Strategic combination of bacteriophages with highly susceptible cells for enhanced intestinal settlement and resistant cell killing.

Avian pathogenic Escherichia coli (APEC) causes enormous economic losses and is a primary contributor to the emergence of multidrug resistance (MDR)-related problems in the poultry industry. Bacteriophage (phage) therapy has been successful in controlling MDR, but phage-resistant variants have rapidly emerged through the horizontal transmission of diverse phage defense systems carried on mobile genetic elements. Consequently, while multiple phage cocktails are recommended for phage therapy, there is a growing need to explore simpler and more cost-effective phage treatment alternatives. In this study, we characterized two novel O78-specific APEC phages, φWAO78-1 and φHAO78-1, in terms of their morphology, genome, physicochemical stability and growth kinetics. Additionally, we assessed the susceptibility of thirty-two O78 APEC strains to these phages. We analyzed the roles of highly susceptible cells in intestinal settlement and fecal shedding (susceptible cell-assisted intestinal settlement and shedding, SAIS) of phages in chickens via coinoculation with phages. Furthermore, we evaluated a new strategy, susceptible cell-assisted resistant cell killing (SARK), by comparing phage susceptibility between resistant cells alone and a mixture of resistant and highly susceptible cells in vitro. As expected, high proportions of O78 APEC strains had already acquired multiple phage defense systems, exhibiting considerable resistance to φWAO78-1 and φHAO78-1. Coinoculation of highly susceptible cells with phages prolonged phage shedding in feces, and the coexistence of susceptible cells markedly increased the phage susceptibility of resistant cells. Therefore, the SAIS and SARK strategies were demonstrated to be promising both in vivo and in vitro.

Rapid Antibacterial Activity Assessment of Chimeric Lysins.

Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

Taxonomy of the order Mononegavirales: update 2018.

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Comparative genomics of QX-like infectious bronchitis viruses in Korea.

To minimize the spread of infectious bronchitis virus (IBV), domestic fowl have been extensively vaccinated with the KM91 strain. However, various IBV QX-like virus strains have become increasingly prevalent in Korea. We conducted comparative genomic analyses of seven QX-like viruses: early viruses (n = 2), new cluster 1 (NC1; recombinants of KM91 and the early QX-like viruses, n = 3) and recurrent viruses (n = 2), to understand their genomic backgrounds. The early and NC1 viruses had KM91-like backgrounds, but the recurrent viruses had QX-like genomic backgrounds. The absence of pure QX-like viruses before the appearance of the early viruses suggests that the viruses were introduced from other countries after recombination, but the NC1 viruses originated in Korea. The recent prevalence of recurrent viruses with different genomic backgrounds and spike genes from the early and the NC1 viruses may indicate the repeated introduction of different infectious bronchitis viruses from other countries and their successful evasion of vaccine immunity in the field. Furthermore, a 1ab gene-based phylogenetic analysis revealed three distinct lineages: North America-Europe, China/Taiwan, and China. KM91 and the early and NC1 viruses were included in the North America-Europe lineage, and the recurrent QX-like viruses were included in the China lineage. The phylogenetic positions of KM91-like 1ab and QX-like spike suggest frequent recombination between the North America-Europe and China lineages. Additional studies on the patterns of recombination, including donor-acceptor relationships, geographical sites, and non-poultry hosts, may be valuable for understanding the evolution of IBVs.

Efficient self-assembly and protective efficacy of infectious bursal disease virus-like particles by a recombinant baculovirus co-expressing precursor polyprotein and VP4.

BACKGROUND: Virus-like particle (VLP) technology is considered one of the most promising approaches in animal vaccines, due to the intrinsic immunogenic properties as well as high safety profile of VLPs. In this study, we developed a VLP vaccine against infectious bursal disease virus (IBDV), which causes morbidity and mortality in chickens, by expressing a baculovirus in insect cells. METHODS: To improve the self-proteolytic processing of precursor polyprotein (PP), we constructed a recombinant baculovirus transfer vector that co-expresses PP and the VP4 protease gene of IBDV. RESULTS: Expression and VLP assembly of recombinant proteins and antigenicity of the VLP were examined by Western blotting, ELISA, and transmission electron microscopy. In animal experiments, vaccination with the recombinant VLP induced strong and uniform humoral immunity and provided complete protection against challenge with very virulent (vv) IBDV in SPF chickens (n = 12). As determined by the bursa of Fabricius (BF)/body weight (B/BW) ratio, the protection against post-challenge bursal atrophy was significantly higher (P < 0.001) in VLP-vaccinated birds than in non-vaccinated controls. CONCLUSIONS: Since the protective efficacy of the VLP vaccine was comparable to that of a commercially available inactivated vaccine, the recombinant VLP merits further investigation as an alternative means of protection against vvIBD.

Genetic evolution of H5 highly pathogenic avian influenza virus in domestic poultry in Vietnam between 2011 and 2013.

In spite of highly pathogenic avian influenza H5N1 vaccination campaigns for domestic poultry, H5N1 viruses continue to circulate in Vietnam. To estimate the prevalence of avian influenza virus in Vietnam, surveillance was conducted between November 2011 and February 2013. Genetic analysis of 312 highly pathogenic avian influenza H5 viruses isolated from poultry in Vietnam was conducted and possible genetic relationships with strains from neighboring countries were investigated. As previously reported, phylogenetic analysis of the avian influenza virus revealed two H5N1 HPAI clades that were circulating in Vietnam. Clade 1.1, related to Cambodian strains, was predominant in the southern provinces, while clade 2.3.2.1 viruses were predominant in the northern and central provinces. Sequence analysis revealed evidence of active genetic evolution. In the gene constellation of clade 2.3.2.1, genotypes A, B, and B(II) existed during the 2011/2012 winter season. In June 2012, new genotype C emerged by reassortment between genotype A and genotype B(II), and this genotype was predominant in 2013 in the northern and central provinces. Interestingly, enzootic Vietnamese clade 2.3.2.1C H5 virus subsequently reassorted with N2, which originated from wild birds, to generate H5N2 highly pathogenic avian influenza, which was isolated from duck in the northeast region. This investigation indicated that H5N1 outbreaks persist in Vietnam and cause genetic reassortment with circulating viruses. It is necessary to strengthen active influenza surveillance to eradicate highly pathogenic avian influenza viruses and sever the link between highly pathogenic avian influenza and other circulating influenza viruses.

Molecular epidemiology of Newcastle disease viruses in Vietnam.

Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNß, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

Immune Cells Are Differentially Affected by SARS-CoV-2 Viral Loads in K18-hACE2 Mice.

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virus-infected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105 PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.

A longitudinal molecular and cellular lung atlas of lethal SARS-CoV-2 infection in K18-hACE2 transgenic mice.

BACKGROUND: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 500 million cases and 6 million deaths worldwide. Previous investigations into the pathophysiology of SARS-CoV-2 primarily focused on peripheral blood mononuclear cells from patients, lacking detailed mechanistic insights into the virus's impact on inflamed tissue. Existing animal models, such as hamster and ferret, do not faithfully replicate the severe SARS-CoV-2 infection seen in patients, underscoring the need for more relevant animal system-based research. METHODS: In this study, we employed single-cell RNA sequencing (scRNA-seq) with lung tissues from K18-hACE2 transgenic (TG) mice during SARS-CoV-2 infection. This approach allowed for a comprehensive examination of the molecular and cellular responses to the virus in lung tissue. FINDINGS: Upon SARS-CoV-2 infection, K18-hACE2 TG mice exhibited severe lung pathologies, including acute pneumonia, alveolar collapse, and immune cell infiltration. Through scRNA-seq, we identified 36 different types of cells dynamically orchestrating SARS-CoV-2-induced pathologies. Notably, SPP1+ macrophages in the myeloid compartment emerged as key drivers of severe lung inflammation and fibrosis in K18-hACE2 TG mice. Dynamic receptor-ligand interactions, involving various cell types such as immunological and bronchial cells, defined an enhanced TGFß signaling pathway linked to delayed tissue regeneration, severe lung injury, and fibrotic processes. INTERPRETATION: Our study provides a comprehensive understanding of SARS-CoV-2 pathogenesis in lung tissue, surpassing previous limitations in investigating inflamed tissues. The identified SPP1+ macrophages and the dysregulated TGFß signaling pathway offer potential targets for therapeutic intervention. Insights from this research may contribute to the development of innovative diagnostics and therapies for COVID-19. FUNDING: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020M3A9I2109027, 2021R1A2C2004501).

Genetic diversity of avian paramyxovirus type 4 isolates from wild ducks in Korea from 2006 to 2011.

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.

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia.

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.

Genetic and antigenic variation of shedding viruses from vaccinated chickens after challenge with virulent Newcastle disease virus.

Newcastle disease virus (NDV) isolation was attempted from La Sota-vaccinated or unvaccinated chickens exposed to the virulent NDV variant E347Kmt. Shedding viruses were purified by plaque assay and then were sequenced for HN and F genes. The amino acid sequences of the F gene of all shedding viruses were identical to the sequence of the challenge virus. However, amino acid substitution occurred at four positions (70, 347, 466, and 517) in the HN protein among shedding viruses from vaccinated and challenged chickens but not from unvaccinated and challenged chickens. Amino acid substitution occurred more frequently at position 347 (K to G or V) in the HN protein compared with the other positions. There was minor antigenic variation between some of mutant viruses shed and challenge virus. However, none of mutant viruses had a significantly lower antigenic R value with La Sota virus compared with challenge virus E347Kmt. Our findings indicate that vaccinal immunity might facilitate an evolutional event through antigenic selection, genetic mutation among virulent virus populations shed from vaccinated flocks, or both.

Phylogenetic analysis and genetic characterization of chicken anemia virus isolates from Cambodia.

Three chicken anemia viruses (CAV) were detected by PCR during screening of field samples from village chickens collected in Cambodia in 2011/2012. Nearly full-length VP1 viral structural protein genes (nt 1-1,293) from the 3 CAV were sequenced and characterized. Phylogenetic analysis revealed that all 3 of the Cambodian CAV were clustered with CAV strains belonging to genotype II and were most closely related to CAV strains from Guangdong province, China. On the amino acid level, major substitutions were observed at 12 residues in the VP1 protein (positions 22, 75, 97, 125, 139, 144, 254, 287, 290, 370, 376, and 413) when compared with published reference CAV strains. In motifs associated with virulence, all Cambodian CAV had virulence-associated motifs composed of 75I, 89T, 125I, 139Q, 141Q, 144Q, and 394Q, which are commonly found in highly virulent genotype II viruses and some genotype III viruses. This is the first report of CAV isolated from village chickens in Southeast Asia as well as Cambodia.

Geographical distribution of low pathogenic avian influenza viruses of domestic poultry in Vietnam and their genetic relevance with Asian isolates.

From the avian influenza virus (AIV) outbreaks and market surveillances in Vietnam during November 2011 and March 2012, a total of 196 AIV were isolated. Although H5N1 highly pathogenic avian influenza (HPAI) was the most prevalent subtype in Vietnam, 57 low pathogenic avian influenza (LPAI) viruses were identified from mainly domestic ducks and some chickens. Of note, various subtypes of LPAI viruses were isolated from domestic ducks in Vietnam: H3 (n = 16), H4 (n = 4), H6 (n = 24), H7 (n = 1), and H9 (n = 10). Geographically, the LPAI viruses were identified in different regions of Vietnam. Phylogenetic analysis of HA and NA genes in LPAIV in Vietnam showed that some H3 (group I) and H4 subtypes AIV clustered with the viruses of several Asian isolates from domestic poultry and wild birds. However, the H6, H9, and some H3 (group II and III) subtypes AIV were closely related to isolates from domestic poultry in Southern China. In addition, whereas the N2 and N6 subtypes AIV belonged to the Eurasian lineage, the N8 subtype AIV was classified to be both of Eurasian and American lineage. These findings revealed that the regional trade and wild birds play a key role transmission of LPAIV in domestic ducks in Vietnam. Further surveillance at the intercountry level is needed to understand the epidemiology of these viruses and to cope with emergence of novel AIV types.

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 co-circulated 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.

Tracing the Evolutionary Pathways of Serogroup O78 Avian Pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry industry, and O78 serogroup APEC strains are prevalent in chickens. In this study, we aimed to understand the evolutionary pathways and relationships between O78 APEC and other E. coli strains. To trace these evolutionary pathways, we classified 3101 E. coli strains into 306 subgenotypes according to the numbers and types of single nucleotide polymorphisms (RST0 to RST63-1) relative to the consensus sequence (RST0) of the RNA polymerase beta subunit gene and performed network analysis. The E. coli strains showed four apparently different evolutionary pathways (I-1, I-2, I-3, and II). The thirty-two Korean O78 APEC strains tested in this study were classified into RST4-4 (45.2%), RST3-1 (32.3%), RST21-1 (12.9%), RST4-5 (3.2%), RST5-1 (3.2%), and RST12-6 (3.2%), and all RSTs except RST21-1 (I-2) may have evolved through the same evolutionary pathway (I-1). A comparative genomic study revealed the highest relatedness between O78 strains of the same RST in terms of genome sequence coverage/identity and the spacer sequences of CRISPRs. The early-appearing RST3-1 and RST4-4 prevalence among O78 APEC strains may reflect the early settlement of O78 E. coli in chickens, after which these bacteria accumulated virulence and antibiotic resistance genes to become APEC strains. The zoonotic risk of the conventional O78 APEC strains is low at present, but the appearance of genetically distinct and multiple virulence gene-bearing RST21-1 O78 APEC strains may alert us to a need to evaluate their virulence in chickens as well as their zoonotic risk.