Prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/esterase gene in dairy calves in China.

Bovine coronavirus (BCoV) is the causative agent of diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract illnesses in cattle across the world. In this study, a total of 190 faecal samples from dairy calves with diarrhoea were collected from 14 farms in six Chinese provinces, and BCoV was detected in 18.95% (36/190) of the samples by reverse transcriptase polymerase chain reaction. Full-length spike, hemagglutinin/esterase (HE), nucleocapsid and transmembrane genes were simultaneously cloned from 13 clinical samples (eight farms in four provinces), and most of the BCoV strains showed a unique evolutionary pattern based on the phylogenetic analysis of these genes. Interesting, 10 of the 13 strains were identified as HE recombinant strains, and these strains had experienced the same recombination event and carried the same recombination sites located between the esterase and lectin domain. They also shared an identical aa variant (F181V) in the R2-loop. Moreover, 9/10 strains displayed another identical aa variant (P, S158A) in the adjacent R1-loop of the HE gene, which differs from the other available BCoV HE sequences in the GenBank database. Our results showed that BCoV is widely circulating in dairy cattle in China, contributing to the diagnosis and control of dairy calves diarrhoea. Furthermore, a BCoV strain that carries a recombinant HE gene has spread in dairy calves in China. To the best of our knowledge, this is the first description of an HE recombination event occurring in BCoV; this is also the first description of the molecular prevalence of BCoV in China. Our findings will enhance current understanding about the genetic evolution of BCoV.

Subtyping of influenza A H1N1 virus using a label-free electrochemical biosensor based on the DNA aptamer targeting the stem region of HA protein.

Rapid subtyping of influenza viruses in clinical laboratories has been increasingly important because three subtypes (seasonal H1N1, H3N2, and 2009 H1N1) of influenza A virus currently disseminated in humans have variable susceptibilities to antiviral drug. Herein, we present DNA aptamers for selective detection of influenza A H1N1 (seasonal and 2009 pandemic H1N1) viruses by targeting recombinant influenza A mini-hemagglutinin (mini-HA) protein (the stable stem region of HA) and whole H1N1 viruses. The dissociation constants (KD) of aptamer candidates V46 and V57 were 19.2 nM and 29.6 nM, respectively, according to electrochemical characterization (differential pulse voltammetry), demonstrating strong binding to mini-HA. In comparison, the KD of the influenza virus antibodies is in the range of 1 µM-10 nM. Aptamer V46 showed higher specificity and binding affinity to the mini-HA protein and H1N1 subtypes, and it was also incorporated into an indium tin oxide-based electrochemical sensor, showing sensitive and specific detection of H1N1 viruses, with a limit of detection (LOD) of 3.7 plaque-forming units per mL. The binding affinity, specificity, and LOD achieved with the electrochemical sensor suggest that it can be used for rapid subtyping of H1N1. We also propose that this aptamer can be used for the neutralization of H1N1 subtypes, suggesting potential therapeutic and diagnostic applications.

Hydrophobic-interaction chromatography for purification of influenza A and B virus.

Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 µg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.

Ion concentration polarization for pre-concentration of biological samples without pH change.

In this paper, a method was developed for pre-concentrating large-volume biological samples for subsequent analysis. We previously developed another pre-concentration device, but it unfortunately altered the pH of the sample when an electric field was applied to the sample reservoir. Changes in the pH are not suitable for subsequent antibody-antigen reactions because of the stability issues that arise based on the target molecule's isoelectric point (pI). Here, this problem was overcome using ion concentration polarization (ICP) with a cation selective membrane (Nafion). Phosphate buffered saline was used as a test solution for the sample. The sample was contained in a reservoir that was not affected by the electric field, and an ICP barrier was formed in front of the reservoir. This device could concentrate microliter-scale samples without changing the pH because the biomolecules were blocked from passing through the ICP barrier while the sample (phosphate buffered saline) was drained. A 40 µL sample was successfully pre-concentrated to 20 µL in a single channel device and 10 µL in a dual channel device, resulting in 2.1-fold and 3.3-fold increases, respectively, in influenza hemagglutinin concentrations. These changes in the concentration were confirmed by ELISA.

Validation of the modified hemagglutination inhibition assay (mHAI), a robust and sensitive serological test for analysis of influenza virus-specific immune response.

BACKGROUND: The hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported. OBJECTIVE: We developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies. STUDY DESIGN: Clinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness. RESULTS: Utilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols. Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components. CONCLUSION: The modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09).

Development of an influenza virus protein array using Sortagging technology.

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.

Epidemiology of canine distemper virus in wild raccoon dogs (Nyctereutes procyonoides) from South Korea.

Raccoon dogs (Nyctereutes procyonoides) are widespread and common in South Korea. In 2011, we obtained serum samples from 102 wild raccoon dogs to survey their exposure to canine distemper virus (CDV). Forty-five of the 102 animals (44.1%) were seropositive. Field cases of canine distemper in wild raccoon dogs from 2010 to 2011 were investigated. Fourteen cases of CDV infection were identified by a commercially available CDV antigen detection kit. These cases were used for virus isolation and molecular analysis. Sequence analysis of hemagglutinin genes indicated that all viruses isolated belonged to the Asia-2 genotype. H protein residues which are related to the receptor and host specificity (residues 530 and 549) were analyzed. A glutamic acid (E) residue is present at 530 in all isolates. At 549, a histidine (H) residue was found in five isolates and tyrosine (Y) residue was found in 6 isolates. Our study demonstrated that CDV infection was widespread in wild raccoon dogs in South Korea.

Influenza virus isolation.

The isolation of influenza viruses is important for the diagnosis of respiratory diseases in lower animals and humans, for the detection of the infecting agent in surveillance programs, and is an essential element in the development and production of vaccine. Since influenza is caused by a zoonotic virus it is necessary to do surveillance in the reservoir species (aquatic waterfowls), intermediate hosts (quails, pigs), and in affected mammals including humans. Two of the hemagglutinin (HA) subtypes of influenza A viruses (H5 and H7) can evolve into highly pathogenic (HP) strains for gallinaceous poultry; some HP H5 and H7 strains cause lethal infection of humans. In waterfowls, low pathogenic avian influenza (LPAI) isolates are obtained primarily from the cloaca (or feces); in domestic poultry, the virus is more often recovered from the respiratory tract than from cloacal samples; in mammals, the virus is most often isolated from the respiratory tract, and in cases of high pathogenic avian influenza (HPAI) from the blood and internal organs of infected birds. Virus isolation procedures are performed by inoculation of clinical specimens into embryonated eggs (primarily chicken eggs) or onto a variety of primary or continuous tissue culture systems. Successful isolation of influenza virus depends on the quality of the sample and matching the appropriate culture method to the sample type.

Quality control of seasonal influenza vaccines.

The purpose of seasonal influenza vaccination is to prevent its spread. The vaccines contain strains of the influenza virus recommended and approved for a particular season. Just like any other medicinal product, all vaccines require marketing approval. Batches of approved vaccines are extensively tested by the manufacturers and additionally controlled by the approving authorities, which issue the quality control certificates. This article not only to describes the legal background of quality control, but also how control test results obtained by a Croatian official control laboratory are compared to manufacturer's results. We have found that testing results can slightly differ depending on methods/analytical procedures used in different laboratories. This investigation has also shown how important it is to test finished medicinal products, independently of testing at intermediate stages, and how retesting by control authorities ensures that marketed vaccines meet quality standards.

New measles virus genotype associated with outbreak, China.

To determine the origin of the virus associated with a measles outbreak in Menglian County, Yunnan Province, People's Republic of China, in 2009, we conducted genetic analyses. Phylogenetic analyses based on nucleoprotein (N) and hemagglutinin (H) gene sequences showed that these Menglian viruses were not closely related to sequences of any World Health Organization (WHO) reference strains representing the 23 currently recognized genotypes. The minimum nucleotide divergence between the Menglian viruses and the most closely related reference strain, genotype D7, was 3.3% for the N gene and 3.0% for the H gene. A search of the databases of GenBank, WHO, and the Health Protection Agency Measles Nucleotide Surveillance showed that the Menglian viruses, together with the 2 older non-Menglian viruses, could be members of a new proposed measles genotype, d11. The new genotype designation will allow for better description of measles transmission patterns, especially in the Southeast Asian and Western Pacific regions.

Vaccinia virus infection in monkeys, Brazilian Amazon.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins.

The fusion peptide is the only universally conserved sequence in the hemagglutinins of all 16 subtypes of influenza A and two genetic lineages of influenza B viruses. Here, peptides selected by bioinformatics approach were modified and conjugated to overcome serious technical hurdles such as the high hydrophobicity and weak immunogenicity of the viral fusion peptides. Antibodies generated against fusion peptides demonstrated remarkable specificity against the viral sequences and robustness of quantitatively analyzing the viral hemagglutinins even under stringent conditions. As quantitatively revealed by antibody-binding experiments, the fusion peptides of diverse hemagglutinins are exposed to the same degree upon unfolding at neutral pH to the physiologically fusogenic state. To our knowledge, this is the first report on the quantitative determination of virtually all influenza vaccines using a single universal antibody.

Native conformation at specific residues in recombinant inclusion body protein in whole cells determined with solid-state NMR spectroscopy.

Inclusion bodies are insoluble aggregates that are formed by bacteria to store excess recombinant protein produced during expression. The structure of the protein in inclusion bodies is poorly understood but it has been hypothesized that the protein may form misfolded beta sheet aggregates. This paper presents an isotopic labeling and solid-state nuclear magnetic resonance approach to determine the secondary structure of individual residues within a recombinant influenza virus "FHA2" protein in inclusion bodies. The inclusion bodies were studied either in the context of the unlysed hydrated E. coli cells or in the hydrated pellet formed from centrifugation of the material insoluble in the cell lysate. The native structure of FHA2 is predominantly helical and native helical structure was also observed for several specific residues in the inclusion body FHA2. This approach will be applicable to structural analysis of many inclusion body proteins and should provide useful information for optimizing solubilization and purification protocols of these proteins.

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge.

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.

Generation of a recombinant Oka varicella vaccine expressing mumps virus hemagglutinin-neuraminidase protein as a polyvalent live vaccine.

We constructed a recombinant varicella-zoster virus (VZV) Oka vaccine strain (vOka) that contained the mumps virus (MuV) hemagglutinin-neuraminidase (HN) gene, inserted into the site of the ORF 13 gene by using the bacterial artificial chromosome (BAC) system in Escherichia coli. Insertion of the HN gene into the VZV genome was confirmed by PCR and Southern blot. The infectious virus reconstituted from the vOka-HN genome (rvOka-HN) had a growth curve similar to the original recombinant vOka without the HN gene. The mumps virus HN protein expressed in rvOka-HN infected cells was expressed diffusely in the cytoplasm, and modification of the protein was similar to that seen in MuV-infected cells. Electron microscopic examination of infected cells revealed that HN was expressed on the plasma membrane of the cells but not in the viral envelope, suggesting that the tropism of rvOka-HN would be unchanged from that of the original vOka strain. Immunization of guinea pigs with rvOka-HN-induced VZV- and HN-specific antibodies. Interestingly, the induced antibodies had a strong neutralizing activity against virus-cell infections of both MuV and VZV. Therefore, the novel varicella vaccine expressing MuV HN protein is suitable as a polyvalent live attenuated vaccine against VZV and MuV infections.

Modulation of airway responses to influenza A/PR/8/34 by Delta9-tetrahydrocannabinol in C57BL/6 mice.

Delta(9)-tetrahydrocannabinol (Delta(9)-THC) has been widely established as a modulator of host immune responses. Accordingly, the objective of the present study was to examine the effects of Delta(9)-THC on the immune response within the lungs and associated changes in the morphology of the bronchiolar epithelium after one challenge with a nonlethal dose of the influenza virus A/PR/8 (PR8). C57BL/6 mice were treated by oral gavage with Delta(9)-THC and/or vehicle (corn oil) for 5 consecutive days. On day 3, mice were instilled intranasally with 50 plaque-forming units of PR8 and/or vehicle (saline) 4 h before Delta(9)-THC exposure. Mice were subsequently killed 7 and 10 days postinfection (dpi). Viral hemagglutinin 1 (H1) mRNA levels in the lungs were increased in a dose-dependent manner with Delta(9)-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4(+) and CD8(+) T cells in Delta(9)-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in Delta(9)-THC-treated mice by 10 dpi. Collectively, these results suggest that Delta(9)-THC treatment increased viral load, as assessed by H1 mRNA levels, through a decrease in recruitment of macrophages and lymphocytes, particularly CD4(+) and CD8(+) T cells, to the lung.

Murine leukemia virus R Peptide inhibits influenza virus hemagglutinin-induced membrane fusion.

The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.

Gripe aviar: situación actual / Avian influenza: situation at present

La presente comunicación, que es continuación y ampliación de trabajos anteriores del autor sobre virus de la gripe o influenza, publicados por la Real Academia Nacional de Farmacia e indicados en la página web de la misma (www.ranf. com), se refiere principalmente a los siguientes aspectos: a) La importancia de la glicoproteína vírica denominada hemaglutinina (HA) en el proceso de fijación selectiva y penetración de este virus en la célula hospedadora; y cómo ciertas modificaciones de la estructura de dicha glicoproteína afectan a la patogenicidad del virus de la gripe aviar subtipo H5N1. b) Los mecanismos de transmisión de la influenza aviar por reagrupamiento («reassortment») de genes de varias procedencias, o por mutación de genes. c) La peligrosidad actual en aves y en seres humanos a causa de dicho virus H5N1. d) Algunos de los posibles remedios y fármacos —excluidos los inhibidores de las neuraminidasa, NA (por ser objeto de otra próxima publicación del autor)— para combatir el riesgo de eventuales epidemias/pandemia

This communication, which follows and amplifies previous papers by the author, published by the Real Academia Nacional de Farmacia, as indicated on its web page (www.ranf.com), is mainly concerned with the following issues: a) The role of the influenza viral glycoprotein named hemagglutinin (HA) in the selective binding and entry of this virus into the host cell; and also how certain modifications in the structure of this glycoprotein influence the pathogenicity of the avian influenza H5N1 subtype virus. b) The mechanisms of avian influenza transmission both by reassortment of genes from several sources and by gene mutation. c) The present danger for birds and humans arising from the infectivity of the H5N1 virus. d) The use of resources and drugs —excluding those related to the inhibition of neuraminidase, NA (since they will be addressed in a forthcoming publication by the author)— to combat the eventual risk of epidemies/ pandemic

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2).

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10(9), 10(6) and 10(3) EID(50)/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10(9), 10(6) and 10(3) EID50, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 106 EID50/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.

Influenza virus assembly and budding in raft-derived microdomains: a quantitative analysis of the surface distribution of HA, NA and M2 proteins.

Influenza virus hemagglutinin (HA) and neuraminidase (NA) are known to associate with lipid rafts, membrane microdomains comprised of densely packed cholesterol and sphingolipids. These specialized membrane regions are believed to be involved in the budding of many enveloped viruses including influenza virus. Quantitative analysis of HA distribution on the surface of virus-infected cells by immunogold staining shows an organization into clusters that grow in size as the expression level of HA increases with time post-infection (p.i.) ( approximately 325-500 nm at 4 h p.i. and approximately 425-600 nm at 6 h p.i.). These HA-containing clusters are likely derived from lipid rafts as they contain a high density of the raft marker ganglioside GM1 and are dependent upon the presence of cholesterol. The clustering of HA is an intrinsic property of the HA protein and occurs in the absence of expression of other viral proteins. NA is also found sequestered within the same microdomains as HA, whereas the M2 ion channel protein does not concentrate within the raft-like microdomains. Quantification of the distribution of surface expressed HA by examining serial sections of virus-infected cells suggests that the HA-containing microdomains give rise to regions of influenza assembly and budding.