. In addition, the expression of HBsAg and HBcAg in ZAP transgenic mouse liver were both significantly less than that of ICR control mouse.
Our findings suggest that ZAP could inhibit HBV replication in vivo in mice, which offers a new target for anti-HBV drug development.
Zinc-finger antiviral protein; Hepatitis B virus; Transgenic mouse; Transcription and replication
BK polyomavirus infects most of the general population. However, its clinical manifestations are almost exclusively seen in immunocompromised patients, particularly in kidney and hematopoietic stem cell transplantation recipients.
A 15-y-old female suffering from common B-cell acute lymphoblastic leukaemia underwent hematopoietic stem cell transplantation. The patient had reactivation of BKPyV infection and developed an haemorrhagic cystitis. Three months after transplant, BKPyV viremia and viruria increased and she developed a severe nephropathy associated to a polyclonal gammopathy with high levels of isolated IgM.
This case report describes a rare and unexpected polyclonal gammopathy developed during a polyomavirus-associated nephropathy confirmed by immunohistochemical and laboratory analyses.
BKPyV; HSCT; Polyclonal gammopathy; Polyomavirus-associated nephropathy
Infectious arthritis in broilers represents an economic and health problem, resulting in severe losses due to retarded growth and downgrading at the slaughterhouse. The most common agents associated with cases of infectious arthritis in poultry are avian reovirus (ARV) and Mycoplasma synoviae (MS). The accurate differentiation and rapid diagnosis of ARV and MS are essential prerequisites for the effective control and prevention of these avian pathogens in poultry flocks. This study thus aimed to develop and validate a duplex real-time PCR assay for the simultaneous detection and quantification of ARV and MS.
Specific primers and probes for each pathogen were designed to target the special sequence of the ARV σC gene or the MS phase-variable surface lipoprotein hemagglutinin (vlhA) gene. A duplex real-time PCR assay was developed, and the reaction conditions were optimized for the rapid detection and quantification of ARV and MS.
The duplex real-time PCR assay was capable of ARV- and MS-specific detection without cross-reaction with other non-targeted avian pathogens. The sensitivity of this assay was 2 × 101 copies for a recombinant plasmid containing ARV σC or MS vlhA gene, and 100 times higher than that of conventional PCR. This newly developed PCR assay was also reproducible and stable. All tested field samples of ARV and/or MS were detectable with this duplex real-time PCR assay compared with pathogen isolation and identification as well as serological tests.
This duplex real-time PCR assay is highly specific, sensitive and reproducible and thus could provide a rapid, specific and sensitive diagnostic tool for the simultaneous detection of ARV and MS in poultry flocks. The assay will be useful not only for clinical diagnostics and disease surveillance but also for the efficient control and prevention of ARV and MS infections.
Duplex real-time PCR assay; Avian reovirus; Mycoplasma synoviae
Schistosomiasis is a significant health problem in more than 70 countries distributed between Africa, Asia and South America, with an infection rate of one in 30 individuals. Data on Schistosomiasis, Hepatitis B virus (HBV) and Hepatitis C virus (HCV) co-infection are scarce; however, there is a high prevalence in countries where schistosomiasis is endemic.
A systematic search was performed on published data from 1980–2014. Published papers in the databases Google, Medline, PubMed, and MiPc library were searched using the keywords epidemiology, pathogenesis and outcomes of HBV, HCV and schistosomiasis and data were extracted from the relevant studies.
The prevalence of HBV/schistosomiasis co-infection in countries where schistosomiasis is endemic was high, ranging between 9.6 to approximately 64% in Egypt, and a maximum of 15.8% among hospitalized patients in Brazil. Concurrent infection between HBV and schistosomiasis is often associated with countries where schistosomiasis is endemic and may lead to chronic liver inflammation. Similarly, HCV infection rates in schistosomiasis populations range from 1% in Ethiopia reaching up to 50% in Egypt.
There is controversy regarding the effects of HBV and HCV on schistosomiasis and vice versa. Vaccination might be a solution to the era of schistosomiasis and co-infection with HBV and HCV.
Schistosomiasis; HBV; HCV; Hepatotropic; Vaccine; Adjuvant
Dengue has emerged as the most significant of arboviral diseases in the 21st century. It is endemic to >100 tropical and sub-tropical countries around the world placing an estimated 3.6 billion people at risk. It is caused by four genetically similar but antigenically distinct, serotypes of dengue viruses. There is neither a vaccine to prevent nor a drug to treat dengue infections, at the present time. The major objective of this work was to explore the possibility of identifying a small molecule inhibitor of the dengue virus protease and assessing its ability to suppress viral replication in cultured cells.
We cloned, expressed and purified recombinant dengue virus type 2 protease. Using an optimized and validated fluorogenic peptide substrate cleavage assay to monitor the activity of this cloned dengue protease we randomly screened ~1000 small molecules from an ‘in-house’ library to identify potential dengue protease inhibitors.
A benzimidazole derivative, named MB21, was found to be the most potent in inhibiting the cloned protease (IC50 = 5.95 μM). In silico docking analysis indicated that MB21 binds to the protease in the vicinity of the active site. Analysis of kinetic parameters of the enzyme reaction suggested that MB21 presumably functions as a mixed type inhibitor. Significantly, this molecule identified as an inhibitor of dengue type 2 protease was also effective in inhibiting each one of the four serotypes of dengue viruses in infected cells in culture, based on analysis of viral antigen synthesis and infectious virus production. Interestingly, MB21 did not manifest any discernible cytotoxicity.
This work strengthens the notion that a single drug molecule can be effective against all four dengue virus serotypes. The molecule MB21 could be a potential candidate for ‘hit-to-lead’ optimization, and may pave the way towards developing a pan-dengue virus antiviral drug.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0248-x) contains supplementary material, which is available to authorized users.
Dengue fever; Dengue virus; NS2b-NS3 protease; Dengue protease inhibitor; Antiviral therapy
Hepatitis A virus (HAV) epidemiology in Tunisia has changed from high to intermediate endemicity in the last decades. However, several outbreaks continue to occur. The last reported sequences from Tunisian HAV strains date back to 2006. In order to provide an updated overview of the strains currently circulating in Tunisia, a large-scale molecular analysis of samples from hepatitis A cases was performed, the first in Tunisia.
Biological samples were collected from patients with laboratory confirmed hepatitis A: 145 sera samples in Tunis, Monastir, Sousse and Kairouan from 2008 to 2013 and 45 stool samples in Mahdia in 2009. HAV isolates were characterised by nested RT-PCR (VP1/2A region) and sequencing. The sequences finally obtained from 81 samples showed 78 genotype IA and 3 genotype IB isolates.
A Tunisian genotype IA sequence dataset, including both the 78 newly obtained IA sequences and 51 sequences retrieved from GenBank, was used for phylogenetic investigation, including analysis of migration pattern among six towns. Virus gene flow from Sfax and Monastir was directed to all other towns; in contrast, the gene flows from Sousse, Tunis, Mahdia and Kairouan were directed to three, two, one and no towns, respectively.
Several different HAV strains co-circulate in Tunisia, but the predominant genotype still continues to be IA (78/81, 96% isolates). A complex gene flow (migration) of HAV genotype IA was observed, with Sfax and Monastir showing gene flows to all other investigated towns. This approach coupled to a wider sampling can prove useful to investigate the factors underlying the spread of HAV in Tunisia and, thus, to implement appropriate preventing measures.
HAV; Sequencing; Phylogenetic analysis; Viral gene flow
Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 184.108.40.206 (previously provisionally referred to as clade 220.127.116.11), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms.
Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 18.104.22.168 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 22.214.171.124 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes.
Laos influenza A(H5N6) virus representative of proposed clade 126.96.36.199, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.
Influenza virus; Avian influenza; Reassortant virus; Molecular diagnosis; H5N6; Clade 188.8.131.52; PCR
Border disease virus (BDV) causes border disease (BD) affecting mainly sheep and goats worldwide. BDV in goat herds suffering diarrhea was recently reported in China, however, infection in sheep was undetermined. Here, BDV infections of sheep herds in Jiangsu, China were screened; a BDV strain was isolated and identified from the sheep flocks in China. The genomic characteristics and pathogenesis of this new isolate were studied.
In 2012, samples from 160 animals in 5 regions of Jiangsu province of China were screened for the presence of BDV genomic RNA and antibody by RT-PCR and ELISA, respectively. 44.4% of the sera were detected positively, and one slowly grown sheep was analyzed to be pestivirus RNA positive and antibody-negative. The sheep kept virus positive and antibody negative in the next 6 months of whole fattening period, and was defined as persistent infection (PI). The virus was isolated in MDBK cells without cytopathic effect (CPE) and named as JSLS12-01. Near-full-length genome sequenced was 12,227 nucleotides (nt). Phylogenetic analysis based on 5'-UTR and Npro fragments showed that the strain belonged to genotype 3, and shared varied homology with the other 3 BDV strains previously isolated from Chinese goats. The genome sequence of JSLS12-01 also had the highest homology with genotype BDV-3 (the strain Gifhorn). Experimental infections of sheep had mild clinical signs as depression and short-period mild fever (5 days). Viremia was detected in 1–7 days post-infection (dpi), and seroconversion began after 14 dpi.
This study reported the genomic and pathogenesis characterizations of one sheep BDV strain, which confirmed the occurrence of BDV infection in Chinese sheep. This sheep derived BDV strain was classified as BDV-3, together with the goat derived strains in China. These results might be helpful for further understanding of BDV infection in China and useful for prevention and control of BDV infections in the future.
BDV; Complete genome sequence; Phylogenetic analysis; Experimental infection
Human challenge models using respiratory viruses such as influenza are increasingly utilised in the development of novel vaccines and anti-viral modalities and can provide preliminary evidence of protection before evaluation in field trials. We describe the results of a clinical study characterising an A/H1N1 influenza challenge virus in humans.
The challenge agent, influenza A/California/2009 (H1N1), was manufactured under cGMP conditions and characterised in accordance with regulatory guidelines. A dose-ascending open-label clinical study was conducted in 29 healthy young adults screened sero-negative to the challenge strain. Subjects were intranasally inoculated with three increasing doses of virus and physician-reported signs, subjected-reported symptoms, viral shedding and immunological responses were monitored.
A dose-dependent increase in clinical signs and symptoms was observed with 75% of subjects developing laboratory-confirmed illness at the highest inoculum (3.5 × 106 TCID50). At the highest dose, physician or subject-reported signs of infection were classified as mild (all subjects), moderate (50%) and severe (16%) with peak symptoms recorded four days after infection. Clinical signs were correlated with nasal mucus weight (P < .001) and subject-reported symptoms (P < .001). Geometric mean peak viral shedding was log10 5.16 TCID50 and occurred three days after inoculation with a median duration of five days. The safety profile was such that physiological responses to viral infection were mainly restricted to the upper airways but were not of such severity to be of clinical concern.
A highly characterised wild-type Influenza A/California/2009 (H1N1) virus manufactured for clinical use was shown to induce a good infectivity profile in human volunteers. This clinical challenge model can be used for evaluating potential efficacy of vaccines and anti-viral therapeutics.
Electronic supplementary material
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Influenza A/H1N1 virus; Challenge agent; Vaccine; Clinical trial
This study reviews the FMDV receptor-binding domain, integrin receptors, and heparan sulfate receptors to provide references for studies regarding the mechanisms underlying FMDV infection.
Foot-and-mouth disease virus; Receptor; Infection
Hepatitis C virus (HCV) infection was recently recognized as an independent risk factor for insulin resistance (IR), the onset phase of type 2 diabetes mellitus (T2DM). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates PI3K/Akt signaling pathway, which is critical for IR development and progression of cirrhosis to hepatocellular carcinoma (HCC). Here, we investigate the role of PTEN in HCV-associated IR and explored the mechanisms by which HCV regulates PTEN.
Western blotting was used to detect the levels of insulin signaling pathway components, including insulin receptor substrate-1 (IRS-1), phosphorylated IRS-1 (pIRS-1) at serine 307 (Ser307), both phosphorylated Akt (pAkt) and total Akt. A time-course experiment measuring activation of the insulin signaling pathway was performed to assess the effect of HCV infection on insulin sensitivity by examining the phosphorylation levels of Akt and GSK3β, a downstream target of Akt. Huh7.5.1 cells were transduced with a lentiviral vector expressing PTEN or PTEN shRNA, and IRS-1 and pIRS-1 (Ser307) levels were determined in both HCV-infected and uninfected cells. The pc-JFH1-core plasmid was constructed to explore the underlying mechanisms by which HCV regulated PTEN and therefore IRS-1 levels.
HCV infection inhibited the insulin signaling pathway by reducing the levels of IRS-1 and pAkt/Akt while increasing phosphorylation of IRS-1 Ser307. In addition, HCV infection decreased the sensitivity to insulin-induced stimulation by inhibiting Akt and GSK3β phosphorylation. Furthermore, PTEN mRNA and protein levels were reduced upon HCV infection as well as transfection with the pc-JFH1-core plasmid. The reduction in IRS-1 level observed in HCV-infected cells was rescued to a limited extent by overexpression of PTEN, which in turn slightly reduced pIRS-1 (Ser307) level. In contrast, IRS-1 level were significantly decreased and phosphorylation of IRS-1 at Ser-307 was strongly enhanced by PTEN knockdown, suggesting that both reduction in IRS-1 level and increase in IRS-1 phosphorylation at Ser307 upon HCV infection occurred in a PTEN-dependent manner.
HCV infection suppresses the insulin signaling pathway and promotes IR by repressing PTEN, subsequently leading to decreased levels of IRS-1 and increased levels of pIRS-1 at Ser307. The findings provide new insight on the mechanism of HCV-associated IR.
Hepatitis C virus; Insulin resistance; IRS-1; pIRS-1 Ser307; PTEN
After the 1968 H3N2 pandemic emerged in humans, H3N2 influenza viruses continuously circulated and evolved in nature. An H3N2 variant was circulating in humans in the 1990s and subsequently introduced into the pig population in the 2000s. This virus gradually became the main subtype of swine influenza virus worldwide. However, there were no reports of infections in dogs with this virus.
In 2013, 35 nasal swabs from pet dogs were positive for Influenza A virus by RT-PCR. Two viruses were isolated and genetically characterized. In the phylogenetic trees of all gene segments, two H3N2 canine isolates clustered with Moscow/10/99 and most H3N2 swine influenza viruses. These results indicated that two H3N2 CIVs possessed high homology with human/swine influenza viruses, which at the same time exhibited some amino acid substitutions in NA, polymerase basic protein 1 (PB1), and nucleoprotein (NP), which probably were related to the interspecies transmission.
These two viruses share the highest homology with swine H3N2, Moscow/99-like viruses, which indicated that these viruses might originate from swine viruses.
Pet dogs; H3N2 subtype; Human-like influenza viruses
Hepatitis C (HCV) is a serious public health issue, and it is estimated that 3% of the world’s population is infected. Patients in hemodialysis units have an increased risk for contracting HCV, and high prevalence rates have been found in hemodialysis units around the world. This study is aimed at determining the prevalence of HCV in patients with terminal chronic renal disease (tCRD) who have been submitted to hemodialysis and peritoneal dialysis in southern Brazil to characterize the most prevalent genotypes, the viral load, and possible risk factors and to assess the validity between the ELISA and RT-PCR detection methods. Of 320 patients from three dialysis units, 318 participated in this study. According to the medical records, 55 patients were reactive to HCV, as determined via ELISA. All 318 samples were submitted to RT-PCR and genotyped using an Abbott Realtime® m2000 system. Data obtained through a questionnaire and chemical variables were associated with the HCV. Results: The prevalence of HCV was 18.24% (58), and the concordance between the HCV serology and the RT-PCR was 94%. Three patients were diagnosed to be negative for HCV using the ELISA assay but positive when using RT-PCR. Genotype 1 was the most prevalent (46.7%) genotype, within which subtype 1a was the most frequent (74.1%). One of the risk factors associated with HCV infection was the length of time that the patient had been undergoing hemodialysis treatments (p < 0.001). Additionally, the viral load was found to vary when tested before and after hemodialysis (p < 0.001). Conclusion: The prevalence of HCV in dialysis units continues to remain high, indicating nosocomial contamination. RT-PCR detected the presence of the hepatitis C virus in patients with a non-reactive serology, which highlights the importance of performing molecular tests on dialysis patients. The variation in the viral load in patients submitted to hemodialysis indicates a possible destruction or gripping of viral particles to the dialyzer membrane.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0238-z) contains supplementary material, which is available to authorized users.
The human polyomavirus BK expresses a 66 amino-acid peptide referred to as agnoprotein. Though mutants lacking agnoprotein are severely reduced in producing infectious virions, the exact function of this peptide remains incompletely understood. To elucidate the function of agnoprotein, we searched for novel cellular interaction partners.
Yeast-two hybrid assay was performed with agnoprotein as bait against human kidney and thymus libraries. The interaction between agnoprotein and putative partners was further examined by GST pull down, co-immunoprecipitation, and fluorescence resonance energy transfer studies. Biochemical and biological studies were performed to examine the functional implication of the interaction of agnoprotein with cellular target proteins.
Proliferating cell nuclear antigen (PCNA), which acts as a processivity factor for DNA polymerase δ, was identified as an interaction partner. The interaction between agnoprotein and PCNA is direct and occurs also in human cells. Agnoprotein exerts an inhibitory effect on PCNA-dependent DNA synthesis in vitro and reduces cell proliferation when ectopically expressed. Overexpression of PCNA restores agnoprotein-mediated inhibition of cell proliferation.
Our data suggest that PCNA is a genuine interaction partner of agnoprotein and the inhibitory effect on PCNA-dependent DNA synthesis by the agnoprotein may play a role in switching off (viral) DNA replication late in the viral replication cycle when assembly of replicated genomes and synthesized viral capsid proteins occurs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-014-0220-1) contains supplementary material, which is available to authorized users.
Agnoprotein; Cell proliferation; DNA polymerase; DNA replication; PCNA; Polyomaviruses
Accumulated evidence gathered over recent decades demonstrated that some members of the Parvoviridae family, in particular the rodent protoparvoviruses H-1PV, the minute virus of mice and LuIII have natural anticancer activity while being nonpathogenic to humans. These studies have laid the foundations for the launch of a first phase I/IIa clinical trial, in which the rat H-1 parvovirus is presently undergoing evaluation for its safety and first signs of efficacy in patients with glioblastoma multiforme. After a brief overview of the biology of parvoviruses, this review focuses on the studies which unraveled the antineoplastic properties of these agents and supported their clinical use as anticancer therapeutics. Furthermore, the development of novel parvovirus-based anticancer strategies with enhanced specificity and efficacy is discussed, in particular the development of second and third generation vectors and the combination of parvoviruses with other anticancer agents. Lastly, we address the key challenges that remain towards a more rational and efficient use of oncolytic parvoviruses in clinical settings, and discuss how a better understanding of the virus life-cycle and of the cellular factors involved in virus infection, replication and cytotoxicity may promote the further development of parvovirus-based anticancer therapies, open new prospects for treatment and hopefully improve clinical outcome.
Oncolytic parvoviruses; Parvovirus-based cancer virotherapy; Parvovirus-based oncolytic vectors; Parvovirus-based combination therapy; Rodent protoparvovirus; Glioblastoma multiforme; Oncolytic virotherapy clinical trial
In recent years, a number of serious disease outbreaks caused by viruses and viroids on greenhouse tomatoes in North America have resulted in significant economic losses to growers. The objectives of this study were to evaluate the effectiveness of commercial disinfectants against mechanical transmission of these pathogens, and to select disinfectants with broad spectrum reactivity to control general virus and viroid diseases in greenhouse tomato production.
A total of 16 disinfectants were evaluated against Pepino mosaic virus (PepMV), Potato spindle tuber viroid (PSTVd), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). The efficacy of each disinfectant to deactivate the pathogen’s infectivity was evaluated in replicate experiments from at least three independent experiments. Any infectivity that remained in the treated solutions was assessed through bioassays on susceptible tomato plants through mechanical inoculation using inocula that had been exposed with the individual disinfectant for three short time periods (0–10 sec, 30 sec and 60 sec). A positive infection on the inoculated plant was determined through symptom observation and confirmed with enzyme-linked immunosorbent assay (PepMV, ToMV, and TMV) and real-time reverse transcription-PCR (PSTVd). Experimental data were analyzed using Logistic regression and the Bayesian methodology.
Statistical analyses using logistic regression and the Bayesian methodology indicated that two disinfectants (2% Virkon S and 10% Clorox regular bleach) were the most effective to prevent transmission of PepMV, PSTVd, ToMV, and TMV from mechanical inoculation. Lysol all-purpose cleaner (50%) and nonfat dry milk (20%) were also effective against ToMV and TMV, but with only partial effects for PepMV and PSTVd.
With the broad spectrum efficacy against three common viruses and a viroid, several disinfectants, including 2% Virkon S, 10% Clorox regular bleach and 20% nonfat dry milk, are recommend to greenhouse facilities for consideration to prevent general virus and viroid infection on tomato plants.
Pepino mosaic virus; Tomato mosaic virus; Tobacco mosaic virus; Potato spindle tuber viroid; Disinfectant; Mechanical transmission; Greenhouse tomato
Despite the easy accessibility and diagnostic utility of PBMCs and their potential to show distinct expression patterns associated with the accelerated disease progression in HIV/HCV co-infection, there has not been a systematic study focusing on the global dysregulations of the biological pathways in PBMCs from HIV, HCV mono- and co-infected individuals. This study aimed at identifying the transcriptome distinctions of PBMCs between these patient groups.
Genome-wide transcriptomes of PBMCs from 10 HIV/HCV co-infected patients, 7 HIV+ patients, 5 HCV+ patients, and 5 HIV/HCV sero-negative healthy controls were analyzed using Illumina microarray. Pairwise comparisons were performed to identify differentially expressed genes (DEGs), followed by gene set enrichment analysis (GSEA) to detect the global dysregulations of the biological pathways between HIV, HCV mono- and co-infection.
Forty-one, 262, and 44 DEGs with fold change > 1.5 and FDR (false discovery rate) <0.05 for the comparisons of HCV versus co-infection, HIV versus co-infection, and HIV versus HCV were identified, respectively. Significantly altered pathways (FDR < 0.05), featured by those involved in immune system, signaling transduction, and cell cycle, were detected. Notably, the differential regulation of cytotoxicity pathway discriminated between HIV, HCV mono- and co-infection (up-regulated in the former versus the latter group: co-infection versus HIV or HCV, HIV versus HCV; FDR <0.001 ~ 0.019). Conversely, the cytokine-cytokine receptor interaction pathway was down-regulated in co-infection versus either HCV (FDR = 0.003) or HIV (FDR = 0.028). For the comparison of HIV versus HCV, the cell cycle (FDR = 0.016) and WNT signaling (FDR = 0.006) pathways were up- and down-regulated in HIV, respectively.
Our study is the first to identify the differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection, which may reflect the distinct patterns of virus-host cell interactions underlying disease progression. Further inspection of cytotoxicity pathway has pinned down to the expression of the KIR genes to be associated with specific patterns of particular virus-host interactions. Between HIV and HCV, the altered cell cycle and WNT signaling pathways may suggest the different impact of HIV and HCV on cell proliferation and differentiation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-014-0236-6) contains supplementary material, which is available to authorized users.
HIV; HCV; HIV/HCV co-infection; Transcriptome; Cytotoxicity pathway
An effective human immunodeficiency virus type 1 (HIV-1) vaccine is expected to have the greatest impact on HIV-1 spread and remains a global scientific priority. Only one candidate vaccine has significantly reduced HIV-1 acquisition, yet at a limited efficacy of 31%, and none have delayed disease progression in vaccinated individuals. Thus, the challenge remains to develop HIV-1 immunogens that will elicit protective immunity. A combination of two independent approaches - namely the elicitation of broadly neutralising antibodies (bNAb) to prevent or reduce acquisition of infection and stimulation of effective cytotoxic T lymphocyte (CTL) responses to slow disease progression in breakthrough infections (recent evidence suggests that CTLs could also block HIV-1 from establishing persistent infection) – is the current ideal. The purpose of this review is to summarise strategies and progress in the design and testing of HIV-1 immunogens to elicit bNAb and protective CTL immune responses. Recent advances in mimicking the functional native envelope trimer structure and in designing structurally-stabilised bNAb epitope forms to drive development of germline precursors to mature bNAb are highlighted. Systematic or computational approaches to T cell immunogen design aimed at covering viral diversity, increasing the breadth of immune responses and/or reducing viable viral escape are discussed. We also discuss a recent novel vaccine vector approach shown to induce extremely broad and persistent T cell responses that could clear highly pathogenic simian immunodeficiency virus (SIV) early after infection in the monkey model. While in vitro and animal model data are promising, Phase II and III human clinical trials are ultimately needed to determine the efficacy of immunogen design approaches.
HIV-1 vaccine; Immunogen design; Broadly neutralising antibodies; T cell immunogens
The most common human papillomavirus (HPV) genotypes isolated from cervical cancer in select African countries are HPV-16, HPV-18, HPV-35, and HPV-45, but the most common genotypes in Zambia are unknown. The overall objective of this study was to assess the potential impact of current HPV vaccines in preventing cervical cancer in Zambia, by determining the combined prevalence of HPV-16 and/or HPV-18 in invasive cervical cancer (ICC) and high-grade pre-cancer [cervical intraepithelial neoplasia 2 or 3 (CIN2/3)] cases.
We compared DNA extraction techniques to determine which assay performs well in the Zambian context, where unbuffered formalin is used to fix specimens. We then tested specimens with the Abbott RealTime High-Risk HPV test to estimate the prevalence of HPV-16/18 in formalin-fixed, paraffin-embedded ICC and CIN2/3 specimens. DNA extraction using heat (without xylene) was more successful than xylene-based extraction. Over 80% of specimens tested using heat extraction and the Abbott RealTime HPV test were positive for HPV. HPV-16 and/or HPV-18 were identified in 65/93 (69.9%) ICC specimens positive for HPV and in 38/65 (58.5%) CIN2/3 specimens positive for HPV.
To our knowledge this is the first report to identify HPV genotypes in cervical cancers in Zambia. A combined HPV-16/18 prevalence of 69.9% in ICC specimens suggests that current vaccines will be highly protective against cervical cancer in Zambia.
Cervical cancer; Formalin-fixed paraffin-embedded; Human papillomavirus; Zambia
Dengue viruses (DENVs) are mosquito-borne viruses which can cause disease ranging from mild fever to severe dengue infection. These viruses are endemic in several tropical and subtropical regions. Multiple outbreaks of DENV serotypes 1, 2 and 3 (DENV-1, DENV-2 and DENV-3) have been reported from the western region in Saudi Arabia since 1994. Strains from at least two genotypes of DENV-1 (Asia and America/Africa genotypes) have been circulating in western Saudi Arabia until 2006. However, all previous studies reported from Saudi Arabia were based on partial sequencing data of the envelope (E) gene without any reports of full genome sequences for any DENV serotypes circulating in Saudi Arabia.
Here, we report the isolation and the first complete genome sequence of a DENV-1 strain (DENV-1-Jeddah-1-2011) isolated from a patient from Jeddah, Saudi Arabia in 2011. Whole genome sequence alignment and phylogenetic analysis showed high similarity between DENV-1-Jeddah-1-2011 strain and D1/H/IMTSSA/98/606 isolate (Asian genotype) reported from Djibouti in 1998. Further analysis of the full envelope gene revealed a close relationship between DENV-1-Jeddah-1-2011 strain and isolates reported between 2004–2006 from Jeddah as well as recent isolates from Somalia, suggesting the widespread of the Asian genotype in this region.
These data suggest that strains belonging to the Asian genotype might have been introduced into Saudi Arabia long before 2004 most probably by African pilgrims and continued to circulate in western Saudi Arabia at least until 2011. Most importantly, these results indicate that pilgrims from dengue endemic regions can play an important role in the spread of new DENVs in Saudi Arabia and the rest of the world. Therefore, availability of complete genome sequences would serve as a reference for future epidemiological studies of DENV-1 viruses.
Dengue virus; Full genome; Phylogenetic analysis; Diversity; Jeddah; Saudi Arabia
H9N2 avian influenza virus (AIV) becomes the focus for its ability of transmission to mammals and as a donor to provide internal genes to form the new epidemic lethal influenza viruses. Residue 627 in PB2 has been proven the virulence factor of H9N2 avian influenza virus in mice, but the detailed data for inflammation difference between H9N2 virus strains with site 627 mutation is still unclear. The inflammasome NLRP3 is recently reported as the cellular machinery responsible for activation of inflammatory processes and plays an important role during the development of inflammation caused by influenza virus infection.
In this study, we investigated the expression pattern of NLRP3 and its related cytokines of IL-1β and TNF-α in BALB/c mice infected by H9N2 AIV strains with only a site 627 difference at both mRNA and protein levels at different time points.
The results showed that the expression level of NLRP3, IL-1β and TNF-α changed in the lung and brain of BALB/c mice after infection by VK627 and rVK627E. The immunohistological results showed that the positive cells of NLRP3, IL-1β and TNF-α altered the positive levels of original cells in tissues and infiltrated inflammatory cells which caused by H9N2 infection.
Our results provided the basic data at differences in expression pattern of NLRP3 and its related cytokines in BALB/c mice infected by H9N2 influenza viruses with only a site 627 difference. This implied that NLRP3 inflammasome plays a role in host response to influenza virus infection and determines the outcome of clinical manifestation and pathological injury. This will explain the variable of pathological presentation in tissues and enhance research on inflammation process of the AIV H9N2 infection.
Avian influenza virus; H9N2; BALB/c mice; NLRP3; IL-1β; TNF-α
Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified.
Epitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle.
In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.
Encephalomyocarditis virus (EMCV); VP1; McAbs; Epitopes
Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.
In this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes.
Seventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).
Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.
Influenza A virus; Nucleoprotein; Yeast two-hybrid; Host restriction
The mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) signaling pathway is involved in viral life cycle. However, the effect of MEK/ERK pathway in enterovirus 71(EV71)-infected immature dendritic cells (iDCs) is still unclear.
Human peripheral blood mononuclear cells (PBMCs) were isolated and induced to generate iDCs. Unifected iDCs and EV71-infected iDCs with a multiplicity of infection (MOI = 5) were analyzed by flow cytometry. Differential gene expressions of MEK/ERK signaling pathway molecules in EV71-infected iDCs were performed by PCR arrays. The phosphorylation of MEK/ERK pathway molecules in EV71-infected iDCs preincubated without or with U0126 (20 μM) at indicated times was detected by Western blot. The concentrations of IL-1α, IL-2, IL-6, IL-12, TNF-α, IFN-α1, IFN-β and IFN-γ in culture supernatant were analyzed by the luminex fluorescent technique.
When iDCs were infected with EV71 for 24 h, the percentage of CD80, CD83, CD86 and HLA-DR expressed on iDCs significantly increased. PCR arrays showed that gene expressions of molecules in MEK/ERK signaling pathway were remarkably upregulated in EV71-infected iDCs. EV71 infection activated both MEK1/2 and ERK1/2, which phosphorylated their downstream transcription factor c-Fos, c-Jun, c-myc and Elk1. Importantly, the treatment of U0126 significantly inhibited MEK/ERK signaling pathway molecules and severely impaired virus replication., Additionally, EV71 infection promoted the expression of son of sevenless (SOS1) and increased the secretion of IL-1α, IL-2, IL-6, IL-12, TNF-α,IFN-β and IFN-γ. Furthermore,the release of IL-1α, IL-2,IL-6 and TNF-α could be effectively suppressed by inhibitor U0126.
Our data suggest that the MEK/ERK signaling pathway plays an important role in EV71-infected iDCs and these molecules may be potential targets for the development of new anti-EV71 drugs.
Enterovirus 71; Virus replication; MEK/ERK; SOS1; Immature dendritic cells
A new subset of T helper (Th) cells, named IL-22-producing Th22 cells, was identified recently. Th22 cells have been implicated in immunity and inflammation. However, the role of these cells in the progression from acute viral myocarditis (AVMC) to dilated cardiomyopathy (DCM) and myocardial fibrosis remains unknown.
BALB/c mice were repeatedly i.p. infected with Coxsackie virus B3 (CVB3) to establish models of AVMC, chronic myocarditis and DCM. On week 2, 12 and 24 post initial injection, the percentage of splenic Th22 cells, the levels of plasma IL-22, cardiac IL-22 receptor (IL-22R) expression, and indicators of myocardial fibrosis were measured. Further, mice with AVMC and chronic myocarditis were treated with an anti-IL-22 neutralizing antibody (Ab). The collagen volume fraction (CVF), the percentage of splenic Th22 cells, plasma IL-22 levels, cardiac IL-22R expression and indicators of myocardial fibrosis were then monitored.
Compared to control mice at the same time points, AVMC, chronic myocarditis and DCM mice have higher percentage of splenic Th22 cells, higher plasma IL-22 levels, increased cardiac IL-22R, as well as increased collagen typeI-A1 (COL1-A1), collagen type III-A1 (COL3-A1) and matrix metalloproteinase-9 (MMP9) expression. However, the expression of tissue inhibitor of metalloproteinase-1(TIMP-1) was decreased. Treatment of AVMC and chronic myocarditis mice with an anti-IL-22 Ab decreased the survival rate and exacerbated myocardial fibrosis. The percentage of splenic Th22 cells, plasma IL-22 levels and cardiac IL-22R expression also decreased in anti-IL-22 Ab treatment group as compared to IgG and PBS treated groups of AVMC and chronic myocarditis mice. Moreover, increased expression of COL1-A1, COL3-A1, MMP9 but decreased expression of TIMP-1 were observed in anti-IL-22 Ab mouse group.
Th22 cells play an important role in the pathogenesis of CVB3-induced mouse chronic myocarditis and DCM. IL-22 is a myocardium-protective cytokine by inhibiting myocardial fibrosis. Therefore, Th 22 cells may be considered as potential therapeutic targets for DCM.
Th22 cells; IL-22; Myocardial fibrosis; Chronic myocarditis; Dilated cardiomyopathy