Multiplex PCR analysis of clusters of unexplained viral respiratory tract infection in Cambodia

Background

Fevers of unknown origin constitute a substantial disease burden in Southeast Asia. In majority of the cases, the cause of acute febrile illness is not identified.

Methods

We used MassTag PCR, a multiplex assay platform, to test for the presence of 15 viral respiratory agents from 85 patients with unexplained respiratory illness representing six disease clusters that occurred in Cambodia between 2009 and 2012.

Results

We detected a virus in 37 (44%) of the cases. Human rhinovirus, the virus detected most frequently, was found in both children and adults. The viruses most frequently detected in children and adults, respectively, were respiratory syncytial virus and enterovirus 68. Sequence analysis indicated that two distinct clades of enterovirus 68 were circulating during this time period.

Conclusions

This is the first report of enterovirus 68 in Cambodia and contributes to the appreciation of this virus as an important respiratory pathogen.

     

NMR-based metabolic characterization of chicken tissues and biofluids: a model for avian research

Introduction

Poultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments.

Objectives

As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus).

Methods

Metabolic extractions were performed prior to ¹H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized.

Results

Nearly 80 metabolites were identified. A cross-comparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice.

Conclusion

This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.

     

Mimotopes selected with neutralizing antibodies against multiple subtypes of influenza A

Background

The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.

Methods

The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).

Results

Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.

Conclusions

Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.

     

Application of chicken microarrays for gene expression analysis in other avian species

Background

With the threat of emerging infectious diseases such as avian influenza, whose natural hosts are thought to be a variety of wild water birds including duck, we are armed with very few genomic resources to investigate large scale immunological gene expression studies in avian species. Multiple options exist for conducting large gene expression studies in chickens and in this study we explore the feasibility of using one of these tools to investigate gene expression in other avian species.

Results

In this study we utilised a whole genome long oligonucleotide chicken microarray to assess the utility of cross species hybridisation (CSH). We successfully hybridised a number of different avian species to this array, obtaining reliable signals. We were able to distinguish ducks that were infected with avian influenza from uninfected ducks using this microarray platform. In addition, we were able to detect known chicken immunological genes in all of the hybridised avian species.

Conclusion

Cross species hybridisation using long oligonucleotide microarrays is a powerful tool to study the immune response in avian species with little available genomic information. The present study validated the use of the whole genome long oligonucleotide chicken microarray to investigate gene expression in a range of avian species.

     

Herpes virus seroepidemiology in the adult Swedish population

Background

Herpes viruses establish a life-long latency and can cause symptoms during both first-time infection and later reactivation. The aim of the present study was to describe the seroepidemiology of Herpes simplex type 1 (HSV1), Herpes simplex type 2 (HSV2), Cytomegalovirus (CMV), Varicella Zoster virus (VZV) and Human herpes virus type 6 (HHV6) in an adult Swedish population (35–95 years of age).

Methods

Presence of antibodies against the respective viruses in serum from individuals in the Betula study was determined with an enzyme-linked immunosorbent assay (ELISA). Singular samples from 535 persons (53.9% women, mean age at inclusion 62.7?±?14.4 years) collected 2003-2005 were analyzed for the five HHVs mentioned above. In addition, samples including follow-up samples collected 1988–2010 from 3,444 persons were analyzed for HSV.

Results

Prevalence of HSV1 was 79.4%, HSV2 12.9%, CMV 83.2%, VZV 97.9%, and HHV6 97.5%. Herpes virus infections were more common among women (p?=?0.010) and a lower age-adjusted HSV seroprevalence was found in later birth cohorts (p?<?0.001). The yearly incidence of HSV infection was estimated at 14.0/1000.

Conclusion

Women are more often seropositive for HHV, especially HSV2. Age-adjusted seroprevalence for HSV was lower in later birth cohorts indicating a decreasing childhood and adolescent risk of infection.

     

Conserved methionine 165 of matrix protein contributes to the nuclear import and is essential for influenza A virus replication

Background

The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif.

Methods

The function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by site-directed mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy.

Results

Mutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p?0,05) and NP (p?0,001) proteins to the nucleus in the cells transfected with the reverse –genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted.

Conclusions

Our finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.

     

Expression pattern of NLRP3 and its related cytokines in the lung and brain of avian influenza virus H9N2 infected BALB/c mice

Background

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.

Methods

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.

Results

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 V_K627 and rV_K627E. 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.

Conclusions

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.

     

The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: Implication for virus-induced apoptosis

Background

Our previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown.

Results

To characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3.

Conclusions

The present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

     

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

Background

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.

Methods

A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.

Results

Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.

Conclusions

In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

     

The so far farthest reaches of the double jelly roll capsid protein fold

Background

During the last two decades, structural biology analyses have shown that viruses infecting hosts far apart in evolution share similar architectural features, prompting a new virus classification based on structural lineages. Until recently, only a few prokaryotic viruses had been described for one of the lineages, whose main characteristic is a capsid protein with a perpendicular double jelly roll.

Main body

Metagenomics analyses are showing that the variety of prokaryotic viruses encoding double jelly roll capsid proteins is much larger than previously thought. The newly discovered viruses have novel genome organisations with interesting implications for virus structure, function and evolution. There are also indications of their having a significant ecological impact.

Conclusion

Viruses with double jelly roll capsid proteins that infect prokaryotic hosts form a large part of the virosphere that had so far gone unnoticed. Their discovery by metagenomics is only a first step towards many more exciting findings. Work needs to be invested in isolating these viruses and their hosts, characterizing the structure and function of the proteins their genomes encode, and eventually access the wealth of biological information they may hold.

     

Assembly and immunogenicity of baculovirus-derived infectious bronchitis virus–like particles carrying membrane,envelope and the recombinant spike proteins

Objective

To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens.

Results

IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation.

Conclusions

This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses.

     

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

Background

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.

Methods

Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.

Results

RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.

Conclusion

IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

     

TarMet: a reactive GUI tool for efficient and confident quantification of MS based targeted metabolic and stable isotope tracer analysis

Introduction

Untargeted and targeted analyses are two classes of metabolic study. Both strategies have been advanced by high resolution mass spectrometers coupled with chromatography, which have the advantages of high mass sensitivity and accuracy. State-of-art methods for mass spectrometric data sets do not always quantify metabolites of interest in a targeted assay efficiently and accurately.

Objectives

TarMet can quantify targeted metabolites as well as their isotopologues through a reactive and user-friendly graphical user interface.

Methods

TarMet accepts vendor-neutral data files (NetCDF, mzXML and mzML) as inputs. Then it extracts ion chromatograms, detects peak position and bounds and confirms the metabolites via the isotope patterns. It can integrate peak areas for all isotopologues automatically.

Results

TarMet detects more isotopologues and quantify them better than state-of-art methods, and it can process isotope tracer assay well.

Conclusion

TarMet is a better tool for targeted metabolic and stable isotope tracer analyses.

     

Promigratory and proangiogenic effects of AdipoRon on bone marrow-derived mesenchymal stem cells: an in vitro study

Objectives

To investigate the effect of AdipoRon on major factors involved in survival, migration and neovascularization of rat bone marrow-derived mesenchymal stem cells.

Results

AdipoRon promoted the MSCs viability. Real-time PCR indicated that the expression of cyclooxygenase-2 (COX-2), hypoxia-inducible factor-1 (HIF-1) C-X-C chemokine receptor type 4 (CXCR4), C–C chemokine receptor type 2 (CCR2), vascular endothelial growth factor matrix metalloproteinase-2 (MMP-2) and MMP-9 were upregulated in AdipoRon-treated MSCs compared to control groups. Prostaglandin E2 (PGE2) level, as well as migration ability of MSCs (scratch assay) was enhanced by AdipoRon preconditioning.

Conclusion

Preconditioning of MSCs with AdipoRon prior to transplantation could enhance cell survival, angiogenesis and migration via activating the COX-2/PGE2/HIF-1 pathway and other contributing factors.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

A conceptual model for optimizing vaccine coverage to reduce vector-borne infections in the presence of antibody-dependent enhancement

Background

Many vector-borne diseases co-circulate, as the viruses from the same family are also transmitted by the same vector species. For example, Zika and dengue viruses belong to the same Flavivirus family and are primarily transmitted by a common mosquito species Aedes aegypti. Zika outbreaks have also commonly occurred in dengue-endemic areas, and co-circulation and co-infection of both viruses have been reported. As recent immunological cross-reactivity studies have confirmed that convalescent plasma following dengue infection can enhance Zika infection, and as global efforts of developing dengue and Zika vaccines are intensified, it is important to examine whether and how vaccination against one disease in a large population may affect infection dynamics of another disease due to antibody-dependent enhancement.

Methods

Through a conceptual co-infection dynamics model parametrized by reported dengue and Zika epidemic and immunological cross-reactivity characteristics, we evaluate impact of a hypothetical dengue vaccination program on Zika infection dynamics in a single season when only one particular dengue serotype is involved.

Results

We show that an appropriately designed and optimized dengue vaccination program can not only help control the dengue spread but also, counter-intuitively, reduce Zika infections. We identify optimal dengue vaccination coverages for controlling dengue and simultaneously reducing Zika infections, as well as the critical coverages exceeding which dengue vaccination will increase Zika infections.

Conclusion

This study based on a conceptual model shows the promise of an integrative vector-borne disease control strategy involving optimal vaccination programs, in regions where different viruses or different serotypes of the same virus co-circulate, and convalescent plasma following infection from one virus (serotype) can enhance infection against another virus (serotype). The conceptual model provides a first step towards well-designed regional and global vector-borne disease immunization programs.

     

Virome analyses of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus

Background

Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30?years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.

Methods

In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.

Results

Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21?nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.

Conclusion

The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

     

Analysis of chicken anemia virus genome: evidence of intersubtype recombination

Background

Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia. CAV putative intergenotypic recombinants have been reported previously. This fact is based on the previous classification of CAV sequences into three genotypes. However, it is unknown whether intersubtype recombination occurs between the recently reported four CAV genotypes and five subtypes of genome sequences.

Results

Phylogenetic analysis, together with a variety of computational recombination detection algorithms, was used to investigate CAV approximately full genomes. Statistically significant evidence of intersubtype recombination was detected in the parent-like and two putative CAV recombinant sequences. This event was shown to occur between CAV subgroup A1 and A2 sequences in the phylogenetic trees.

Conclusions

We revealed that intersubtype recombination in CAV genome sequences played a role in generating genetic diversity within the natural population of CAV.