Age is a determinant factor in the susceptibility of domestic ducks to H5 clade 2.3.2.1c and 2.3.4.4e high pathogenicity avian influenza viruses.

High pathogenicity avian influenza (HPAI) is a viral disease with devastating consequences for the poultry industry worldwide. Domestic ducks are a major source of HPAI viruses in many Eurasian countries. The infectivity and pathogenicity of HPAI viruses in ducks vary depending on host and viral factors. To assess the factors influencing the infectivity and pathogenicity of HPAI viruses in ducks, we compared the pathobiology of two HPAI viruses (H5N1 clade 2.3.2.1c and H5N6 clade 2.3.4.4e) in 5- and 25-week-old ducks. Both HPAI viruses caused mortality in a dose-dependent manner (104, 106, and 108 EID50) in young ducks. By contrast, adult ducks were infected but exhibited no mortality due to either virus. Viral excretion was higher in young ducks than in adults, regardless of the HPAI strain. These findings demonstrate the age-dependent mortality of clade 2.3.2.1c and clade 2.3.4.4e H5 HPAI viruses in ducks.

Research Note: Evaluation of the cross protective efficacy of a subtype B aMPV vaccine against virulent subtype A aMPV by different administration routes.

Avian metapneumovirus (aMPV) causes respiratory and reproductive diseases in birds, including chickens. In the chicken industry, live vaccines against aMPV subtypes A and B, which are the major aMPV subtypes, are widely used to control disease caused by aMPV. In this study, we evaluated the cross protective efficacy of a live aMPV subtype B vaccine administered via 3 different routes (nasal, spray, and oral) against virulent aMPV subtype A in chickens. At 3 wk after vaccination of 1-wk-old specific-pathogen-free chickens, we measured the serological responses. On the same day, we challenged the birds with aMPV subtype A. Protection was evaluated by viral gene detection and histopathological examination at 3 and 5 days postchallenge. Although there were differences in the serological responses according to administration route, all vaccinated birds showed complete protection at 5 days postchallenge. Regardless of administration route, genome of challenge virus was not detected in vaccinated group, and there were significant differences between vaccinated birds and control group. Overall, our results demonstrated that a subtype B aMPV vaccine can provide cross protection against virulent subtype A aMPV in chickens.

Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles.

In recent decades "saliva" has emerged as an important non-invasive biofluid for diagnostic purposes in both human and animal health sectors. However, with the rapid evolution of molecular detection technologies, the limitation has been the lack of an efficient method for the facile amplification of target RNA from such a complex matrix. Herein, we demonstrate the novel application of hydrogel microparticles of primer-immobilized networks (PIN) for direct quantitative reverse transcription PCR (dirRT-qPCR) of viral RNA from saliva samples without prior RNA purification. Each of these highly porous PIN particles operates as an independent reactor. They filter in micro-volumes of the analyte solution. Viral RNA is captured and converted to complementary DNA (cDNA) through the RT step using covalently incorporated RT primers. The PIN with cDNA of the viral target will be ready for subsequent highly specific qPCR. Preceded by heat-treatment for viral lysis, we were able to conduct PIN dirRT-qPCR with 95% efficiency of the matrix (M) gene for influenza A virus (IAV) and 5' untranslated region (5' UTR) for chicken coronavirus spiked into saliva samples. The addition of reverse transcriptase enzyme (RTase) and 10% dilution of the matrix improved the assay sensitivity considerably. PIN particles' compatibility with microfluidic PCR chip technology has significantly reduced total sample processing time to 50 min, instead of an average of 120 min that are normally used by other assays. We anticipate this technology will be useful for other viral RNA targets by changing the incorporated RT primer sequences and can be adapted for onsite diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00065-0.

Thermal Image Scanning for the Early Detection of Fever Induced by Highly Pathogenic Avian Influenza Virus Infection in Chickens and Ducks and Its Application in Farms.

Highly pathogenic avian influenza (HPAI) is considered as one of the most devastating poultry diseases. It is imperative to immediately report any known outbreaks to the World Organization for Animal Health. Early detection of infected birds is of paramount importance to control virus spread, thus minimizing the associated economic loss. In this study, thermal imaging camera devices were used to detect change in the maximum surface temperature (MST) of chickens (n = 5) and ducks (n = 2) as an early indicator of experimental HPAI infection. The MST of both chickens and ducks increased at least 24 h before the manifestation of clinical signs of HPAI infection, depending on the severity of the infection. The basal MST was recorded for broiler chickens housed under small pen and normal farm conditions without intentional infection. A threshold cutoff of MST was established based on the circadian rhythm of normal MST. This study suggests that thermal imaging of chickens and ducks is a promising tool to screen any potential HPAI-infected flock in order to expedite HPAI diagnosis.

Evaluation of insulated isothermal PCR devices for the detection of avian influenza virus.

Highly pathogenic avian influenza viruses (HPAIVs) and low pathogenic avian influenza viruses (LPAIVs) represent important threats to the poultry industry and global human health. Due to the high rates of avian influenza virus (AIV) transmission, controlling AIV outbreaks is challenging. HPAIV is known to be transmitted from wild birds to domestic ducks, from which it can be transmitted to layer and broiler chickens. Therefore, surveillance of AIV in domestic ducks and chickens in advance of outbreaks can prevent its spread and enable timely implementation of disease control measures. Certain molecular diagnostic tools can be applied in the field for faster AIV detection. In this study, we evaluated the AIV-detection ability of two insulated isothermal PCR (iiPCR) devices, POCKIT™Micro DUO Nucleic Acid Analyzer (POCKIT DUO) and POCKIT™ Central Nucleic Acid Analyzer (POCKIT Central). We found that the analytical, in vivo and clinical performances of the two POCKIT devices were comparable to those of real-time reverse transcription PCR. Due to their brief protocols and short detection times, POCKIT DUO and POCKIT Central represent promising molecular diagnostic devices for the reliable detection of AIV.

Efficacy of a novel avian metapneumovirus live vaccine candidate based on vaccination route and age.

This article describes a series of animal studies for the development of an avian metapneumovirus (aMPV) live vaccine. Although aMPV causes continual economic loss in the poultry industry, there are no live aMPV vaccines available in Korea. Furthermore, information is limited with respect to standard field practices for vaccinations at an early age. Here, the development of an aMPV live vaccine was attempted, and its efficacy was investigated with respect to the vaccination route and age to develop a method for controlling aMPV. Before vaccine development, an animal challenge model was established using the aMPV field isolate to identify the most effective time and site for collecting samples for evaluation. After attenuation of the virulent aMPV in Vero cells, a safety and efficacy test was conducted for the vaccine candidate. As a novel aMPV live vaccine candidate, aMPV K655/07HP displayed sufficient safety in day-old chicks with 10 vaccine doses. The efficacy test using 1-week-old chicks showed weaker humoral immune response than that in 4-week-old chicks. However, the candidate vaccine provided complete protection against infection caused by the challenge virus for all ages of vaccinated chicks. In conclusion, an effective aMPV challenge model was established for studying aMPV in chickens, which offered important, insightful information. The safety and efficacy study suggested that the new aMPV candidate vaccine could be used to effectively reduce the economic losses incurred because of aMPV infection.

The 3D8 single chain variable fragment protein suppresses Newcastle disease virus transmission in transgenic chickens.

BACKGROUND: The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G2 transgenic chickens to evaluate suppression of viral transmission. RESULT: The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences (p < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV. CONCLUSIONS: Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.

C-di-GMP with influenza vaccine showed enhanced and shifted immune responses in microneedle vaccination in the skin.

A microneedle is a biomedical device which consists of multiple micron scale needles. It is widely used in various fields to deliver drugs and vaccines to the skin effectively. However, when considering improved vaccine efficacy in microneedle vaccination, it is important to find an appropriate adjuvant that is able to be used in transdermal delivery. Herein, we demonstrated the applicability of c-di-GMP, which is a stimulator of interferon genes (STING) agonist, as an adjuvant for influenza microneedle vaccination. Thus, 2 and 10 µg of GMP with the influenza vaccine were coated onto a microneedle, and then, BALB/c mice were immunized with the coated microneedle to investigate the immunogenicity and protection efficacy of the influenza microneedle vaccination. As a result, the adjuvant groups had an enhanced IgG response, IgG subtypes and HI titer compared to the vaccine only group. In addition to the humoral immunity, the use of an adjuvant has also been shown to improve the cellular immune response. In a challenge study, adjuvant groups had a 100% survival rate and rapid weight recovery. Taken together, this study confirms that GMP is an effective adjuvant for influenza microneedle vaccination. Graphical abstract.

Introduction of Avian Influenza A(H6N5) Virus into Asia from North America by Wild Birds.

An avian influenza A(H6N5) virus with all 8 segments of North American origin was isolated from wild bird feces in South Korea. Phylogenetic analysis suggests that this virus may have been introduced into Asia by wild birds, highlighting the role of wild birds in the dispersal of these viruses.

Different pathogenicity of two strains of clade 2.3.4.4c H5N6 highly pathogenic avian influenza viruses bearing different PA and NS gene in domestic ducks.

H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) have been disseminated to wide geographic regions since 2014. In 2016, five distinct genotypes (C-1 to C-5) of clade 2.3.4.4c H5N6 HPAIVs were detected in South Korea. In this study, we evaluated the pathogenicity, susceptibility to infection, and transmissibility of the two strains representing the C-1 and C-4 genotypes of the H5N6 viruses, which have different PA and NS gene, in domestic ducks. Although the susceptibility to infection of domestic ducks to the two strains was similar, the C-4 genotype virus induced higher mortality in ducks than C-1 genotype virus. A higher titer of viral shedding were detected in ducks challenged with the C-4 genotype virus compared with the C-1 genotype virus. These results indicated that the reassortment of HPAIVs with prevailing low pathogenic avian influenza viruses could effect on the pathogenicity in ducks.

New Reassortant Clade 2.3.4.4b Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2017-18.

We isolated new reassortant avian influenza A(H5N6) viruses from feces of wild waterfowl in South Korea during 2017-18. Phylogenetic analysis suggested that reassortment occurred between clade 2.3.4.4b H5N8 and Eurasian low pathogenicity avian influenza viruses circulating in wild birds. Dissemination to South Korea during the 2017 fall migratory season followed.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017.

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Isolation and genomic characterization of a novel avian orthoreovirus strain in Korea, 2014.

In this study, we isolated a novel avian reovirus (ARV) strain, K738/14, from a broiler chicken with viral arthritis in South Korea. Genome sequence comparisons showed relatively low nucleotide identity with previously identified ARV strains. Phylogenetic analyses suggested multiple reassortment events between reovirus strain S1133 and reoviruses of Hungarian, Chinese, and US origin had occurred. In addition, recombination analyses showed evidence of intra-segmental recombination in the M2 and S2 genes. Based on our genetic analyses, multiple reassortment events, intra-segmental recombination, and accumulation of point mutations have possibly contributed to the emergence of this novel genotype of ARV, identified in Korea.

Molecular Characterization of Avian Paramyxovirus Types 4 and 8 Isolated from Wild Migratory Waterfowl in Mongolia.

Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

Effect of zymosan and poly (I:C) adjuvants on responses to microneedle immunization coated with whole inactivated influenza vaccine.

Microneedles are the micrometer size devices used for the delivery of vaccines and biotherapeutics. In order to increase the vaccine efficacy and reduce the antigen dose, there is a significant need to find some adjuvants for the microneedle vaccination. In this study, zymosan, which is the cell wall preparation of Saccharomyces cerevisiae, or poly (I:C) was coated on a microneedle with inactivated influenza virus, and then immunized into BALB/c mouse to determine the immunogenicity, protection and synergetic effect between two adjuvants. As a result, the group administered with zymosan and vaccine antigen showed significantly stronger IgG response, HI titer and IgG subtypes without any adverse effects, compared to the group immunized with the vaccine antigen alone. Also, there were enhanced cellular immune responses in the group received adjuvant with vaccine antigen. In addition, they showed superior protection and lung viral reduction against lethal viral challenge. Taken together, this study confirms that zymosan can be used as an immunostimulant for microneedle vaccination.

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission.

The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken ß-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

Experimental infection with highly pathogenic H5N8 avian influenza viruses in the Mandarin duck (Aix galericulata) and domestic pigeon (Columba livia domestica).

Wild birds play a major role in the evolution, maintenance, and dissemination of highly pathogenic avian influenza viruses (HPAIV). Sub-clinical infection with HPAI in resident wild birds could be a source of dissemination of HPAIV and continuous outbreaks. In this study, the pathogenicity and infectivity of two strains of H5N8 clade 2.3.4.4 virus were evaluated in the Mandarin duck (Aix galericulata) and domestic pigeon (Columba livia domestica). None of the birds experimentally infected with H5N8 viruses showed clinical signs or mortality. The H5N8 viruses efficiently replicated in the virus-inoculated Mandarin ducks and transmitted to co-housed Mandarin ducks. Although relatively high levels of viral shedding were noted in pigeons, viral shedding was not detected in some of the pigeons and the shedding period was relatively short. Furthermore, the infection was not transmitted to co-housed pigeons. Immunohistochemical examination revealed the presence of HPAIV in multiple organs of the infected birds. Histopathological evaluation showed the presence of inflammatory responses primarily in HPAIV-positive organs. Our results indicate that Mandarin ducks and pigeons can be infected with H5N8 HPAIV without exhibiting clinical signs; thus, they may be potential healthy reservoirs of the H5N8 HPAIV.

Optimization of inactivated H5N9 highly pathogenic avian influenza vaccine and inactivated Salmonella enterica serovar Typhimurium vaccine with antigen dose and prime-boost regimen in domestic ducks.

Owing to the increase in the number of diseases affecting ducks and the demand for food safety by consumers, vaccination has become one of the factors that influence duck meat productivity. The highly pathogenic avian influenza (HPAI) virus is one of the most prevalent and causes one of the most lethal diseases in domestic ducks, and Salmonella enterica serovar Typhimurium is a food-borne pathogen persistent in the domestic duck population. To better understand the optimal usage of HPAI and S. enterica serovar Typhimurium vaccines, we aimed to determine antigen dose, oil and gel adjuvant usage with prime-boost regimen, and vaccination age, inducing the best immune response in ducks, without an effect on body weight gain. In the case of the inactivated H5N9 vaccine, a single dose of vaccine was inadequate to induce proper antibody titer when administered to day-old ducks, which necessitates boost vaccination. Administration of the oil-adjuvanted H5N9 vaccine administration in day-old and 2-week-old ducks resulted in a lower body weight at the time of slaughtering, compared to that of gel-adjuvanted H5N9 vaccine. However, gel-adjuvanted H5N9 vaccine failed to induce proper immune response to an extent recommend by OIE-World Organization for Animal Health. In the case of the Salmonella enterica serovar Typhimurium vaccine, a moderate or low dose of vaccine was appropriate for day-old ducks receiving the gel prime-oil boost vaccination. Single vaccination with oil adjuvants affects the mean body weight of 7-week-old ducks, suggesting that the gel adjuvant is more suitable for meat production. We expect that the use of adjuvants in a prime-boost regimen and at antigen doses set in this study will be helpful to maximize body weight in the case of domestic duck production at the actual farm site.

Complete Genome Sequence of an Avian Paramyxovirus Type 4 Strain Isolated from Domestic Duck at a Live Bird Market in South Korea.

We report here the first full-genome sequence of an avian paramyxovirus type 4 (APMV-4) strain isolated from a domestic mallard duck at a live bird market in South Korea. Phylogenetic analyses provide genetic information on a new genetic clade, APMV-4, isolated from a domestic duck and evidence of APMV-4 exchange between poultry and wild birds.

Effective humoral immune response from a H1N1 DNA vaccine delivered to the skin by microneedles coated with PLGA-based cationic nanoparticles.

Intradermal DNA vaccination is a promising method of immunization that overcomes some practical drawbacks of conventional intramuscular vaccinations. However, it is difficult to deliver DNA vaccines to target cells in the skin and polyplexes. This study outlines the development of an intradermal pH1N1 DNA vaccine delivery platform using microneedles (MNs) coated with a polyplex containing poly lactic-co-glycolic acid/polyethyleneimine (PLGA/PEI) nanoparticles (NPs). Stainless steel MNs with enhanced hydrophilicity have been manufactured by silanization, which improves coating efficiency. MNs coated with the polyplex encapsulating pDNA vaccine were prepared by optimizing the N/P ratio, with a 6:1 ratio showing the highest transfection efficiency in mammalian cells. Polyplexes were coated on MNs without severe aggregation of the polyplex in the dry form. The coated polyplex rapidly dissolved in porcine skin (within 5min) and induced a greater humoral immune response than that of intramuscular polyplex delivery or naked pH1N1 DNA vaccine delivery by a dry-coated MN. These results indicate that intradermal delivery of pDNA vaccines within a cationic polyplex coated on MNs has potential in skin immunizations.