Hemagglutination Units Research Articles

Antibody Response of Mice to Bali Isolate of Canine Parvovirus Propagated in Madin-Darby Canine Kidney Cell Culture

Canine parvovirus (CPV) infection is still common among dogs, leading to severe disease with high mortality. The potential of a local isolate of CPV as an effective vaccine to prevent the disease warrants investigation. This study aimed to determine the antibody response in mice against a Bali isolate of CPV propagated in the Madin-Darby Canine Kidney (MDCK) cell culture. The virus was purified using polyethylene glycol (PEG)-6000 and mixed with an Aluminum hydroxide adjuvant. Fifteen 7-week female mice were divided into three treatment groups: treatment group 1 (PEG-purified virus and Adjuvant), treatment group 2 (crude unpurified virus and adjuvant), and treatment group 3 (adjuvant without virus), with five replicates per group. The Bali isolate of CPV was successfully replicated in MDCK cells, achieving a titer of 210-211 hemagglutination (HA) units after eight serial passages through the cell culture. The virus was confirmed as CPV by immunocytochemistry test using a monoclonal antibody and hemagglutination inhibition (HI) test using chicken anti-CPV polyclonal antibody. Following the first immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (5.6) was significantly higher than those immunized with crude unpurified CPV (4.2) and adjuvant without CPV (1.4). Similarly, after the second immunization, the antibody endpoint titer in mice immunized with PEG-purified CPV (7.6) also remained significantly higher than those immunized with crude unpurified CPV (6.4) and adjuvant without CPV (0.8). Significant increases in antibody endpoint titer were observed after the second immunization in mice immunized with PEG-purified CPV and crude unpurified CPV, but not in those given adjuvant without CPV. The Bali isolate of CPV propagated in MDCK cell culture induced a robust antibody response in mice, suggesting it’s a potential as an alternative vaccine candidate for preventing CPV infection in dogs.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

An oleanic acid decorated gold nanorod for highly efficient inhibition of hemagglutinin and visible rapid detection of the influenza virus

Accurate diagnosis and effective antiviral treatments are urgently needed for the prevention and control of flu caused by influenza viruses. In this study, a novel oleanic acid (OA) functionalized gold nanorod OA-AuNP was prepared through a convenient ligand-exchange reaction. As hemagglutinin (HA) on the viral surface binds strongly to the multiple OA molecules on the surface of the nanoparticle, the prepared OA-AuNP was found to exhibit potent antiviral activity against a wide range of influenza A virus strains. Furthermore, the change in color resulting from the specific binding between HA and OA and the resultant aggregation of the OA-AuNP can be visually observed or measured by UV–vis spectra with a detection limit of 2 and 0.18 hemagglutination units (HAU), respectively, which is comparable to the commercially available influenza colloid gold rapid diagnostic kits. These findings demonstrate the potential of the OA-AuNP for the development of novel multivalent antiviral conjugates and the diagnosis of influenza virus.

Comparative study of HA and HNB staining RT-LAMP assays for peste des petits ruminants virus detection in West African Dwarf goats.

Peste des petits ruminants (PPR) cause severe economic losses to many countries of the world where the disease is endemic. It has been targeted for global eradication by 2030 following the successful eradication of rinderpest in 2011. The proposed eradication program would benefit from efficient and relatively reliable diagnostic tools for early PPR virus (PPRV) detection. A total of 33 eight to 12 months old West African Dwarf (WAD) goats were used. Nineteen goats infected by commingling with two PPR virus-positive animals formed the infected group (PPRV-infected goats) while 14 non-infected goats formed the control group (CTG). The suitability of hydroxyl naphthol blue (HNB) staining of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and haemagglutination (HA) assays was compared for their sensitivity to detect the PPRV in PPRV-infected goats and non-infected CTG. PPR disease severity in WAD goats at different days post infection (dpi) was evaluated by clinical scoring and haemagglutination titre (HAT). HNB staining RT-LAMP reaction and HA showed sensitivities of 100% and 73.68%, respectively, for PPRV detection. Expression of PPR clinical signs began from 3 dpi, attained peak at 5 dpi, thereafter showed irregular patterns till 24 dpi. Evaluation of HAT in PPRV-infected goats at 12 dpi ranged from 2 to 64 haemagglutination units (HAU), while CTG goats had 0 HAU. In conclusion, HA could be a good tool for rapid diagnosis of PPRV in a developing country setting. However, HNB staining RT-LAMP assay demonstrated high sensitivity for accurate diagnoses of PPRV and as an important diagnostic tool when precise phenotyping is desired.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Generation of a high yield vaccine backbone for influenza B virus in embryonated chicken eggs

Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens’ eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.

Features of the Synthesis of Extracellular Cytotoxic Lectin Bacillus subtilis IMV B-7724, Depending on the Cultivation Conditions in the Laboratory Fermenter

The level of oxygen mass transfer (KV) is an important parameter influencing the growth rate of aerobic microorganisms and the synthesis of metabolites. It is mainly determined by the agitation and the aeration rates in the fermenter. Aim. To study changes in pH, optical density (OD), and hemagglutinating (lectin) activity (HAA) of culture fluid (CF) of Bacillus subtilis strain IMV B-7724, a producer of extracellular cytotoxic lectin (ECL), during its cultivation in a laboratory fermenter at different agitation and aeration rates as well as to determine and compare the HAA, carbohydrate specifi city, and cytotoxic properties of the corresponding samples of the preparation isolated from CF. Methods. Batch antifoam-free fermentations were performed by culturing the strain in the modified Gause medium with galactose in two identical lab-scale fermenters with a working volume of 2.5 L at 37ºC for 48—72 h according to three fermentation variants. Variant 1: n — 400 rpm for the whole cultivation, the air supply to the CF — through a sparger at 0.1 vvm until the 39th h with further gradual decrease, KV — 4.2±0.3 g O2·L−1·h−1. Variant 2: n — 400 rpm for the first 24 h, then a gradual decrease to 200 rpm, air supply — through a sparger at 0.1 rpm for the first 12 h, followed by its switching into the fermenter free space, corresponding KV — from 4.2±0.3 to 0.3±0.1 g O2·L−1·h−1. Variant 3: n — 400 rpm and air supply to the fermenter free space during the whole cultivation, KV — 4.0±0.3 g O2·L−1·h−1. A number of biological properties of strain CF and isolated lectin samples were evaluated by biochemical, spectrophotometric, immunological, and culture methods. Statistical analysis was performed using Student’s t-test. Results. The maximum increase in the OD of CF relative to the initial values (28 and 21-fold) at the end of the period of the rapid growth of the strain (at 9th h), the μmax values of 0.33 and 0.41 h−1, and pH not lower than 6.7 and 6.3 units were observed for fermentation variants 1 and 2, respectively. In the case of variant 2, the HAA of CF reached 32 hemagglutinating units (HAU), and the samples isolated from it had a lectin activity of 512±64 HAU, whereas for variant 1 such values were lower:16 and 32±8 HAA, respectively; carbohydrate specificity of preparations to bovine submandibular gland mucin was the same, i.e. 0.2±0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μmax, and significant acid formation (15-fold at the 9th h, 0.25 h−1, and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2—4 HAU) and was absent in the corresponding isolated samples. The probable reason for such differences was the limited mass transfer in the CF due to the isolating effect of the foam layer on its surface formed as a result of intensive agitation. Conclusions. The rapid growth of the strain and an increase in the HAA of CF were observed during cultivation in a lab-scale fermenter by maintaining the maximum level of oxygen mass transfer with air supply into the CF through a sparger until the maximum OD was reached and the subsequent gradual decrease in the specifi ed level during further cultivation started.

Dose immunogenicity study of a plant-produced influenza virus-like particle vaccine in layer hens

Avian influenza poses one of the largest known threats to global poultry production and human health, but effective poultry vaccines can reduce infections rates, production losses and prevent mortalities, and reduce viral shed to limit further disease spread. The antigenic match between a vaccine and the circulating field influenza A viruses (IAV) is a critical determinant of vaccine efficacy. Here, an Agrobacterium tumefaciens-mediated transient tobacco plant (Nicotiana benthamiana) system was used to rapidly update an H6 influenza subtype virus-like particle (VLP) vaccine expressing the hemagglutininn (HA) protein of South African H6N2 IAVs circulating in 2020. Specific pathogen free White Leghorn layer hens vaccinated twice with ≥125 hemagglutinating unit (HAU) doses elicited protective antibody responses associated with prevention of viral shedding, i.e. hemaglutination inhibition (HI) mean geometric titres (GMTs) of ≥7 log2, for at least four months before dropping to approximately 5–6 log2 for at least another two months. A single vaccination with a 250 HAU dose induced significantly higher HI GMTs compared lower or higher doses, and was thus the optimal dose for chickens. Use of an adjuvant was essential, as the plant-produced H6 HA VLP alone did not induce protective antibody responses. Plant-produced IAV VLPs enable differentiation between vaccinated and infected animals (DIVA principle), and with sucrose density gradient-purified yields of 20,000 doses per kg of plant material, this highly efficacious, safe and economical technology holds enormous potential for improving poultry health in lower and middle-income countries.

Importance of Influenza Anti-Hemagglutinin Antibodies During the SARS-CoV-2 Pandemic in the 2019/2020 Epidemic Season in Poland.

BackgroundThe aim of this study was to determine the level of anti-hemagglutinin antibodies in the serum of recovered patients during the SARS-CoV-2 pandemic in the 2019/2020 epidemic season in Poland, and the course of COVID-19.Material/MethodsThe material for the study consisted of the sera of COVID-19 convalescents obtained from the following 9 Regional Blood Donation and Blood Supply Centers located in 8 voivodeships. The hemagglutination inhibition reaction assay (HAI) using 8 viral hemagglutination units was used to determine antibody levels, in accordance with WHO recommendations.ResultsThis research confirms that a patient’s declared severity of the course of SARS-CoV-2 infection is influenced by the patient’s age and concomitant diseases. There was no statistically significant correlation between the level of anti-hemagglutinin antibodies and the severity of the course of a SARS-CoV-2 infection. Based on the serological tests conducted, it can be unequivocally concluded that both vaccinated and influenza-infected patients had a response rate in line with the requirements of the European Commission and the Committee for Medicinal Products for Human Use hemagglutinin antibodies for 4 influenza virus antigens tested.ConclusionsPatients who confirmed their antibody levels with the Commission of the European Communities and the Committee for Propriety Medicinal Products (CPMP) requirements had a mild COVID-19 course. The results of our research emphasize the role of anti-hemagglutinin antibodies in the course of SARS-CoV-2 infection. COVID-19 convalescents have a higher response rate against all 4 types of anti-hemagglutinin antibodies analyzed.

Movable Layer Device for Rapid Detection of Influenza a H1N1 Virus Using Highly Bright Multi-Quantum Dot-Embedded Particles and Magnetic Beads.

Preventing the rapid spread of viral infectious diseases has become a major concern for global health. In this study, we present a microfluidic platform that performs an immunoassay of viral antigens in a simple, automated, yet highly sensitive manner. The device uses silica particles embedded with highly bright quantum dots (QD2) and performs the immunoassay with a vertically movable top layer and a rotating bottom layer. Through the motion of the layers and the surface tension in the liquids, reagents move from top chambers to bottom chambers and mix homogeneously. A tip in the top layer with a mobile permanent magnet moves the immune complexes comprising the magnetic beads, virus particles, and QD2 between the bottom chambers. In this way, our automated device achieves a highly sensitive magnetic bead-based sandwich immunoassay for the influenza A H1N1 virus within 32.5 min. The detection limit of our method is 5.1 × 10−4 hemagglutination units, which is 2 × 103 times more sensitive than that of the conventional hemagglutination method and is comparable to PCR. Our device is useful for the rapid and sensitive detection of infectious diseases in point-of-care applications and resource-limited environments.

Semi-Quantification of Lectins in Rice (Oryza sativa L.) Genotypes via Hemagglutination

Lectins are unique glycoproteins that react with specific sugar residues on cell surfaces resulting in agglutination. They offer enormous applications in therapeutics, diagnostics, medicine, and agriculture. Rice lectins are naturally expressed during biotic and abiotic stresses suggesting their importance in stress resistance physiology. The objective of this study was to determine the presence and relative concentration of lectins in different accessions of rice obtained from IABGR/NARC Islamabad mainly originated from Pakistan. About 210 rice accessions including 02 local varieties and 05 transgenic seeds were screened for seed lectins using a hemagglutination (HA) assay with 5% Californian bred rabbits’ erythrocytes. A protein concentration of 3–8 mg/100 mg of seed flour was measured for all the rice accessions; the highest was 8.03 mg for accession 7600, while the lowest noted was 3.05 mg for accession 7753. Out of 210 accessions, 106 showed the highest HA activity. These 106 genotypes were further screened for titer analysis and specific activity. The highest titer and specific activity were observed for accession 7271 as 1024 and 236 hemagglutination unit (HAU), respectively. The selected accessions’ relative affinity and HA capability were evaluated using blood from four different sources: human, broiler chicken, local rabbit, and Californian-breed rabbit. The highest HA activity was observed with Californian-breed rabbit RBCs. The lectin assay was stable for about 1–2 h. After the required investigations, the accessions with higher lectin concentration and HA capability could be used as a readily available source of lectins for further characterization and utilization in crop improvement programs.

Detection of peste des petits ruminants virus in pneumonic lungs from clinically apparently healthy camels slaughtered at Tambul slaughterhouse, Central Sudan.

The study investigated the presence and prevalence of peste des petits ruminants (PPR) viral antigens among camels in Tambul area, Gezira State, Central Sudan, regardless of its sex, age and breed, and their possible contribution in the epidemiology of the disease in the Sudan. Hundred pneumonic lung tissues were aseptically collected from clinically apparently healthy camels showed no signs of illness at ante‐mortem examination, from Tambul slaughterhouse, Tambul area, Gezira State, Central Sudan, between November and December 2018. Samples were collected based on presence of the pneumonic signs, at the tissue level, including congestion of the lungs, presence of abscesses, fragility, changes in colour and thickness of the tissue. In order to detect PPR viral antigen, haemagglutination (HA) test was employed on lung tissue homogenate, using chicken RBCs suspension, which gave a positive reaction in 17–19 min. PPRV antigen was detected in 98 of camel samples with an overall antigenic prevalence of 98%. Of note, the HA titres achievable ranged from 4 to 256 HA units (HAU) with mean titre of 14.4 HAU, whereas apparently most of the samples achieved HA titres of 8 HAU. The results demonstrated presence of PPR viral antigens associated with pneumonia in camels indicating exposure of these camels to PPRV and probably presence of subclinical infection. Infection of species other than small ruminants suggests the fact that camels are potential hosts for PPRV and might play a role (or not) in the epidemiology of the disease. Further studies are needed to demonstrate if camels are able to transmit PPRV for in‐contact small ruminants or other animal species.

Melatonin modulates mitophagy, innate immunity and circadian clocks in a model of viral-induced fulminant hepatic failure.

The haemorrhagic disease virus (RHDV) is a non‐cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV‐infected rabbits. This study investigated whether protection against viral‐derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 104 haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV‐induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV‐induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.

A strip test for the optical determination of influenza virus H3 subtype using gold nanoparticle coated polystyrene latex microspheres.

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. Graphical abstract .

Development of an antigen-capture enzyme-linked immunosorbent assay and immunochromatographic strip based on monoclonal antibodies for detection of H6 avian influenza viruses.

Continuous surveillance has shown that H6 subtype avian influenza viruses (AIVs) are prevalent in poultry and occasionally break the species barrier to infect humans. It is therefore necessary to establish a specific, rapid and sensitive method to screen H6 AIVs. In this study, a panel of monoclonal antibodies (mAbs) against the hemagglutinin (HA) of an H6 AIV isolate was produced. The purified mAbs have high affinity and specificity for H6 AIVs. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic strip were developed based on two mAbs (1D7 and 1F12). The AC-ELISA results showed high sensitivity with a limit of detection (LOD) of 3.9 ng/ml for H6 HA protein and 0.5 HAU (HA units)/100µl for live H6 subtype AIVs. The average recovery of the AC-ELISA with allantoic fluid, respiratory specimens, and cloacal swabs was 91.907 ± 1.559%, 82.977 ± 1.497% and 73.791 ± 2.588%, respectively. The intra- and inter-assay coefficient of variation was less than 10%. The LOD of immunochromatographic strip was 1 HAU when evaluated by the naked eye, and the detection time was less than 10 min without any equipment. Storage at room temperature or 4°C for 30days or 60days had no effect on sensitivity and specificity of the strip. Thus, the AC-ELISA and immunochromatographic strips described here could be a secondary method to diagnose H6 AIV infections with high specificity, sensitivity, and stability.

Development of a colloidal gold-based immunochromatographic strip test using two monoclonal antibodies to detect H7N9 avian influenza virus.

H7N9 low pathogenic avian influenza viruses (AIVs) emerged in China in 2013 and mutated into highly pathogenic strains in 2017, causing disease in infected birds and humans. Thus, the development of rapid, specific, and sensitive detection methods is urgently required. Herein, two specific monoclonal antibodies against H7N9 AIV were produced to develop a colloidal gold-based immunochromatographic test strip to detect H7N9 AIV. High specificity, repeatability, and sensitivity were achieved, with a detection limit of two hemagglutinin units or 102.55 50% tissue culture infective dose. This assay may represent a powerful tool to rapidly detect H7N9 influenza viruses in the future.

Hemagglutination Assay for Influenza Virus.

The hemagglutination assay (HA) is a tool used to screen cell culture supernatant or amniotic-allantoic fluid harvested from embryonated chicken eggs for hemagglutinating agents, such as type A influenza. The HA is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonated chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative as one hemagglutinating unit (HAU) is equal to approximately 5-6 log10 of virus. It is inexpensive and relatively simple to conduct. Several factors (e.g., the quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in the interpretation of the test each time it is run. This chapter will describe the methods validated and used by the US National Veterinary Services Laboratories for screening and identification of hemagglutinating viruses.

Process development for pandemic influenza VLP vaccine production using a baculovirus expression system

BackgroundInfluenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform.ResultsAn influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 μL to 512 HAU/50 μL).ConclusionsIn this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.

An integrated self-driven microfluidic device for rapid detection of the influenza A (H1N1) virus by reverse transcription loop-mediated isothermal amplification

Influenza A is amongst the most infectious diseases of the past decade. Although rapid and accurate detection can efficiently prevent its spread, the sensitivity and specificity of current rapid influenza diagnostic tests (RIDTs) are relatively low. In this study, a self-driven microfluidic device was developed to rapidly detect the H1N1 influenza virus via a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. The device was capable of 1) virus isolation via H1N1-specific aptamers conjugated magnetic beads, 2) virus lysis, 3) isothermal nucleic acid amplification, and 4) colorimetric detection of the virus. The limit of detection was measured to be only 3 × 10−4 hemagglutinating units (HAU)/reaction, which is sensitive enough for clinical applications. The entire procedure could be performed in only 40 min by capillary forces through a novel polydimethylsiloxane surface treatment in cooperation with hydrophobic soft valves. This is the first time that a simple, self-driven passive microfluidic chip has been demonstrated to be capable of performing sample pretreatment, RT-LAMP, and H1N1 virus detection on a single chip. This microfluidic chip may therefore serve a promising RIDT with high sensitivity and high specificity for point-of-care diagnostics in the near future.