Type Virus Research Articles

Apolipoprotein A-I Is a Prognosticator of Nasopharyngeal Carcinoma in the Era of Intensity-modulated Radiotherapy.

Background: In the era of intensity-modulated radiotherapy (IMRT), distant metastasis remains the major cause of death from nasopharyngeal carcinoma (NPC). This study aimed to evaluate the clinical value of pretreatment serum lipid profiles in predicting clinical outcome of NPC.Methodology / Principal Findings: A total of 1927 consecutive patients who had untreated NPC and completed radical IMRT between Jan. 2010 and Dec. 2011 were retrospectively reviewed. Pretreatment serum lipid indexes including total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, apolipoprotein A-I (apoAI) and apolipoprotein B were analyzed for their association with survivals, together with the clinical features (age, sex, pathological type, anemia, chemotherapy sequence and Epstein-Barr virus deoxyribonucleic acid). Hazard ratio (HR) and 95% confidence interval (CI) were calculated for each independent prognosticator. After univariate and multivariate survival analysis, low apoAI level (< 1.125 mmol/L) appeared to predict poor 5-year overall survival (OS), disease-free survival (DFS) and distant-metastasis-free survival (DMFS).The HRs were 1.549 (95% CI, 1.137-2.109), 1.293 (95% CI, 1.047-1.597) and 1.288 (95% CI, 1.022-1.623), respectively. Subgroup survival analysis showed that the apoAI maintained predicting independence for OS, DFS and DMFS in patients with locally advanced NPC, even in those treated with concurrent chemoradiotherapy.Conclusions / Significance: NPC patient with low serum level of pretreatment apoAI might be at risk of distant metastasis. Treatment aiming to eradicate distant metastasis might improve survival of these patients.

Усиление иммуногенности вируса гриппа A путем подавления иммуносупрессорной функции белка NS1

Укорочение неструктурного белка NS1 является перспективным методом создания аттенуированных высокоиммуногенных вирусов гриппа. Однако механизмы врожденного иммунитета, обуславливающие повышенную иммуногенность штаммов с модифицированным NS1 в настоящее время изучены недостаточно. Целью данной работы было сравнение продукции цитокинов, динамики изменения популяционного состава клеток врожденного иммунитета и уровня адаптивного Т-клеточного иммунного ответа после иммунизации мышей двумя вариантами вируса гриппа А/Puerto Rico/8/1934 (A/PR8): вирусом дикого типа А/PR8/full NS и вирусом с укороченным до 124 аминокислотных остатков белком NS1 (А/PR8/NS124). В данном исследовании для компенсации различий в репродуктивной активности исследуемых штаммов в респираторном тракте был выбран интраперитонеальный способ введения вирусов. Уровень интерферона β (IFNβ), фактора некроза опухолей α (TNFα), моноцитарного хемоаттрактантного белка 1 (MCP1), интерлейкина 6 (IL6) и IL27 в перитонеальных смывах мышей, иммунизированных А/PR8/NS124, был существенно выше, чем в группе, получившей А/PR8/full NS. В то же время группа А/PR8/NS124 характеризовалась замедленным привлечением моноцитов и нейтрофилов в перитонеальную полость и более выраженным снижением относительного содержания дендритных клеток по сравне-нию с А/PR8/full NS. Важно, что уровень экспрессии активационного маркера CD86 на клетках, экспрессирующих молекулы главного комплекса гистосовместимости II (MHCII+) перитонеальной полости мышей, иммунизированных штаммом А/PR8/NS124, имел более высокие значения по сравнению с группой А/PR8/full NS. Анализ адаптивного иммунного ответа показал, что иммунизация штаммом А/PR8/NS124 приводит к формированию повышенного содержания вирус-специфических CD8+-эффекторных Т-лимфоцитов, характеризующихся одновременной продукцией IFNγ, IL2 и TNFα. Мы предполагаем, что повышенная продукция цитокинов, усиленная миграция дендритных клеток, а также сохранение высокого уровня экспрессии CD86 на антигенпрезентирующих клетках (АПК) мышей через 24 ч после иммунизации штаммом А/PR8/NS124 приводит к более эффективной презентации антигенов вируса гриппа и, как следствие, к усилению вирусспецифического Т-клеточного иммунного ответа.

Enhancement of the immunogenicity of influenza A virus by the inhibition of immunosuppressive function of NS1 protein

ABSTRACT The truncation of the nonstructural NS1 protein is a novel approach for the generation of immunogenic attenuated influenza viruses. However, the innate immune mechanisms that cause the increased immunogenicity of influenza viruses with altered NS1 proteins are poorly understood. The goal of this study was to compare the immune responses in mice immunized with two variants of the influenza A/Puerto Rico/8/1934 (A/PR8) virus: the wild type virus (A/PR8/full NS) and the variant with the NS1 protein shortened to 124 amino acid residues (A/PR8/NS124). The investigated parameters of immunity included cytokine production, the dynamic variation of the innate immune cell populations, and the rate of the influenza-specific T-cell responses. An intraperitoneal route of immunization was chosen due to the variability in the replication capacity of the investigated viruses in the respiratory tract. The levels of interferon β (IFNβ), tumor necrosis factor α (TNFα), monocyte chemo-attractant protein 1 (MCP1), interleukin 6 (IL6), and IL27 in peritoneal washings of mice immunized with A/PR8/NS124 were significantly higher compared to the mice immunized with the wild-type virus. The A/PR8/NS124 treated group showed a delayed attraction of monocytes and neutrophils as well as a more pronounced reduction in the percentage of dendritic cells in the peritoneal cavity. The expression level of the CD86 activation marker on the cells expressing the molecules of the major histocompatibility complex II (MHCII+) was significantly higher in mice immunized with A/PR8/NS124 than in the group immunized with A/PR8/full NS. Finally, immunization with A/PR8/NS124 led to an increased formation of influenza-specific CD8+ effector T-cells characterized by the simultaneous production of IFNγ, IL2, and TNFα. We hypothesize that elevated cytokine production, enhanced dendritic cell migration, and increased CD86 expression on antigen-presenting cells upon immunization with A/PR8/NS124 lead to a more effective presentation of viral antigens and, therefore, promote an increased antigen-specific CD8+ immune response.

Isolation and Characteristics of the Arkansas-Type Infectious Bronchitis Virus in China.

Two infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0712/11 (I0712/11) and γCoV/ck/China/I0108/17 (I0108/17), were isolated from diseased chicken flocks in different provinces in China and genotyped as Arkansas (Ark)-type viruses with three other Chinese Ark field strains, the Jilin vaccine strain, and the American Ark- and Ark DPI-like viruses. Complete genomic sequence analysis and pairwise comparison of nucleotide sequences encoding the S1 subunit of the spike protein and other structural and accessory proteins revealed that Chinese Ark field isolates were genetically closely related to the Jilin vaccine and American ArkDPI11 strains, although extensive nucleotide changes were found across the genomes of Chinese Ark field isolates. This suggests that Chinese Ark-type isolates are derived from the Jilin vaccine, and have diverged and evolved independently by point mutations since introduction into China. It is also possible that the Chinese Ark viruses have arisen as a result of different introductions of American ArkDPI11-like strains from the United States; this hypothesis requires further investigation. Pathogenicity testing showed that Chinese Ark viruses had comparable virulence to that of the Massachusetts-type M41 strain, although they had lower affinity for the kidneys of chickens than the M41 strain had. Although Ark-type viruses are not widespread in China, surveillance and updating the currently applied vaccination strategy for sound protection against IBV disease are important because this type of virus has caused heavy economic losses in the United States.

Особливості застосування екстракту алое у технології культивування герпесвірусів

In the article there are studied peculiarities of aloe extract application in the cultivation technology of herpes viruses on the re-cultivating culture of testicular piglets cells. We were faced with the following tasks: to select a cell culture and aloe extract concentration to determine the toxic dose of the drug; to study the manifestation of the cytopathic effect in cell cultures infected with herpesvirus at different drug concentrations ; to determine infectious virus activity after its passage on the cells culture with the use of aloe extract. The work was carried out at the faculty of veterinary medicine, in the educational-research laboratory of epizootology department of microbiology, pharmacology and epizootology of the Zhytomyr National Agroecological University. The research materials included: the re-cultivating culture of testicular piglets, horse herpes virus of the first type and sterile aloe extract. Cultivation of crops, infection with the virus and determination of its concentration after contact with culture and the introduction of aloe extract were carried out under sterile conditions according to the generally accepted methods. The results were recorded daily, paying attention to the environment color changing, the integrity of the monolayer and the presence of cells in the medium. Along with the infected cells we were observing additional, uninfected, cultures of cells. The Incubation process was stopped at the destruction of 90-100% cell monolayer. As a result of this work, it is proved that the aloe extract in a dose of 0.14-0.28 mg per cm³ of growth medium allows to observe for the 2nd day of replanting a solid dense monolayer in the mattress without color changing , as well as enhanced growth and regeneration of cells and the metabolism of the epithelial tissue of the re-cultivating culture of testicular piglets, which makes it possible to shorten the time to grow a new monolayer for further virological and immunological studies. The use of this drug helps to increase cells concentration in the mattresses, which, in turn, can increase the concentration of the virus to 1-2 log2 for the use of similar amounts of growth medium without aloe extract. Such modified way to increase the biomass of cells is suitable for the further multiplication of viruses and the production of diagnostic antigens with lower economic costs. The use of this methodical approach will allow to solve a number of tasks related with accumulation of viruses and the production of drugs for the diagnosis and prevention of viral infections.

Inhibition of apoptosis in BHV-1-infected cells depends on Us3 serine/threonine kinase and its enzymatic activity

Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers.To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors.

Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains

Lumpy skin disease (LSD) is a transboundary viral disease of cattle with severe economic impact. Immunization of cattle with homologous live attenuated vaccines poses a number of diagnostic problems, as it has been associated with adverse reactions resembling disease symptoms. The latter hampers clinical diagnosis and poses challenges in virus identification. To this end, a duplex quantitative real-time PCR method targeting the GPCR gene was developed and validated, for the concurrent detection and differentiation of wild type and vaccine Lumpy skin disease virus (LSDV) strains. The method was evaluated in three laboratories. The evaluation included a panel of 38 poxvirus isolates/strains and the analytical characteristics of the method were determined. Amplification efficiencies were 91.3% and 90.7%, for wild type and vaccine LSDV, respectively; the limit of detection was 8 DNA copies for both targets and the inter-assay CV was 0.30% for wild type and 0.73% for vaccine LSDV. The diagnostic performance was assessed using 163 LSDV-positive samples, including field specimens and samples from experimentally vaccinated/infected animals. The method is able to confirm diagnosis in suspect cases, it differentiates infected from vaccinated animals (DIVA) and can be regarded as an important tool for effective LSD surveillance and eradication during vaccination campaigns.

A novel bladder cancer - specific oncolytic adenovirus by CD46 and its effect combined with cisplatin against cancer cells of CAR negative expression

BackgroundConditionally replicative oncolytic adenoviruses (CRAds) display significant anti-tumor effects. However, the traditional adenovirus of serotype 5 (Ad5) entering cancer cells via coxsackie virus and adenovirus receptor (CAR) can’t be utilized for bladder cancer with low expression of CAR, which limits the application of Ad5.MethodsWe utilized Ad5/F11p containing the chimeric fiber gene encoding the Ad5 fiber tail domain and Ad11p fiber shaft and knob domains to construct bladder cancer-specific chimeric type viruses Ad5/F11p-PSCAE-UPII-E1A, which can infect bladder cancer cells mediated by CD46 molecule. We carried out series of experiments in vitro to research anti-tumor effect of Ad5/F11p-PSCAE-UPII-E1A and the interaction in combination with cisplatin.ResultsThe results demonstrated Ad5/F11p-PSCAE-UPII-E1A could infect bladder cancer cells (T24, EJ and 5637) in a CAR-independent way, and exert anti-tumor effect by blocking the cancer cells in G1 phase and inducing apoptosis. Ad5/F11p-PSCAE-UPII-E1A plus cisplatin enhanced the anti-proliferative effect and increased the number of apoptotic cells compared with viruses or cisplatin alone. Ad5/F11p-PSCAE-UPII-E1A plus cisplatin could upregulate the proteins expression of p53, Bax, and cleaved caspase-3, and downregulated Bcl-2 protein expression in T24, EJ and 5637 cells.ConclusionWe constructed a bladder cancer-specific oncolytic adenovirus and provided new combination treatment strategies for bladder cancer.

Genome characterization, antigenicity and pathogenicity of a novel infectious bronchitis virus type isolated from south China

In 2014, three infectious bronchitis virus (IBV) strains, designated as γCoV/ck/China/I0111/14, γCoV/ck/China/I0114/14 and γCoV/ck/China/I0118/14, were isolated and identified from chickens suspected to be infected with IBV in Guangxi province, China. Based upon data arising from S1 sequence and phylogenetic analyses, the three IBV isolates were genetically different from other known IBV types, which represented a novel genotype (GI-29). Virus cross-neutralization tests, using γCoV/ck/China/I0111/14 as a representative, showed that genotype GI-29 was antigenically different from all other known IBV types, thus representing a novel serotype. Complete genomic analysis showed that GI-29 type viruses were closely related to and might originate from a GX-YL5-like virus by accumulation of substitutions in multiple genes. These GI-29 viral genomes are still evolving and diverging, particularly in the 3′ region, although we cannot rule out the possibility of recombination events occurring. For isolate γCoV/ck/China/I0114/14, we found that recombination events had occurred between nsps 2 and 3 in gene 1 which led to the introduction of a 4/91 gene fragment into the γCoV/ck/China/I0114/14 viral genome. In addition, we found that the GI-29 type γCoV/ck/China/I0111/14 isolate was a nephropathogenic strain and high pathogenic to 1-day-old specific pathogen-free (SPF) chickens although cystic oviducts were not observed in the surviving layer chickens challenged with γCoV/ck/China/I0111/14 isolate.

Primary Human Renal-Derived Tubular Epithelial Cells Fail to Recognize and Suppress BK Virus Infection.

BK polyomavirus (BKV)-associated nephropathy is a threat to kidney allograft survival affecting up to 15% of renal transplant patients. Previous studies revealed that tubular epithelial cells (TEC) show a limited response towards BKV infection. Here we investigated the interplay between BKV and TEC in more detail. In particular, we questioned whether BKV suppresses and/or evades antiviral responses. Human primary TEC and peripheral blood mononuclear cells were infected with BKV Dunlop strain or other viruses. Moreover, TEC were stimulated with genomic double-stranded (ds)DNA or IFN. Viral replication and cellular responses were measured using quantitative real time PCR and multiplex assay. BKV infection of primary human TEC did not induce an antiviral response, whereas infection with influenza A virus, herpes simplex virus 1, or cytomegalovirus induced a strong antiviral response measured by upregulation of interferon-stimulated genes, such as CXCL10 and DAI. In addition, intracellular delivery of dsDNA or stimulation with IFN did elicit a rapid and pronounced response. However, BKV infection did not affect dsDNA-induced gene expression, indicating BKV did not modulate the antiviral response. Prestimulation of primary TEC with IFNα or dsDNA did not hamper replication of BKV, whereas influenza and herpes simplex virus 1 replication were clearly reduced. In contrast, BKV infection of leukocytes did elicit an antiviral response. BKV specifically evades innate immunity in TEC and is not susceptible to an intrinsic interferon response, which may facilitate latent presence of the virus in this cell type.

The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast.

Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.

Virulence of an H5N8 highly pathogenic avian influenza is enhanced by the amino acid substitutions PB2 E627K and HA A149V

A novel reassortant H5N8 highly pathogenic avian influenza (HPAI) virus was recently identified in Asia, Europe, and North America. The H5N8 HPAI virus has raised serious concerns regarding the potential risk for human infection. However, the molecular changes responsible for allowing mammalian infection in H5N8 HPAI viruses are not clear. The objective of this study was to identify amino acid substitutions that are potentially associated with the adaptation of H5N8 HPAI viruses to mammals. In this study, an avian-origin H5N8 virus was adapted to mice through serial lung-to-lung passage. The virulence of mouse-adapted virus was increased and adaptive mutations, HA (A149V) and PB2 (E627K), were detected after the ninth passage in each series of mice. Reverse genetics were used to generate reassortants of the wild type and mouse-adapted viruses. Substitutions in the HA (A149V) and PB2 (E627K) proteins led to enhanced viral virulence in mice, the viruses displayed expanded tissue tropism, and increased replication kinetics in mammalian cells. Continued surveillance in poultry for amino acid changes that might indicate H5N8 HPAI viruses pose a threat to human health is required.

Assessing the oseltamivir-induced resistance risk and implications for influenza infection control strategies.

BackgroundOseltamivir-resistant mutants with higher drug resistance rates and low trans-mission fitness costs have not accounted for influenza (sub)type viruses. Predicting the impacts of neuraminidase inhibitor therapy on infection rates and transmission of drug-resistant viral strains requires further investigation.ObjectivesThe purpose of this study was to assess the potential risk of oseltamivir-induced resistance for influenza A (H1N1) and A (H3N2) viruses.Materials and methodsAn immune-response-based virus dynamic model was used to best fit the oseltamivir-resistant A (H1N1) and A (H3N2) infection data. A probabilistic risk assessment model was developed by incorporating branching process-derived probability distribution of resistance to estimate oseltamivir-induced resistance risk.ResultsMutation rate and sensitive strain number were key determinants in assessing resistance risk. By increasing immune response, antiviral efficacy, and fitness cost, the spread of resistant strains for A (H1N1) and A (H3N2) were greatly decreased. Probability of resistance depends most strongly on the sensitive strain number described by a Poisson model. Risk of oseltamivir-induced resistance increased with increasing the mutation rate for A (H1N1) only. The ≥50% of resistance risk induced by A (H1N1) and A (H3N2) sensitive infected cells were 0.4 (95% CI: 0.28–0.43) and 0.95 (95% CI 0.93–0.99) at a mutation rate of 10−6, respectively. Antiviral drugs must be administrated within 1–1.5 days for A (H1N1) and 2–2.5 days for A (H3N2) virus infections to limit viral production.ConclusionProbabilistic risk assessment of antiviral drug-induced resistance is crucial in the decision-making process for preventing influenza virus infections.

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.

Is chickenpox so bad, what do we know about immunity to varicella zoster virus, and what does it tell us about the future?

Varicella and zoster continue to cause significant morbidity and even mortality in children and adults. Complications include bacterial superinfection, central nervous system manifestations such as meningitis, encephalitis, and cerebellar ataxia, and pain syndromes especially post herpetic neuralgia. Many developed countries but not all, are now administering live attenuated varicella vaccine routinely, with a decrease in the incidence of disease, providing personal and herd immunity. There is some controversy, however, in some countries concerning whether a decrease in the circulation of wild type virus will result in loss of immunity to VZV in persons who have already had varicella. This manuscript reviews the complications of varicella and zoster in detail, the reasons for development of vaccines against these diseases, complications of vaccinations, and mechanisms by which immunity to this virus develops and is maintained. There are strong indications that the best way to control disease and spread of this virus is by vaccination against both.

Prevention and control of emergent infectious disease with high specific antigen sensor

This study aims to evaluate the application of a new type of high specificity antigen sensor in detecting the viruses in sudden infectious diseases. Influenza A (H1N1) virus immunosensor was used for the respective determination of the six kinds of antigens of H1N1, H3N2 viral protein, HA protein of H7N9, influenza B virus, adenovirus, and EV71 virus of same dilution degree on the Screen Printed Carbon Electrode (SPCE), so as to test the specificity of the detection method. In addition, various batches of chick embryo allantoic saliva dilution simulation samples were also detected on their recovery (accuracy), repeatability (precision), and stability. The results were as follows: the linear equation was y = 121.33x + 168; the slope of the linear equation was 121.33 nA/HA unit, representing the sensitivity; correlation coefficient was R2=0.9921 > 0.90. Using Statistical Analysis System (SAS) software, we found that: the W values of seven sets of data after Shapiro–Wilk detection were 0.853, 0.991, 0.901, 0.906, 0.825, 0.974, and 0.992, respectively; P values were 0.247, 0.831, 0.386, 0.405, 0.174, 0.691, and 0.821, respectively, all of which were greater than 0.05, suggesting that normality was met. The results of homogeneity test for variance were as follows: F = 2.44, P = 0.0775 > 0.05, suggesting that homogeneity of variance was met. The parametric test results were as follows: F = 19114.0, P < 0.0001, suggesting that there were obvious differences between testing data of the seven groups. The determination recovery rate of electrochemical immunosensor was 80–110%. Relative Standard Deviation (RSD) values of repeatability (precision) test of H1N1 influenza virus electrochemical immunosensor were 7.74%, 3.54%, and 2.01%, all of which were smaller than 10%. The signal response of H1N1 electrochemical immune biological sensor could still maintain more than 85% of the original signal within 30 days of storage. In conclusion, H1N1 electrochemical immune biosensor has good specificity and the test results are not affected by other viruses of the same type. Besides, it has good accuracy which can realize the accurate determination of A (H1N1) influenza virus in actual detection. Thus, the requirement of precision measurement of A (H1N1) flu virus detection can be met. Therefore, H1N1 electrochemical immune biosensors can be used in actual detection with good stability.

Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.

Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry.

To determine the genetic and epidemiological relationship of infectious bronchitis virus (IBV) isolates from commercial poultry to attenuated live IBV vaccines we conducted a phylogenetic network analysis on the full-length S1 sequence for Arkansas (Ark), Massachusetts (Mass) and Delmarva/1639 (DMV/1639) type viruses isolated in 2015 from clinical cases by 3 different diagnostic laboratories. Phylogenetic network analysis of Ark isolates showed two predominant groups linked by 2 mutations, consistent with subpopulations found in commercial vaccines for this IBV type. In addition, a number of satellite groups surrounding the two predominant populations were observed for the Ark type virus, which is likely due to mutations associated with the nature of this vaccine to persist in flocks. The phylogenetic network analysis of Mass-type viruses shows two groupings corresponding to different manufacturers vaccine sequences. No satellite groups were observed for Mass-type viruses, which is consistent with no persistence of this vaccine type in the field. At the time of collection, no vaccine was being used for the DMV/1639 type viruses and phylogenetic network analysis showed a dispersed network suggesting no clear change in genetic distribution. Selection pressure analysis showed that the DMV/1639 and Mass-type strains were evolving under negative selection, whereas the Ark type viruses had evolved under positive selection. This data supports the hypothesis that live attenuated vaccine usage does play a role in the genetic profile of similar IB viruses in the field and phylogenetic network analysis can be used to identify vaccine and vaccine origin isolates, which is important for our understanding of the role live vaccines play in the evolutionary trajectory of those viruses.

Comparison of humoral and T-cell-mediated immune responses to a single dose of Bovela® live double deleted BVDV vaccine or to a field BVDV strain

The objective of this study was to determine and compare the humoral and cellular immune responses of calves exposed to a single dose of Bovela® bovine viral diarrhea virus (BVDV) live double deleted vaccine or a field strain virus (FSV) of BVDV type 2 (strain 890). Thirty seronegative, colostrum-deprived 5 month-old Holstein steer calves that tested negative for persistent BVDV by ear notch immunohistochemistry and seronegative to BVDV types 1 and 2 were used. Calves were screened by multi-parameter flow cytometry (MP-FCM) 1 week before vaccination to ensure that they were negative for T cell responses to the BVDV types 1 and 2 viruses in the Bovela® vaccine. Calves were assigned to 3 treatment groups: control (PBS), FSV inoculated, and Bovela® vaccinated. The humoral response was tested by standard serum virus neutralization (SVN) test to BVDV types 1 (Singer strain) and 2 (strain 125). The response by CD4, CD8, and gamma delta (γδ TCR) T cells was evaluated by MP-FCM using individual BVDV types 1 and 2 from Bovela® vaccine as recall antigens at 5, 6, and 7 weeks after vaccination. Activation markers used were upregulation of surface CD25 (IL-2R), intracellular interferon gamma (IFNγ) and intracellular interleukin 4 (IL-4). Each T cell subset was evaluated for increased expression of each activation marker compared to non-antigen stimulated cells of the same animal. All Bovela® vaccinated and FSV inoculated calves produced SVN antibodies to both BVDV types 1 and 2 while control animals remained seronegative throughout the study. The mean (weeks 5, 6, and 7) T cell recall responses to Bovela® BVDV type 1 and type 2 recall antigens were numerically higher in all three T cell subsets (CD4, CD8, and γδ TCR) for all three activation markers (CD25, IFNγ, and IL-4) when compared to either the control animals or to the FSV inoculated animals. These differences were often, but not always, statistically significant (P<0.05)

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014–2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or Ct-value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious.