SARS‑CoV‑2 Omicron variant causes brain infection with lymphoid depletion in a mouse COVID‑19 model / 한국실험동물학회지

Background@#The Omicron variant has become the most prevalent SARS-CoV-2 variant. Omicron is known to induce milder lesions compared to the original Wuhan strain. Fatal infection of the Wuhan strain into the brain has been well documented in COVID-19 mouse models and human COVID-19 cases, but apparent infections into the brain by Omicron have not been reported in human adult cases or animal models. In this study, we investigated whether Omicron could spread to the brain using K18-hACE2 mice susceptible to SARS-CoV-2 infection. @*Results@#K18-hACE2 mice were intranasally infected with 1 × 105 PFU of the original Wuhan strain and the Omicron variant of SARS-CoV-2. A follow-up was conducted 7 days post infection. All Wuhan-infected mice showed > 20% body weight loss, defined as the lethal condition, whereas two out of five Omicron-infected mice (40%) lost > 20% body weight. Histopathological analysis based on H&E staining revealed inflammatory responses in the brains of these two Omicron-infected mice. Immunostaining analysis of viral nucleocapsid protein revealed severe infection of neuron cells in the brains of these two Omicron-infected mice. Lymphoid depletion and apoptosis were observed in the spleen of Omicron-infected mice with brain infection. @*Conclusion@#Lethal conditions, such as severe body weight loss and encephalopathy, can occur in Omicron-infected K18-hACE2 mice. Our study reports, for the first time, that Omicron can induce brain infection with lymphoid depletion in the mouse COVID-19 model.

Receptor binding motif surrounding sites in the Spike 1 protein of infectious bronchitis virus have high susceptibility to mutation related to selective pressure

Background@#To date, various genotypes of infectious bronchitis virus (IBV) have cocirculated and in Korea, GI-15 and GI-19 lineages were prevailing. The spike protein, particularly S1 subunit, is responsible for receptor binding, contains hypervariable regions and is also responsible for the emerging of novel variants. @*Objective@#This study aims to investigate the putative major amino acid substitutions for the variants in GI-19. @*Methods@#The S1 sequence data of IBV isolated from 1986 to 2021 in Korea (n = 188) were analyzed. Sequence alignments were carried out using Multiple alignment using Fast Fourier Transform of Geneious prime. The phylogenetic tree was generated using MEGA-11 (ver.11.0.10) and Bayesian analysis was performed by BEAST v1.10.4. Selective pressure was analyzed via online server Datamonkey. Highlights and visualization of putative critical amino acid were conducted by using PyMol software (version 2.3). @*Results@#Most (93.5%) belonged to the GI-19 lineage in Korea, and the GI-19 lineage was further divided into seven subgroups: KM91-like (Clade A and B), K40/09-like, QX-like (I-IV).Positive selection was identified at nine and six residues in S1 for KM91-like and QX-like IBVs, respectively. In addition, several positive selection sites of S1-NTD were indicated to have mutations at common locations even when new clades were generated. They were all located on the lateral surface of the quaternary structure of the S1 subunits in close proximity to the receptor-binding motif (RBM), putative RBM motif and neutralizing antigenic sites in S1. @*Conclusions@#Our results suggest RBM surrounding sites in the S1 subunit of IBV are highly susceptible to mutation by selective pressure during evolution.

Laboratory information management system for COVID-19 non-clinical efficacy trial data / 한국실험동물학회지

Background@#As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. @*Results@#In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. @*Conclusions@#This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Genetic characterization of H9N2avian influenza virus previously unrecognized in Korea

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.

Genetic characterization of H9N2avian influenza virus previously unrecognized in Korea

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.

Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.

Patterns of antibiotic resistance in Escherichia coli isolated from fresh and recycled poultry litter

The isolation rate of Escherichia (E.) coli in poultry litter was investigated at 44 broiler farms, 20 that used fresh litter and 24 that used recycled litter. The patterns of resistance to antibiotics of the E. coli isolates were compared. In litter sampled before the rearing period, the isolation rate of E. coli was higher at farms that used fresh litter; E. coli was present in the litter in 94.5% (35 out of 37 flocks tested) of the farms that used fresh litter vs. 51.2% (21 out of 41 flocks) of the farms that used recycled litter. The susceptibility of the 93 isolates of E. coli to 13 antibiotics was studied. Before the rearing period, E. coli isolates from the farms that recycled litter showed higher resistance rates than isolates from farms that replaced litter with fresh litter. Comparing the antibiotic resistance patterns of isolates from litter sampled before and at the end of the rearing period, the antibiotic resistance rates at the end of the rearing period increased dramatically compared with rates before the rearing period.

Newcastle disease virus vectored vaccines as bivalent or antigen delivery vaccines

Recent advances in reverse genetics techniques make it possible to manipulate the genome of RNA viruses such as Newcastle disease virus (NDV). Several NDV vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. The safety, immunogenicity, and protective efficacy of these NDV-vectored vaccines have been evaluated in pre-clinical and clinical studies. The vaccines are safe in mammals, humans, and poultry. Bivalent NDV-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. These bivalent vaccines confer solid protective immunity against NDV and other foreign antigens. In most cases, NDV-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. This review summarizes the development of NDV-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use.

Correlations in the results of virus neutralization test, hemagglutination inhibition test, and enzyme-linked immunosorbent assay to determine infectious bronchitis virus vaccine potency

The virus neutralization (VN) test was used to determine potency of the infectious bronchitis (IB) vaccine. The results of VN, hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA) were compared with those of the IBV M41. The r² values between VN and HI titers and the ELISA antibody titer were 0.8782 and 0.0336, respectively, indicating a high correlation between VN and HI, but not VN and ELISA. The Cohen's kappa coefficient between the VN titer of 2 log₁₀ and HI titer of 5 log₂ was 0.909. Our results showed that VN could be replaced with HI for testing the potency of IBV M41.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Recharacterization of Morphological and Genetic Feature of Getah Virus Isolated from South Korea

Three QIAG93 strains, QIAG9301, QIAG9302 and QIAG9303 that have been identified as Getah virus (GETV) are analyzed in this study. The morphological features of three virus isolates were observed by using electron microscopy, suggesting that the QIAG9301, QIAG9302 and QIAG9303 isolate can be classified as tentative member of Alphavirus species in the Semliki Forest complex. The full length of the structural polyprotein gene of each QIAG93 isolate (QIAG9301, QIAG9302 and QIAG9303) was determined that are identical in size, comprising 3759 nucleotides that encoded 1253 amino acids. The sequence analysis of the structural polyprotein gene, including the C, E3, E1, 6K and E2 domain, showed that each QIAG93 isolate shares >98.9% sequence identity. The phylogenetic analysis and evolutionary distance (ED) estimation based on the structural polyprotein gene sequence showed that the QIAG9301 isolate is closely related to GETV South Korea strain (99.9% sequence identity and ED value 0.001) and Chinese GETV YN0540 strain (99.3% sequence identity ED value 0.007) than other Alphavirus species analyzed in this study. Both QIAG9032 and QIAG9303 isolate exhibited genetically close relationship with Mongolian GETV LEIV17741MPR strain (at least 99.3% sequence identity and mean ED value 0.0065). Therefore, our findings will be valuable for molecular epidemiological analyses of GETV in Korea and contribute to a further study on pathogenicity of three QIAG93 isolates in animals.

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Characterization and comparison of the pathogenicity of viscerotropic velogenic Newcastle disease virus isolates in Korea / 대한수의학회지

A total of 18 Newcastle disease virus (NDV) isolates that were recovered from 1949 through 1997 were characterized and pathotyped. All viruses were highly virulent as determined by intracerebral pathogenicity indices > or = 1.81 in day-old. These pathotypes are typical for viscerotropic velogenic NDV (VVNDV) pathotype viruses. Some differences were observed for the chicken red blood cell elution rate and thermostability of the hemagglutinin at 56degrees C. Three antigenic groups were identified by a hemagglutination-inhibition assay using NDV monoclonal antibodies. And the predominant gross lesions were as follows: discharge from the nasal cavity, tracheal mucus, petechial hemorrhage in the heart fat, kidney urates and hemorrhage with or without necrosis in the gastrointestinal tract. Severe hemorrhagic or necrotic lesions were also noted in the lymphoid organs and were localized primarily in the spleen and cecal tonsil. However, differences in the occurrence and frequency of the gross lesions were observed between the virus strains. Among them, NDV strains that induced neurological symptoms belonged only to genotype VI. This strain had spread throughout Korea during the late 1980s to the 1990s, which suggests that specific VVNDVs genotypes might result in neurological symptoms.

Generation and Biological Characterization of a Neutralization-Resistant Mutant of Newcastle Disease Virus

A neutralization-resistant mutant of Newcastle disease virus (NDV) Kr005 strain belonging to class II genotype VII was generated using a neutralizing monoclonal antibody and its biological effects were assessed. The mutant showed single amino acid substitution (E to K) at position 347 of the hemagglutinin-neuraminidase (HN) protein (E347K mutant). The E347K mutant exhibited marked rounding of the cells and few syncytia in infected chicken embryofibroblast (CEF) cells. The hemadsorption and neuraminidase activities of the E347K mutant of the wild-type virus were 118% and 166%, respectively. The mutant produced a rapid elution pattern whereas the wild type had a slow elution pattern. Growth kinetics studies showed that the E347K mutant produced an 80-times higher yield of extracellular virus in CEF cells compared with the wild-type virus. The time-course virus titer showed a marked increase in mutant-infected cells from 6 h to 12 h post infection (pi), which was consistent with the titer pattern time-course for NA activity. The E347K mutant virus showed a slight decrease in virulence compared to the wild-type virus, but there was no change in pathotype when measured by in vivo pathogenicity testing. These results suggest that an E347K mutation in HN protein might be associated with increased NA activity and subsequent enhancement of virus release from infected cells without change in viral pathotype.

Development of an attenuated vaccine strain from a korean respiratory type infectious bronchitis virus / 대한수의학회지

An attenuated vaccine strain AVR1/08 of Korean respiratory type of infectious bronchitis virus (IBV) was developed by 89th passages of IBV D85/06 strain in chicken eggs. The AVR1/08 strain had higher virus titer at least 20 times (10(1.3)) than the parent virus D85/06 by egg inoculation method. The AVR1/08 strain had a single point mutation (S to Y) at position 56 of spike protein of IBV compared to parent virus IBV D85/06 strain. The mutation was observed consistently at viruses after 47th passage in chicken eggs. The AVR1/08 strain showed no virulence even after 6 passages in chickens and all chickens inoculated induced anti-IBV antibody 14 days after vaccination. The AVR1/08 strain had broad protective efficacy against QX type Korean nephropathogenic virus (Q43/06 strain), KM91 type Korean nephropathogenic virus (KM91 strain) and Korean respiratory virus (D85/06 strain). In contrast, Massachusetts (Mass) type attenuated vaccine strain H120 showed protection of 37.5 to 50% against these three viruses. Our results indicate that the AVR1/08 strain has potential as an attenuated vaccine effective in controlling IBVs circulating in Korea.

Development of Competitive ELISA for Detection of Avian Metapneumovirus Antibodies in Chicken

Avian metapneumovirus (aMPV) causes an acute and highly contagious upper respiratory tract infection in turkeys and chickens. In this study, a competitive ELISA (C-ELISA) was developed for the detection of antibodies to aMPV in chicken sera and/or their egg yolks. This assay is based on the competitive binding of monoclonal antibody with serum antibodies to recombinant aMPV N protein expressed by a recombinant baculovirus. The C-ELISA showed specificity and sensitivity of 100% and 98.0%, respectively, when compared to the virus neutralization test. In specific pathogen-free chickens experimentally infected with aMPV SC1509 strain, the C-ELISA started to detect antibodies to aMPV as early as 5 days post infection from birds infected with aMPV, while a commercial ELISA kit detected first 10 days post infection. The C-ELISA was similar or superior to a commercial ELISA kit when serum and egg yolk samples collected from chickens on six outbreak farms were tested for diagnosis. The C-ELISA developed in the present work provides a short turnaround time and can be a useful diagnostic and screening tool for aMPV infection in the field.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

Pathogenicity and antigenicity of a new variant of Korean nephropathogenic infectious bronchitis virus

Despite the existence of an active vaccination program, recently emerged strains of nephropathogenic infectious bronchitis virus (IBV) in Korea have caused significant economic losses in the poultry industry. In this study, we assessed the pathogenic and antigenic characteristics of a K-IIb type field strain of IBV that emerged in Korea since 2003, such as Kr/Q43/06. Specific pathogen free 1-week-old chickens exhibited severe respiratory symptoms (dyspnea) and nephropathogenic lesions (swollen kidneys with nephritis and urate deposits) following challenge with the recent IBV field strain. The antigenic relatedness (R value), based on a calculated virus neutralization index, of the K-IIb type field strain and K-IIa type strain KM91 (isolated in 1991) was 30%, which indicated that the recent strain, Kr/Q43/06, is a new variant that is antigenically distinct from strain KM91. This report is the first to document the emergence of a new antigenic variant of nephropathogenic IBV in chicken from Korea.

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 +/- 0.09 and 0.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.

Isolation and Characterization of Avian Metapneumovirus from Broiler Breeder Chickens in Korea

Avian metapneumovirus (AMPV) is an emerging pathogen causing respiratory and reproductive illness in poultry worldwide. To demonstrate the presence of AMPV in domestic chickens in Korea, we attempted to isolate AMPV from affected chickens. A cytopathic agent was isolated using chicken tracheal ring culture from dead chickens from a broiler breeder farm with reduced egg production in Korea. This agent, termed SC1509 strain, subsequently passed in Vero cells with distinct cytopathic effects. The SC1509 strain was confirmed as avian metapneumovirus (AMPV) using both RT-PCR test and monoclonal antibody-based immunofluorescence assay. Sequence analysis based on the G glycoprotein revealed that the SC1509 strain had 22.5 to 96.0% nucleotide sequence identity and 11.1 to 92.7% predicted amino acid sequence identity with previously published AMPV strains, particularly with the highest sequence homology (95.8 to 96% for nucleotides and 92.2 to 92.7% for amino acids) to European strains belonging to genotype B. The SC1509 strain was phylogenetically clustered with genotype B viruses, confirming that the SC1509 strain belongs to genotype B. This is the first report of genotype B avian metapneumovirus from chickens in Korea.