Lower Viral Titers Research Articles

Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge.

Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR(-/-)) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR(-/-) mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses

To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028. In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous monitoring of their mammalian pathogenicity.

PS-329 Tlr3 Mediated Expressions Of Cytokines And Socs1 In Respiratory Syncytial Virus Persistent Cells

Background Respiratory syncytial virus (RSV) persistent HEp-2 cells are a heterogeneous mixture of viral antigen-positive and -negative variants, and the mechanism through which viral replication evades the innate immune response and becomes latent remains unclear. Materials and methods RSV persistently infected HEp-2 cells were isolated and the clones were passaged. By using siRNA silence of RIG-I or TRL3, protein levels of SOCS1, SOCS3 and STAT1/2 in the viral persistent cells were checked by western blot, cytokines concentrations in the supernatant of were determined by ELISA, and antiviral genes expression was detected by RT-PCR. Results The RSV persistent cells always differentiated into two distinct populations characterised by viral permission or resistance respectively. The viral persistent cells produced a low viral titer, resisted wild-type RSV superinfection, and secreted high levels of IFN-β, Mip-α, IL-8 and Rantes. TLR3, RIG-I and SOCS1 were found to be upregulated. The silence of TLR3 decreased the expression of SOCS1 and the secretion of cytokines. Conclusion RSV persistent cells are in an inflammatory state that the upregulation of SOCS1 is related to the TLR3 induced signalling pathway, which could be associated with viral persistence.

WFDC1 Is a Key Modulator of Inflammatory and Wound Repair Responses

WFDC1/ps20 is a whey acidic protein four-disulfide core member that exhibits diverse growth and immune-associated functions invitro. Invivo functions are unknown, although WFDC1 is lower in reactive stroma. A Wfdc1-null mouse was generated to assess core functions. Wfdc1-null mice exhibited normal developmental and adult phenotypes. However, homeostasis challenges affected inflammatory and repair processes. Wfdc1-null mice infected with influenza A exhibited 2.75-log-fold lower viral titer relative to control mice. Wfdc1-null infected lungs exhibited elevated macrophages and deposition of osteopontin, a potent macrophage chemokine. In wounding studies, Wfdc1-null mice exhibited an elevated rate of skin closure, and this too was associated with elevated deposition of osteopontin and macrophage recruitment. Wfdc1-null fibroblasts exhibited impaired spheroid formation, elevated adhesion to fibronectin, and an increased rate of wound closure invitro. This was reversed by neutralizing antibody to osteopontin. Osteopontin mRNA and cleaved protein was up-regulated in Wfdc1-null cells treated with lipopolysaccharide or polyinosinic-polycytidylic acid coordinate with constitutively active matrix metallopeptidase-9 (MMP-9), a protease that cleaves osteopontin. These data suggest that WFDC1/ps20 modulates core host response mechanisms, in part, via regulation of osteopontin and MMP-9 activity. Release from WFDC1 regulation is likely a key component of inflammatory and repair response mechanisms, and involves the processing of elevated osteopontin by activated MMP-9, and subsequent macrophage recruitment.

Regulation of virus-induced inflammatory response by Dunaliella salina alga extract in macrophages

Previous reports have suggested that many constituents within various algal samples are able to attenuate LPS-induced inflammatory effects. To date no report has been published on the regulation of virus-induced inflammatory response of Dunaliella salina carotenoid extract. In the present study, the anti-inflammatory effect of D. salina carotenoid extract on pseudorabies virus (PRV)-infected RAW 264.7 macrophages was investigated.We evaluated the anti-inflammatory effect of D. salina carotenoid extract on PRV-infected RAW 264.7 cells by measuring cell viability, cytotoxicity, production of inflammatory mediators such as NO, iNOS, COX-2, pro-inflammatory cytokines and anti-virus replication by plaque assay.We found down-regulation of the expression of the iNOS, COX-2 and pro-inflammatory genes IL-1β, IL-6, TNF-α, and MCP-1 in a dose-dependent manner. Although there was no effect on viral replication, there were tendencies toward lower virus titer and tendencies toward higher cell survival. Most importantly, we found that inhibition of TLR9, PI3K and Akt phosphorylation plays a crucial role in the extract-mediated NF-κB regulation by modulating IKK-IκB signaling in PRV-infected RAW264.7 cells. These results indicate that D. salina carotenoid extracts inhibited inflammation by inhibition of NF-κB activation by TLR9 dependent via PI3K/Akt inactivation.

Protective efficacy of intranasally administered bivalent live influenza vaccine and immunological mechanisms underlying the protection

Previously we reported the generation of a new potential live attenuated influenza vaccine (LAIV) named SIV/606 that expresses H1 and H3 HAs. We also demonstrated intratracheal vaccination of SIV/606 conferred protection against infections with both H1 and H3 swine influenza virus subtypes in pigs. Here we vaccinated pigs with SIV/606 intranasally, which is a more suitable route for LAIV, and evaluated vaccine efficacy. Intranasal vaccination of SIV/606 induced serum IgG antibody responses against both H1N1 and H3N2 SIVs and high titer of virus neutralizing antibodies against H1N1 SIV but not against H3N2 SIV. When we challenged the pigs with H1N1 and H3N2 SIVs, we observed marked reduction of lung lesions and viral titer in lung tissue in vaccinated pigs. Our analyses also showed that vaccinated pigs had more IFN-γ secreting cells in trachea–bronchial lymph nodes. Our studies demonstrated that intranasal vaccination of SIV/606 is efficacious for H1N1 and H3N2 SIVs infections. Moreover, our results may help explaining the protection from H3N2 SIV infection despite the low viral neutralizing antibody titer.

Hemagglutinin mutation D222N of the 2009 pandemic H1N1 influenza virus alters receptor specificity without affecting virulence in mice

The D222N hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pdmH1N1) infections. The impact of this mutation was investigated in vitro and in vivo using recombinant viruses. The recombinant D222N virus grew to significantly lower viral titers than the WT in A549 but not in MDCK cells. A dose-dependent glycan array analysis with the D222N virus showed a modest increase in the binding avidity to human-like (α-2,6 sialylated glycan) receptors and avian-like (α-2,3 sialylated glycan) receptors than the WT virus. The D222N HA mutation resulted in slight weight loss, lower lung titers, inflammatory cytokines and alveolar inflammation in mice than the WT virus. This may or may not be associated with severe clinical outcomes reported in humans.

Tu2022 Optimizing Outcomes of Complex Ventral Hernia Repairs Using a Sandwich Reconstruction Technique With Novel Hybrid-VAC Closure

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.

Tu2021 Interferon Expressing Oncolytic Adenovirus for Esophageal Cancer

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.

CpG immunization protects against a lethal influenza challenge and generates antigen specific memory T cells (VAC2P.932)

Abstract Despite extensive research, influenza A virus (IAV) remains a major cause of mortality and healthcare expenditure. Emerging pandemics from highly pathogenic IAV strains such as H5N1 and H1N1 highlight the need for universal, cross protective IAV vaccines. Current vaccine formulations generate strain-specific neutralizing antibodies primarily against the outer coat proteins hemagglutinin and neuraminidase. In contrast to these highly mutable proteins, internal proteins of IAV are more conserved and are therefore a favorable target for developing vaccines that induce strong T cell responses in the absence of humoral immunity. Here, we found that intranasal administration of a TLR 9 agonist (CpG) and inactivated x31 (H3N2) protects mice from a heterosubtypic challenge with a lethal dose of PR8 virus (H1N1). Vaccinated mice also showed less weight loss and lower viral titers compared to control mice following a lethal PR8 challenge. Both CD4 and CD8 effector functions were measured five days post challenge with significant increases in the number of Granzyme B positive cells as well as antigen specific IFN-γ cells. Additionally, vaccinated mice had higher memory recall responses following ex vivo restimulation with NP specific peptides as measured by IL-2 and IFN-γ production. Thus, CpG is a potent inducer of antiviral immunity highlighted by robust T cell responses underscoring the potential of this vaccination strategy as a prophylactic measure against pandemic IAV strains.

Use of different cell lines for in vitro cultures of bovine respiratory syncytial virus

This study compared the use of different cell lines for in vitro cultures of bovine respiratory syncytial virus (BRSV). The BRSV 375 strain and 3 nasal swabs obtained from Simmental calves were used for this study. The culture was performed on 3 cell lines: bovine kidney cells (LLC-PK1), bovine tracheal cells (TBTR) and primary chicken embryo-related cells (CER). A comparative analysis of titres was performed using a microplate agglutination test with human group O erythrocytes and bovine erythrocytes. The presence of BRSV in all cell lines was confirmed using the reverse transcriptase polymerase chain reaction (RT-PCR) method.The first small refractile changes in the LLC-PK1 cells occurred at 48h after infection. Syncytial changes were noted 4 days after incubation. Large refractile cell changes were observed on day 3 of growth in the TBTR culture. Syncytia were observed on the second day after infection in subsequent passages. The cytopathic effect in the CER cells occurred 24h after infection, and syncytia appeared after 3 passages. Changes in syncytia indicate an adaptation of the virus for the infection of cells other than tracheal cells in primary and secondary cultures. The highest viral titre was obtained using the TBTR line. The titres obtained in the LLC-PK1 and CER cultures averaged 101.86/ml. The low virus titres in all culture types suggest the need for research aimed at the optimisation of culture conditions.

Quantitative Influenza Follow-Up Testing (QIFT)—A Novel Biomarker for the Monitoring of Disease Activity at the Point-of-Care

BackgroundInfluenza infections induce considerable disease burden in young children. Biomarkers for the monitoring of disease activity at the point-of-care (POC) are currently lacking. Recent methodologies for fluorescence-based rapid testing have been developed to provide improved sensitivities with the initial diagnosis. The present study aims to explore the utility of second-generation rapid testing during longitudinal follow-up of influenza patients (Rapid Influenza Follow-up Testing = RIFT). Signal/control fluorescent readouts (Quantitative Influenza Follow-up Testing = QIFT) are evaluated as a potential biomarker for the monitoring of disease activity at the POC.Methods and FindingsRIFT (SOFIA) and QIFT were performed at the POC and compared to blinded RT-PCR at the National Reference Centre for Influenza. From 10/2011-4/2013, a total of 2048 paediatric cases were studied prospectively; 273 cases were PCR-confirmed for influenza. During follow-up, RIFT results turned negative either prior to PCR (68%), or simultaneously (30%). The first negative RIFT occurred after a median of 8 days with a median virus load (VL) of 5.6×10∧3 copies/ml and cycle threshold of 37, with no evidence of viral rebound. Binning analysis revealed that QIFT differentiated accurately between patients with low, medium and high viral titres. QIFT increase/decrease showed 88% agreement (sensitivity = 52%, specificity = 95%) with VL increase/decrease, respectively. QIFT-based viral clearance estimates showed similar values compared to PCR-based estimates. Variations in viral clearance rates were lower in treated compared to untreated patients. The study was limited by use of non-invasive, semi-quantitative nasopharyngeal samples. VL measurements below the limit of detection could not be quantified reliably.ConclusionsDuring follow-up, RIFT provides a first surrogate measure for influenza disease activity. A “switch” from positive to negative values may indicate a drop in viral load below a critical threshold, where rebound is no longer expected. QIFT may provide a useful tool for the monitoring of disease burden and viral clearance at the POC.

Seroprevalence of HBV infection and viral loads in outpatients attending a district hospital located in Mardan, Pakistan

Abstract Hepatitis B is a major public health problem in Pakistan like other developing countries. HBV infection is a more contagious disease than HCV and HIV infection. The aim of the current study is to compare seroprevalence of HBV infection in outpatients who attended a district headquarter hospital of district Mardan, Pakistan. A total number of 270 patients were selected for this research work. Serum of patients were tested by HBsAg and ICT strip method and those found positive were sent to GCMBDR to confirmed for HBV DNA by Real Time PCR. Out of the total 270 screened patients, HBV DNA was confirmed in 94(34.81%) patients using Real Time PCR of which 56(59.6%) were male while 38(40.4%) were female outpatients. All the positive patients were divided into three categories on the bases of viral load categories such as low ( 10,000,000 IU/ml). On the bases of these categories 50 %( n=47) patients were found with low viral load, 33 %( n=31) with intermediate and 17 %( n=16) were found having high viral load. Female patients were slightly more susceptible to HBV infection compared to male patients. The highest HBV infection seroprevalence was found in patients of 21-40 age group. Majority of the sick subjects were however seen with low viral titers.

Disruption of Vector Transmission by a Plant-Expressed Viral Glycoprotein

Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.

Evaluating the virucidal efficacy of hydrogen peroxide vapour

SummaryBackgroundSurface contamination has been implicated in the transmission of certain viruses, and surface disinfection can be an effective measure to interrupt the spread of these agents.AimTo evaluate the in-vitro efficacy of hydrogen peroxide vapour (HPV), a vapour-phase disinfection method, for the inactivation of a number of structurally distinct viruses of importance in the healthcare, veterinary and public sectors. The viruses studied were: feline calicivirus (FCV, a norovirus surrogate); human adenovirus type 1; transmissible gastroenteritis coronavirus of pigs (TGEV, a severe acute respiratory syndrome coronavirus [SARS-CoV] surrogate); avian influenza virus (AIV); and swine influenza virus (SwIV).MethodsThe viruses were dried on stainless steel discs in 20- or 40-μL aliquots and exposed to HPV produced by a Clarus L generator (Bioquell, Horsham, PA, USA) in a 0.2-m3 environmental chamber. Three vaporized volumes of hydrogen peroxide were tested in triplicate for each virus: 25, 27 and 33 mL.FindingsNo viable viruses were identified after HPV exposure at any of the vaporized volumes tested. HPV was virucidal (>4-log reduction) against FCV, adenovirus, TGEV and AIV at the lowest vaporized volume tested (25 mL). For SwIV, due to low virus titre on the control discs, >3.8-log reduction was shown for the 25-mL vaporized volume and >4-log reduction was shown for the 27-mL and 33-mL vaporized volumes.ConclusionHPV was virucidal for structurally distinct viruses dried on surfaces, suggesting that HPV can be considered for the disinfection of virus-contaminated surfaces.

Enforced expression of microRNA-21 influences the replication of varicella-zoster virus by triggering signal transducer and activator of transcription 3.

Varicella-zoster virus (VZV) causes chronic pain and serious complications, including zoster paresis. However, the mechanism of VZV replication, a critical part of VZV pathogenesis, remains largely unknown and was investigated in the present study. The upregulation of microRNA-21 (miR-21) was identified following VZV infection in vitro by quantitative polymerase chain reaction. The hypothesis that the overexpression of miR-21 activates the signal transducer and activator of transcription 3 (STAT3) signaling pathway was validated by measuring the mRNA expression levels of STAT3 and the anti-apoptotic protein survivin in human malignant melanoma (MeWo) and human embryonic lung fibroblast (HELF) cell lines transfected with miR-21-mimic and comparing them with those in cells transfected with miR-control. To further study the interaction of miR-21, STAT3 and VZV replication, the effects of miR-21 overexpression and STAT3 knockdown were evaluated. Higher virus titers were detected when miR-21 was upregulated in vitro. Moreover, it was identified that significantly lower virus titers were present in MeWo cells in which STAT3 was knocked down. In addition, the overexpression of miR-21 did not stimulate VZV replication in the MeWo cell line when the STAT3 gene was silenced. Therefore, the observations of the present study indicate that the enforced expression of miR-21 promotes the replication of VZV by activating STAT3 in vitro.

Culicoides species abundance and potential over-wintering of African horse sickness virus in the Onderstepoort area, Gauteng, South Africa.

In South Africa, outbreaks of African horse sickness (AHS) occur in summer; no cases are reported in winter, from July to September. The AHS virus (AHSV) is transmitted almost exclusively by Culicoides midges (Diptera: Ceratopogonidae), of which Culicoides imicola is considered to be the most important vector. The over-wintering mechanism of AHSV is unknown. In this study, more than 500 000 Culicoides midges belonging to at least 26 species were collected in 88 light traps at weekly intervals between July 2010 and September 2011 near horses in the Onderstepoort area of South Africa. The dominant species was C. imicola. Despite relatively low temperatures and frost, at least 17 species, including C. imicola, were collected throughout winter (June-August). Although the mean number of midges per night fell from > 50 000 (March) to < 100 (July and August), no midge-free periods were found. This study, using virus isolation on cell cultures and a reverse transcriptase polymerase chain reaction (RT-PCR) assay, confirmed low infection prevalence in field midges and that the detection of virus correlated to high numbers. Although no virus was detected during this winter period, continuous adult activity indicated that transmission can potentially occur. The absence of AHSV in the midges during winter can be ascribed to the relatively low numbers collected coupled to low infection prevalence, low virus replication rates and low virus titres in the potentially infected midges. Cases of AHS in susceptible animals are likely to start as soon as Culicoides populations reach a critical level.

Relationship between apoptosis and the BH2 domain sequence of the VP5 peptide of infectious pancreatic necrosis virus

ABSTRACTObjective. To determine whether the level of apoptosis induced by infectious pancreatic necrosis virus (IPNV) is related to the amino acid sequence of the BH2 domain of the VP5 protein and the level of infectivity. Materials and methods. Three IPNV strains were used, the VP2 protein gene was amplified for genotyping and the VP5 sequence was also obtained. The infectivity of the strains was calculated using the viral titer obtained at 12, 24, 36 and 45 hpi in CHSE-214 cells. The percentage of apoptosis in infected cells was visualized by TUNEL assay and immunohistochemistry (caspase 3 detection). Results. The V70/06 and V33/98 strains corresponded to genotype Sp, while V112/06 to VR-299; the amino acid analysis of the V70/06 strain allows its classification as middle virulent strain and V33/98 and V112/06 strains as low virulent ones; infection with the V112/06 strain produced a lower viral titer (p<0.05). The VP5 gene of the 3 strains showed four homologous domains to Bcl-2, however, the BH2 domain was truncated in V70/06 and V33/98 (12 kDa), being complete (15kDa) in V112/06, which also showed the Trp155 residue, equivalent to Trp188 considered as a critical factor for the function of Bcl-2. The average apoptosis was below 12%, showing no differences between strains (p>0.05). Conclusions. The results showed that the differences in the BH2 sequence of the VP5 protein, infectivity and the VP2 sequence are not associated with the modulation of apoptosis.

Immunomodulatory effects of Newcastle disease virus AF2240 strain on human peripheral blood mononuclear cells.

Immunotherapy has raised the attention of many scientists because it hold promise to be an attractive therapeutic strategy to treat a number of disorders. In this study, the immunomodulatory effects of low titers of Newcastle disease virus (NDV) AF2240 on human peripheral blood mononuclear cells (PBMC) were analyzed. We evaluated cytokine secretion and PBMC activation by cell proliferation assay, immunophenotyping and enzyme linked immunosorbent assay. The proliferation of the human PBMC was measured to be 28.5% and 36.5% upon treatment with 8 hemaglutinin unit (HAU) and 2 HAU of NDV respectively. Interestingly, the percentage of cells with activating markers CD16 and CD56 were increased significantly. Furthermore, the intracellular perforin and granzyme levels were also increased upon virus infection. Human PBMC treated with NDV titer 8 HAU was found to stimulate the highest level of cytokine production including interferon-γ, interleukin-2 and interleukin-12. The release of these proteins contributes to the antitumor effect of PBMC against MCF-7 breast cancer cells. Based on the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, activated human PBMC showed high cytolytic efficiency towards human breast tumor cells. In summary, NDV was able to stimulate PBMC proliferation, cytokine secretion and cytolytic activity.

Avian influenza virus RNA extraction.

The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types; enriched (e.g. virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter two well established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented.