ABSTRACT
INTRODUCTION: The avian influenza (AI) virus causes a highly contagious disease which is common in wild and domestic birds and sporadic in humans. Mutations and genetic reassortments among the 8 negative-sense RNA segments of the viral genome alter its pathogenic potential, demanding well-targeted, active surveillance for infection control. METHODS: Wild duck fecal samples were collected during the 2018 bird health annual surveillance in South Korea for tracking variations of the AI virus. One low-pathogenic avian influenza H5N3 reassortment virus (A/mallard duck/South Korea/KNU18-91/2018 [H5N3]) was isolated and genomically characterized by phylogenetic and molecular analyses in this study. RESULTS: It was devoid of polybasic amino acids at the hemagglutinin (HA) cleavage site and exhibited a stalk region without deletion in the neuraminidase (NA) gene and NA inhibitor resistance-linked E/D627K/N and D701N marker mutations in the PB2 gene, suggesting its low-pathogenic AI. It showed a potential of a reassortment where only HA originated from the H5N3 poultry virus of China and other genes were derived from Mongolia. In phylogenetic analysis, HA was different from that of the isolate of H5N3 in Korea, 2015. In addition, this novel virus showed adaptation in Madin-Darby canine kidney cells, with 8.05 ± 0.14 log10 50% tissue culture infectious dose (TCID50) /mL at 36 h postinfection. However, it could not replicate in mice well, showing positive growth at 3 days postinfection (dpi) (2.1 ± 0.13 log10 TCID50/mL) but not at 6 dpi. CONCLUSIONS: The HA antigenic relationship of A/mallard duck/South Korea/KNU18-91/2018 (H5N3) showed differences toward one of the old low-pathogenic H5N3 viruses in Korea. These results indicated that a novel reassortment low-pathogenic avian influenza H5N3 subtype virus emerged in South Korea in 2018 via novel multiple reassortments with Eurasian viruses, rather than one of old Korean H5N3 strains.
Subject(s)
Influenza A virus , Influenza in Birds , Animals , Animals, Wild , Dogs , Ducks , Influenza A virus/genetics , Mice , PhylogenyABSTRACT
The aim of the study was to investigate the genetic and immunogenic features of commercial vaccines against infectious bronchitis virus (IBV), which is a major contagious pathogen of poultry. Although numerous vaccines have been developed based on the genetic characteristics of field strains, the continual emergence of variants decreases vaccine efficacy and cross-protection. To address this issue, we compared the S1 gene sequences of three IBV vaccines commercially available in Korea with those of various field isolates. Phylogenetic analysis showed that the vaccine strains clustered into two different lineages. Comparison of commercial vaccines with their parental viruses showed that most of the genetic variability occurred around hypervariable regions (HVRs). Conversely, antigenic stimulation with commercial vaccines and regional IBV variants was not sufficient to alter major immune cell phenotypes. Our study suggests that vaccines should be selected carefully based on their genetic background because genetic variability can affect the antigenicity of vaccines and host immune responses.
Subject(s)
Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Vaccines , Animals , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Phylogeny , Viral Vaccines/geneticsABSTRACT
Repeated interspecies transmission of H9N2 virus from poultry to humans and human infections transmitted via aerosols highlight the need for a highly sensitive, rapid diagnostic system for the detection of this virus. However, no such test exhibiting high performance has been developed. In this study, the performance of a smartphone-based rapid fluorescent diagnostic system (SRFDS) was optimized for the diagnosis of an H9N2-virus-infected animal. To suppress the nonspecific reactivity of the bioconjugate in oropharyngeal (OP) and cloacal (CL) samples derived from chickens, different blocking reagents were tested, and a mixture of casein and sucrose was found to be optimal. To assess the performance of SRFDS, OP and CL samples were obtained from specific-pathogen-free chickens and used for comparison of this method with real-time reverse transcription PCR (rRT-PCR) at time points of three, five, and seven days postinfection (dpi). The limit of detection of SRFDS was found to be 7.5 PFU/mL, which was 138-fold higher than that of a conventional colloidal-gold-based avian influenza rapid diagnostic test. In the animal study, the presence of viral antigen was monitored with SRFDS, and the relative sensitivity (relative to rRT-PCR results) was 94.44 % (17/18) and 95.23 % (20/21) in OP and CL specimens, respectively. The specificity of SRFDS was 100 %. These results imply that the diagnostic performance of SRFDS might be comparable to that of rRT-PCR for diagnosis of H9N2 in chickens and that this test can be used as a highly sensitive rapid diagnostic method in field studies on broiler poultry and wild birds.
Subject(s)
Diagnostic Tests, Routine/methods , Influenza A Virus, H9N2 Subtype/isolation & purification , Influenza in Birds/diagnosis , Poultry Diseases/diagnosis , Smartphone/statistics & numerical data , Animals , Antigens, Viral/chemistry , Antigens, Viral/metabolism , Chickens , Diagnostic Tests, Routine/instrumentation , Fluorescence , Influenza A Virus, H9N2 Subtype/chemistry , Influenza A Virus, H9N2 Subtype/genetics , Influenza A Virus, H9N2 Subtype/metabolism , Influenza in Birds/virology , Poultry Diseases/virology , Sensitivity and Specificity , Specific Pathogen-Free OrganismsABSTRACT
A new avian hepatitis E virus (HEV) GI-B was identified in broiler breeders with hematomas, liver rupture, and splenomegaly, along with excessive abdominal fat, in Korea. Previously, genotype 1 had been identified in avian HEV strains in Korea. Complete sequence analyses revealed that the new avian HEV clustered in genotype 2, which has been identified in the USA and Spain; the GI-B isolate was closely related to the USA prototype avian HEV isolated from a chicken with hepatitis-splenomegaly syndrome. Although some HEV genotypes show a geographical distribution pattern, the discovery of genotype 2 in addition to genotype 1 in Korea suggests that the geographical grouping might be reconsidered. These findings have important implications for understanding the global epidemiology and spread of avian HEV.
Subject(s)
Chickens/virology , Hepatitis E/virology , Hepatitis, Viral, Animal/virology , Hepevirus/genetics , Poultry Diseases/virology , Splenomegaly/virology , Amino Acid Sequence , Animals , Genotype , Phylogeny , Republic of Korea , SpainABSTRACT
A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in leghorn male hepatoma (LMH) chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation competent when transfected into LMH cells and infectious when injected intrahepatically into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and helped characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were injected intrahepatically into chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity.
Subject(s)
DNA, Complementary/genetics , DNA, Viral/genetics , Hepatitis, Viral, Animal/virology , Hepevirus/genetics , Hepevirus/physiology , RNA Virus Infections/veterinary , Virus Replication , Animal Experimentation , Animals , Chickens , Genotype , Hepatitis, Viral, Animal/pathology , Hepatocytes/virology , Hepevirus/classification , Male , Poultry Diseases/pathology , Poultry Diseases/virology , RNA Virus Infections/pathology , RNA Virus Infections/virologyABSTRACT
Chicken parvovirus (ChPV) is one of the causative agents of viral enteritis. Recently, the genome of the ABU-P1 strain of ChPV was fully sequenced and determined to have a distinct genomic composition compared with that of vertebrate parvoviruses. However, no comparative sequence analysis of coding regions of ChPVs was possible because of the lack of other sequence information. In this study, we obtained the nucleotide sequences of all genomic coding regions of three ChPVs by polymerase chain reaction using 13 primer sets, and deduced the amino acid sequences from the nucleotide sequences. The non-structural protein 1 (NS1) gene of the three ChPVs showed 95.0 to 95.5% nucleotide sequence identity and 96.5 to 98.1% amino acid sequence identity to those of NS1 from the ABU-P1 strain, respectively, and even higher nucleotide and amino acid similarities to one another. The viral proteins (VP) gene was more divergent between the three ChPV Korean strains and ABU-P1, with 88.1 to 88.3% nucleotide identity and 93.0% amino acid identity. Analysis of the putative tertiary structure of the ChPV VP2 protein showed that variable regions with less than 80% nucleotide similarity between the three Korean strains and ABU-P1 occurred in large loops of the VP2 protein believed to be involved in antigenicity, pathogenicity, and tissue tropism in other parvoviruses. Based on our analysis of full-length coding sequences, we discovered greater variation in ChPV strains than reported previously, especially in partial regions of the VP2 protein.
Subject(s)
Chickens/virology , Genetic Variation , Parvovirus/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers/genetics , Molecular Sequence Data , Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Sequence Homology , Species SpecificityABSTRACT
Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.
Subject(s)
DNA Virus Infections , Genome, Viral , Phylogeny , Ranavirus , Urodela , Animals , Ranavirus/genetics , Ranavirus/isolation & purification , Ranavirus/classification , Urodela/virology , Republic of Korea/epidemiology , DNA Virus Infections/veterinary , DNA Virus Infections/virology , DNA Virus Infections/epidemiology , Open Reading Frames , Whole Genome SequencingABSTRACT
Fowl adenovirus-4 (FAdV-4) is a highly contagious virus that causes acute and lethal hepatitis. It leads to substantial economic losses in the poultry industry. Among the structural proteins of FAdV-4, hexon and fiber2 are associated with immunopathogenesis. A frameshift mutation was generated in the fiber2 protein by seral passages in the Leghorn male hepatoma (LMH) cell line. Immunization using the attenuated virus (80 times passaged) before the virulent FAdV-4 challenge protected hosts from the infection and cleared the invading virus. In immunized animals, activated CD4+ and CD8+ T cell populations were larger during the FAdV-4 challenge. The change in the B cell population was similar. Myeloid cells were highly increased during FAdV-4 infection after the immunization, but the immunization inhibited the expansion in both liver and spleen. The functional gene expression for immune modulation was strongly associated with immune cell changes in the liver, however, this association was not strong in the spleen. The present findings imply that genetic modification by cellular adaptation regulates immune cell phenotype and function in the target organ. In addition, we suggest the attenuated virus as a protective strategy against the novel FAdV-4 strains.
ABSTRACT
The S2 glycoprotein and membrane (M) protein genes and S1 glycoprotein and nucleocapsid (N) genes of 11 Korean infectious bronchitis virus (IBV) isolates were amplified by RT-PCR, cloned, and sequenced. The resultant nucleotide sequences were compared with the published sequences for non-Korean IBV strains. Korean IBV isolates formed two independent subclusters within the phylogenetic tree based on S2 glycoprotein gene sequences. However, four and two different clusters were formed in the phylogenetic tree based on S1 glycoprotein and M gene sequences, respectively. In particular, Korean IBV K446-01 and K203-02 strains appeared to be the result of recombination between an indigenous Korean IBV strains and a vaccine strain (Massachusetts serotype) currently used in Korea. The recent IBV isolate, K026-10, formed a new subgroup that was closely related to traditional Korean IBV group in a phylogenetic tree based on the S1 and S2 genes, but it was grouped into the traditional Korean IBV cluster in a phylogenetic tree based on the M and N genes. Our data show that field IBVs in Korea are continuing to evolve and that vaccine strains might actually play a critical role in the appearance of new IBV strains via recombination in the field.
Subject(s)
Coronavirus Infections/veterinary , Infectious bronchitis virus/classification , Infectious bronchitis virus/genetics , Phylogeny , Poultry Diseases/virology , Recombination, Genetic , Animals , Base Sequence , Chickens , Coronavirus Infections/virology , Glycoproteins/genetics , Infectious bronchitis virus/isolation & purification , Molecular Sequence Data , Nucleocapsid Proteins/genetics , Republic of Korea , Sequence Analysis, DNA , Viral Envelope Proteins/geneticsABSTRACT
Thirteen isolates of avian paramyxovirus type 4 (APMV-4) isolated from wild ducks in Korea from 2006 to 2011 were genetically characterized by sequence analysis of the N-terminal region of the APMV-4 fusion (F) protein gene. The results revealed that the amino acid sequence homology within Korean isolates was 97.5 % or greater. The homologies of the Korean isolates with the APMV-4/duck/HK/D3/75 and APMV-4/duck/BE/15129/07 strains were 86.9-88.0 and 95.5-96.1 %, respectively. All Korean isolates had sequence motifs of (116)DIQPR↓F(121) at the F0 cleavage site. Phylogenetic analysis based on the N-terminal region of the F protein gene of APMV-4 isolates revealed that all 2006-2011 Korean isolates formed a single genotypic cluster that was phylogenetically different from APMV-4/duck/HK/D3/75 or APMV-4/duck/BE/15129/07 strains. Korean APMV-4 isolates were more closely related to APMV-4/goose/ZA/N1468/10 (isolated in South Africa) than to the Belgium APMV-4 virus. Korean APMV-4 isolates were further divided into at least two subgroups (A and B) based on phylogenetic analysis. Subgroup A viruses were isolated throughout Korea, whereas subgroup B viruses were detected only in isolates from Cheju island in 2011, suggesting that Korean APMV-4 exhibits marked genetic diversity and differs from viruses currently circulating in Europe and other locations.
Subject(s)
Animals, Wild/virology , Avulavirus Infections/veterinary , Avulavirus/genetics , Avulavirus/isolation & purification , Bird Diseases/virology , Ducks/virology , Genetic Variation , Amino Acid Sequence , Animals , Avulavirus/classification , Avulavirus Infections/virology , Molecular Sequence Data , Phylogeny , Republic of Korea , Sequence Homology, Amino Acid , Viral Fusion Proteins/geneticsABSTRACT
Fowl adenovirus 4 (FAdV-4) is a major avian virus that induces fatal diseases in chicken such as, hydropericardium and hepatitis. The viral structure consists of hexon, penton, fiber-1, and fiber-2 which are associated with immunopathogenesis. In this study, we investigated the genetic modification of a FAdV-4 strain after continuous passages in a cell line and evaluated the pathogenicity associated with mutations. We used the FadV-4 KNU14061 strain, which was isolated from layers in 2014. The virus went through 80 passages in the Leghorn male hepatoma (LMH) cell line. The full genetic sequence was identified, and we found a frameshift in the fiber-2 amino acid sequence after the initial thirty passages. To examine whether the frameshift in the fiber-2 gene affects the pathogenicity in chicken, we inoculated LMH80 (80 times passaged) and LMH10 (10 times passaged) into 3-day-old chickens and examined the pathogenesis. LMH10 infection via intramuscular route induced fatal pathology, but LMH80 did not. Furthermore, LHM80 pre-treatment protected hosts from the LMH10 challenge. Thus, the genetic modification isolated by serial passage lowered pathogenicity and the resulting virus acted as an attenuated vaccine that can be a FAdV-4 vaccine strain candidate.
ABSTRACT
Short chain fatty acids (SCFAs) are major gut metabolites that are involved in the regulation of dysfunction in immune responses, such as autoimmunity and cytokine storm. Numerous studies have reported a protective action of SCFAs against infectious diseases. This study investigated whether SCFAs have protective effect for immunity during fowl adenovirus-4 (FAdV-4) infection. We examined whether SCFA mixture (acetate, propionate, and butyrate) administration could protect against intramuscular challenge of a virulent viral strain. SCFA treatment promoted MHCII-expressing monocytes, the active form of T cells, and effector molecules in both peripheral and lymphoid tissues. It also boosted the production of immune molecules involved in pathogen elimination by intraepithelial lymphocytes and changed the intestinal microbial composition. We suggest that gut metabolites influence the gut microbial environment, and these changes stimulate macrophages and T cells to fight against the intramuscular challenge of FAdV-4.
Subject(s)
Butyrates , Fatty Acids, Volatile , Fatty Acids, Volatile/metabolism , Propionates , Macrophages/metabolism , Adenoviridae/metabolismABSTRACT
Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57 % of chicken flocks and 28 % of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97 % nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.
Subject(s)
Hepatitis, Viral, Animal/epidemiology , Hepevirus/classification , Hepevirus/isolation & purification , Poultry Diseases/epidemiology , RNA Virus Infections/veterinary , Animals , Chickens , Cluster Analysis , Enzyme-Linked Immunosorbent Assay , Genome, Viral , Hepatitis, Viral, Animal/virology , Hepevirus/genetics , Hepevirus/immunology , Molecular Sequence Data , Phylogeny , Poultry Diseases/virology , RNA Virus Infections/epidemiology , RNA Virus Infections/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Republic of Korea/epidemiology , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Seroepidemiologic StudiesABSTRACT
A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either 112GKQGRL117 (n = 8) or 112GRQGRL117 (n = 2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n = 8) and II (n = 2). Genotype I viruses were most closely related to the NDV V4 strain (n = 7) or the NDV Ulster 2C strain (n = 1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.
Subject(s)
Chickens , Ducks , Newcastle Disease/epidemiology , Newcastle disease virus/genetics , RNA, Viral/genetics , Animals , Molecular Sequence Data , Newcastle Disease/virology , Newcastle disease virus/classification , Newcastle disease virus/isolation & purification , Phylogeny , Republic of Korea/epidemiology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus-KNU2019-48 (A/Mallard (Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))-was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59-69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID 50/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.
Subject(s)
Influenza A virus , Influenza in Birds , Animals , Ducks , Neuraminidase/genetics , Phylogeny , Republic of KoreaABSTRACT
In-solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on surface proteins of myxovirus particles, not only the hemagglutinin and neuraminidase of influenza virus but also the hemagglutinin-neuraminidase of Newcastle disease virus (NDV). Imbalances in viral protein composition cause ion suppression of tryptic fragments from low-abundant target proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic peptides from several proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply detergent-based (gel-free) partitioning of whole viruses into soluble surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic peptides from separated viral proteins. In this study, we achieved type/subtype of avian influenza virus (AIV) within 5 h, based on 4 major proteins, by significantly reducing ion suppression and signal overlap from various protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.
Subject(s)
Influenza A virus/isolation & purification , Influenza in Birds/virology , Newcastle disease virus/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acid Sequence , Animals , Birds , Influenza A virus/chemistry , Molecular Sequence Data , Reproducibility of ResultsABSTRACT
An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza virus (AIV), occurred in a chicken farm and caused severe economic losses due to mortality and diarrhea. AIV was isolated and identified in a sample from an affected native Korean chicken. Genetic analysis of the isolate revealed a high sequence similarity to genes of novel reassortant H9N2 viruses isolated from slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge studies demonstrated that the replication kinetics and pathogenicity of the isolate were considerably altered due to adaptation in chickens. Vaccine protection studies indicated that commercial vaccine was not able to prevent virus shedding and clinical disease when chickens were challenged with the isolate. These results suggest that the novel H9N2 virus possesses the capacity to replicate efficiently in the respiratory system against vaccination and to cause severe disease in domestic chickens. The results also highlight the importance of appropriate updating of vaccine strains, based on continuous surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.
Subject(s)
Chickens , Influenza A Virus, H9N2 Subtype/isolation & purification , Influenza in Birds/virology , Animals , Disease Outbreaks/veterinary , Influenza A Virus, H9N2 Subtype/genetics , Influenza Vaccines/immunology , Influenza in Birds/epidemiology , Influenza in Birds/prevention & control , Republic of Korea/epidemiologyABSTRACT
The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.
Subject(s)
Influenza A Virus, H5N2 Subtype/classification , Influenza A Virus, H5N2 Subtype/genetics , Reassortant Viruses/classification , Reassortant Viruses/genetics , Animals , Animals, Wild/virology , Birds/virology , Disease Models, Animal , Dogs , Ducks/virology , Feces/virology , Geese/virology , Influenza A Virus, H5N2 Subtype/isolation & purification , Influenza A Virus, H5N2 Subtype/pathogenicity , Influenza A virus/genetics , Influenza in Birds/epidemiology , Influenza in Birds/virology , Japan , Madin Darby Canine Kidney Cells , Mammals , Mice , Molecular Epidemiology , Phylogeny , Poultry/virology , Reassortant Viruses/isolation & purification , Reassortant Viruses/pathogenicity , Republic of Korea/epidemiology , Virulence , Virus ReplicationABSTRACT
Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018-2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins-the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs.
Subject(s)
Evolution, Molecular , Influenza A virus/genetics , Influenza in Birds/virology , Viral Zoonoses/virology , Animals , Animals, Wild/virology , Antigens, Viral/genetics , Birds/virology , Genome, Viral/genetics , Influenza A virus/isolation & purification , Influenza A virus/pathogenicity , Influenza in Birds/epidemiology , Influenza in Birds/transmission , Mutation , Phylogeny , RNA, Viral/genetics , Reassortant Viruses/genetics , Reassortant Viruses/isolation & purification , Reassortant Viruses/pathogenicity , Republic of Korea/epidemiology , Viral Zoonoses/epidemiology , Viral Zoonoses/transmission , Virulence/geneticsABSTRACT
Parabens are synthetic chemicals widely used as preservatives in cosmetics, pharmaceuticals, and foods. Although parabens, i.e., ethyl- and methyl-parabens, are considered relatively safe, study of possible health hazards has been undertaken due to the frequent exposure to parabens and their accumulation in the body. In this study, we elucidated the effect of parabens on inflammasome induction of inflammatory responses in innate immunity, such as interleukin (IL)-1ß maturation and gasdermin D (GSDMD)-mediating pyroptosis. Parabens attenuated the inflammatory responses to intracellular lipopolysaccharide (LPS) triggering of non-canonical (NC) inflammasome activation, but did not alter canonical inflammasome (i.e., NLRP3, NLRC4 and AIM2) responses. The NC inflammasome is assembled by the interaction of murine caspase (Casp)-11 (Casp4/5 in human) with cytosolic LPS, inducing endotoxin sepsis. Parabens selectively inhibited NC inflammasome activation in both human and murine macrophages and diminished the peritoneal IL-1ß production in LPS-injected mice. Parabens blocked the cleavage of GSDMD, Casp1, and Casp4, but did not change the expression of Casp11 or the activity of Casp1. Taken together, the results indicate that parabens could disrupt Gram-negative pathogen infection through the inhibition of NC inflammasome activation.