High Pathogenicity Research Articles

Dopamine, an exogenous quorum sensing signaling molecule or a modulating factor in Pseudomonas aeruginosa?

Pseudomonas aeruginosa is recognized globally as an opportunistic pathogen of considerable concern due to its high virulence and pathogenicity, especially in immunocompromised individuals. While research has identified several endogenous quorum sensing (QS) signaling molecules that enhance the virulence and pathogenicity of P. aeruginosa, investigations on exogenous QS signaling molecules or modulating factors remain limited. This study found that dopamine serves as an exogenous QS signaling molecule or modulating factor of P. aeruginosa PAO1, enhancing the production of virulence factors and biofilms. Compared to the control group, treatment with 40 μM dopamine resulted in a 33.1 % increase in biofilm formation, 68.1 % increase in swimming mobility, 63.1 % increase in swarming mobility, 147.2 % increase in the signaling molecule 3-oxo-C12-HSL, and 50.5 %, 28.5 %, 27.0 %, and 33.2 % increases in the virulence factors alginate, rhamnolipids, protease, and pyocyanin, respectively. This study further explored the mechanism of dopamine regulating the biofilm formation and virulence of P. aeruginosa PAO1 through transcriptome and metabolome. Transcriptomic analysis showed that dopamine promoted the expression of virulence genes psl, alg, lasA, rhlABC, rml, and phz in P. aeruginosa PAO1. Metabolomic analysis revealed changes in the concentrations of tryptophan, pyruvate, ethanolamine, glycine, 3-hydroxybutyric acid, and alizarin. Furthermore, KEGG enrichment analysis of altered genes and metabolites indicated that dopamine enhanced phenylalanine, tyrosine, and tryptophan in P. aeruginosa PAO1. The results of this study will contribute to the development of novel exogenous QS signaling molecules or modulating factors and advance our understanding of the interactions between P. aeruginosa and the host environment.

Comparative genomics analysis to explore the biodiversity and mining novel target genes of Listeria monocytogenes strains from different regions.

As a common foodborne pathogen, infection with L. monocytogenes poses a significant threat to human life and health. The objective of this study was to employ comparative genomics to unveil the biodiversity and evolutionary characteristics of L. monocytogenes strains from different regions, screening for potential target genes and mining novel target genes, thus providing significant reference value for the specific molecular detection and therapeutic targets of L. monocytogenes strains. Pan-genomic analysis revealed that L. monocytogenes from different regions have open genomes, providing a solid genetic basis for adaptation to different environments. These strains contain numerous virulence genes that contribute to their high pathogenicity. They also exhibit relatively high resistance to phosphonic acid, glycopeptide, lincosamide, and peptide antibiotics. The results of mobile genetic elements indicate that, despite being located in different geographical locations, there is a certain degree of similarity in bacterial genome evolution and adaptation to specific environmental pressures. The potential target genes identified through pan-genomics are primarily associated with the fundamental life activities and infection invasion of L. monocytogenes, including known targets such as inlB, which can be utilized for molecular detection and therapeutic purposes. After screening a large number of potential target genes, we further screened them using hub gene selection methods to mining novel target genes. The present study employed eight different hub gene screening methods, ultimately identifying ten highly connected hub genes (bglF_1, davD, menE_1, tilS, dapX, iolC, gshAB, cysG, trpA, and hisC), which play crucial roles in the pathogenesis of L. monocytogenes. The results of pan-genomic analysis showed that L. monocytogenes from different regions exhibit high similarity in bacterial genome evolution. The PCR results demonstrated the excellent specificity of the bglF_1 and davD genes for L. monocytogenes. Therefore, the bglF_1 and davD genes hold promise as specific molecular detection and therapeutic targets for L. monocytogenes strains from different regions.

Phenotypic and Molecular Characterization of Vancomycin and Linezolid Resistance in Clinical Isolates of Staphylococcus aureus

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections and poses a significant global challenge due to its high pathogenicity and rapid spread among patients. Vancomycin and linezolid are key antibiotics for treating staphylococcal MRSA infections. Objectives: This study aimed to investigate resistance to vancomycin and linezolid in clinical isolates of S. aureus. Methods: In this study, 270 S. aureus isolates were collected from patients admitted to hospitals in Tehran and Qazvin. Strain identification was performed using biochemical methods and femA gene amplification. Resistance to methicillin and vancomycin was determined using disk diffusion, minimum inhibitory concentration (MIC) determination, and E-test methods. Methicillin resistance was also assessed by detecting the presence of the mecA and mecC genes. Resistance to vancomycin and linezolid was evaluated by examining the presence of vanA, B, C, and cfr genes using PCR and sequencing. Results: Out of 270 S. aureus isolates, 152 (56.3%) were identified as MRSA, with 144 (94.7%) of these MRSA isolates containing the mecA gene. Two (0.7%) VRSA isolates were observed, and the vanA gene was detected in both. Additionally, four VISA (1.4%) isolates were identified, but the vanA gene was not detected in any of the VISA strains. None of the isolates contained the mecC, vanB, or vanC genes. Among the 10 (3.7%) linezolid-resistant isolates, none contained the cfr gene. Conclusions: The results of this study demonstrated a high prevalence of MRSA isolates, which can lead to treatment failure with beta-lactam antibiotics. The emergence and spread of VRSA and VISA isolates pose a serious concern for the healthcare system, particularly in the treatment of MRSA infections. Therefore, the findings emphasize the need for appropriate monitoring and control measures to prevent the emergence and transmission of MRSA and VRSA strains. Vancomycin and linezolid remain suitable drugs for treating these infections.

Detection and Characterization of Virulent Pseudomonas aeruginosa genes in Fresh Fish at Al-Diwaniya City Market, Iraq.

Fish meat is more susceptible to pathogen contamination than other protein-rich foodstuffs due to its high deterioration. Pseudomonas aeruginosa is considered one of the most common microbial strains responsible for the deterioration of fish and seafood products. the study's goals are to detecting virulence variables Pseudomonas aeruginosa and estimating the level of contamination in a fish sample, In this research, 120 fresh fish samples, gathered from a the local Diwaniyah city market local fish meat sample. including (60 marine fish and 60 common carp (Cyprinus carpio). The isolates were subjected to numerous biochemical, microbiological, and molecular analyses, and the results showed that about 26 samples were contaminated with Pseudomonas spp. performed on 21.6% of the total sample. Polymerase chain reaction (PCR) was used to amplify 539 bp, 511 bp, and 495 bp, for 16S DNA, Tox A, and Tox S respectively. A total of 21 Tox A genes were identified in 26 P. aeruginosa isolates, whereas the ToxS gene was detected in 17 of the same isolates. According to the results Pseudomonas aeruginosa isolated from fish samples contains multiple virulence factor genes, indicating high pathogenicity, which may affect consumer health because these variants exhibit the capability to increase the pathogenesis of the microorganisms with the severity of the infection and cause economic losses

A Simulation Study to Reveal the Epidemiology and Aerosol Transmission Characteristics of Botrytis cinerea in Grape Greenhouses.

Most previously studies had considered that plant fungal disease spread widely and quickly by airborne fungi spore. However, little is known about the release dynamics, aerodynamic diameter, and pathogenicity threshold of fungi spore in air of the greenhouse environment. Grape gray mold is caused by Botrytis cinerea; the disease spreads in greenhouses by spores in the air and the spore attaches to the leaf and infects plant through the orifice. In this study, 120 μmol/L propidium monoazide (PMA) were suitable for treatment and quantitation viable spore by quantitative real-time PCR, with a limit detection of 8 spores/mL in spore suspension. In total, 93 strains of B. cinerea with high pathogenicity were isolated and identified from the air samples of grapevines greenhouses by a portable sampler. The particle size of B. cinerea aerosol ranged predominately from 0.65-3.3 μm, accounting for 71.77% of the total amount. The B. cinerea spore aerosols were infective to healthy grape plants, with the lowest concentration that could cause disease being 42 spores/m3. Botrytis cinerea spores collected form six greenhouse in Shandong Province were quantified by PMA-qPCR, with a higher concentration (1182.89 spores/m3) in May and June and a lower concentration in July and August (6.30 spores/m3). This study suggested that spore dispersal in aerosol is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases.

CFTR mutations and phenotypic correlations in people with cystic fibrosis: a retrospective study from a single centre in south India

Emerging data reveal higher-than-expected prevalence of cystic fibrosis (CF) among non-European populations worldwide including in the Indian subcontinent. Systematic analyses of the CFTR mutation profile, and genotype-phenotype correlations among people with CF from south, east, or northeast India have not been reported before. We wanted to identify CFTR mutations in people with CF, and highlight novel variants, selective phenotypic correlations, and regional variances within India. A retrospective study was conducted at Christian Medical College, Vellore, India (single tertiary referral hospital) from September 2010 to August 2022, involving 120 people with CF from (i) four south Indian states (Tamil Nadu, Andhra Pradesh, Kerala, Karnataka), (ii) in and nearby regions of West Bengal, India and (iii) Bangladesh. Comprehensive CFTR mutation analyses were done by Next-Generation Sequencing, and variants were categorized per American College of Medical Genetics guidelines and compared with validated Locus-specific databases. Demographic characteristics, mutation profile, novel mutations, selective phenotype correlations, and regional variances were assessed. In 120 people with CF, 55 CFTR variants were identified, including six novel variants. F508del was the predominant mutation, yet with a lower allele frequency than reported among European populations (27% versus 70%). Phenotypic correlations suggested high mutational pathogenicity causing severe multi-organ morbidity, and death in 27%. Milder variants associated with pancreatic sufficiency were also evident in 23% of people with CF. Statistically significant regional variances were noted in genotype frequency, and clinical phenotype among people with CF from the two regions. Hotspot exons and introns that could potentially help create targeted mutation panels were identified. The identification of 55 different CFTR variants among 120 people with CF describes the diversity of mutations noted in India, while also revealing the challenges that providers may encounter in timely diagnosis and treatment of CF. However, these single-centre data have specific limitations and cannot be generalised to all people with CF from India or to those of non-European origin. Our data on regional CFTR mutations contribute to the emerging national registry on CF epidemiology in India, help formulate diagnostic and newborn screening algorithms, help optimise clinical care, and highlight urgency to improve access to life-changing modulator therapy. Cystic Fibrosis Foundation, USA (towards the CF-India Demonstration Project) and Christian Medical College, Vellore, India.

Comparative resistomics analysis of multidrug-resistant Chryseobacteria.

Chryseobacteria consists of important human pathogens that can cause a myriad of nosocomial infections. We isolated four multidrug-resistant Chryseobacterium bacteria from activated sludge collected at domestic wastewater treatment facilities in the New York Metropolitan area. Their genomes were sequenced with Nanopore technology and used for a comprehensive resistomics comparison with 211 Chryseobacterium genomes available in the public databases. A majority of Chryseobacteria harbor 3 or more antibiotic resistance genes (ARGs) with the potential to confer resistance to at least two types of commonly prescribed antimicrobials. The most abundant ARGs, including β-lactam class A (blaCGA-1 and blaCIA) and class B (blaCGB-1 and blaIND) and aminoglycoside (ranA and ranB), are considered potentially intrinsic in Chryseobacteria. Notably, we reported a new resistance cluster consisting of a chloramphenicol acetyltransferase gene catB11, a tetracycline resistance gene tetX, and two mobile genetic elements (MGEs), IS91 family transposase and XerD recombinase. Both catB11 and tetX are statistically enriched in clinical isolates as compared to those with environmental origins. In addition, two other ARGs encoding aminoglycoside adenylyltransferase (aadS) and the small multidrug resistance pump (abeS), respectively, are found co-located with MGEs encoding recombinases (e.g., RecA and XerD) or transposases, suggesting their high transmissibility among Chryseobacteria and across the Bacteroidota phylum, particularly those with high pathogenicity. High resistance to different classes of β-lactam, as well as other commonly used antimicrobials (i.e., kanamycin, gentamicin, and chloramphenicol), was confirmed and assessed using our isolates to determine their minimum inhibitory concentrations. Collectively, though the majority of ARGs in Chryseobacteria are intrinsic, the discovery of a new resistance cluster and the co-existence of several ARGs and MGEs corroborate interspecies and intergenera transfer, which may accelerate their dissemination in clinical environments and complicate efforts to combat bacterial infections.

FOUR-WEEK ORAL ADMINISTRATION OF BALOXAVIR MARBOXIL AS AN ANTI-INFLUENZA VIRUS DRUG SHOWS NO TOXICITY IN CHICKENS.

High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens (Gallus domesticus, order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens (n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.

Assessment of Survival Kinetics for Emergent Highly Pathogenic Clade 2.3.4.4 H5Nx Avian Influenza Viruses.

High pathogenicity avian influenza viruses (HPAIVs) cause high morbidity and mortality in poultry species. HPAIV prevalence means high numbers of infected wild birds could lead to spill over events for farmed poultry. How these pathogens survive in the environment is important for disease maintenance and potential dissemination. We evaluated the temperature-associated survival kinetics for five clade 2.3.4.4 H5Nx HPAIVs (UK field strains between 2014 and 2021) incubated at up to three temperatures for up to ten weeks. The selected temperatures represented northern European winter (4 °C) and summer (20 °C); and a southern European summer temperature (30 °C). For each clade 2.3.4.4 HPAIV, the time in days to reduce the viral infectivity by 90% at temperature T was established (DT), showing that a lower incubation temperature prolonged virus survival (stability), where DT ranged from days to weeks. The fastest loss of viral infectivity was observed at 30 °C. Extrapolation of the graphical DT plots to the x-axis intercept provided the corresponding time to extinction for viral decay. Statistical tests of the difference between the DT values and extinction times of each clade 2.3.4.4 strain at each temperature indicated that the majority displayed different survival kinetics from the other strains at 4 °C and 20 °C.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Analysis of the parasitological situation regarding eimeriosis in cattle in the Russian Federation

The purpose of the study is to review the spread of bovine eimeriosis in the territory of the Russian Federation. Objectives of the study: to analyze the epizootic situation of bovine eimeriosis in the territory of the Russian Federation. Bovine coccidiosis is widespread in many regions of Russia and in other countries of the world. The pathogens are more than 10 species of eimeria, which have a specific localization in the body of animals, as well as morphological differences from each other. Three species (E. zuernii, E. bovis and E. alabamensis) are characterized by the highest pathogenicity and are most often associated with the clinical manifestation of the disease. Mostly young animals aged from 2 weeks to 1 year are ill, and adult animals have an asymptomatic form of invasion. In many animals, eimeria causes dangerous diseases, accompanied by a decrease in productivity, the birth of weak offspring, often the death of young animals, and additional feed costs per unit of production. In livestock complexes, the incidence of calves with eimeriosis is 10–100%, and mortality is 25–60%. The maximum infection of calves and adult animals is observed in the spring and autumn periods, and in some regions in the autumn-winter period. It was found that with an increase in the density of calves in the machine, their infection with eimeria increases significantly. The invasion of calves was minimal when kept in individual machines. Given that parasites have a complex developmental biology, one of the most importantmeasures in the complex of measures ensuring the prevention and elimination of animal diseases with coccidiosis is currently still pharmacotherapy and prevention, contributing not only to the liberation of animals from parasites, but also to prevent the dispersion of the invasive origin in the environment and to prevent the threat of new infection.

Pathogenicity and transmission of novel highly pathogenic H7N2 variants originating from H7N9 avian influenza viruses in chickens

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses.

The Milk of Cows Immunized with Trivalent Inactivated Vaccines Provides Broad-Spectrum Passive Protection against Hand, Foot, and Mouth Disease in Neonatal Mice.

Hand, foot, and mouth disease (HFMD) is a contagious viral infection predominantly affecting infants and young children, caused by multiple enteroviruses, including Enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A10 (CA10), and Coxsackievirus A6 (CA6). The high pathogenicity of HFMD has garnered significant attention. Currently, there is no specific treatment or broad-spectrum preventive measure available for HFMD, and existing monovalent vaccines have limited impact on the overall incidence or prevalence of the disease. Consequently, with the emergence of new viral strains driven by vaccine pressure, there is an urgent need to develop strategies for the rapid response and control of new outbreaks. In this study, we demonstrated the broad protective effect of maternal antibodies against three types of HFMD by immunizing mother mice with a trivalent inactivated vaccine targeting EV71, CA16, and CA10, using a neonatal mouse challenge model. Based on the feasibility of maternal antibodies as a form of passive immunization to prevent HFMD, we prepared a multivalent antiviral milk by immunizing dairy cows with the trivalent inactivated vaccine to target multiple HFMD viruses. In the neonatal mouse challenge model, this immunized milk exhibited extensive passive protection against oral infections caused by the three HFMD viruses. Compared to vaccines, this strategy may offer a rapid and broadly applicable approach to providing passive immunity for the prevention of HFMD, particularly in response to the swift emergence and spread of new variants.

Unraveling factors responsible for pathogenic differences in Lassa virus strains.

Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a severe hemorrhagic disease with potential for lethal outcomes. Apart from acute symptoms, LF survivors often endure long-term complications, notably hearing loss, which significantly impacts their quality of life and socioeconomic status in endemic regions of West Africa. Classified as a Risk Group 4 agent, LASV poses a substantial public health threat in affected areas. Our laboratory previously developed a novel lethal guinea pig model of LF utilizing the clinical isolate LASV strain LF2384. However, the specific pathogenic factors underlying LF2384 infection in guinea pigs remained elusive. In this study, we aimed to elucidate the differences in the immunological response induced by LF2384 and LF2350, another LASV isolate from a non-lethal LF case within the same outbreak. Through comprehensive immunological gene profiling, we compared the expression kinetics of key genes in guinea pigs infected with LASV LF2384 and LF2350. Our analysis revealed differential expression patterns for several immunological genes, including CD94, CD19-2, CD23, IL-7, and CIITA, during LF2384 and LF2350 infection. Moreover, through the generation of recombinant LASVs, we sought to identify the specific viral genes responsible for the observed pathogenic differences between LF2384 and LF2350. Our investigations pinpointed the L protein as a crucial determinant of pathogenicity in guinea pigs infected with LASV LF2384.

A Luciferase Immunosorbent Assay Based on Attachment Glycoprotein for the Rapid and Easy Detection of Nipah Virus IgG Antibodies.

Nipah virus (NiV) is a virulent zoonotic disease whose natural host is the fruit bat (Pteropus medius), which can coexist with and transmit the virus. Due to its high pathogenicity, wide host range, and pandemic potential, establishing a sensitive, specific, and rapid diagnostic method for NiV is key to preventing and controlling its spread and any outbreaks. Here, we established a luciferase immunosorbent assay (LISA) based on the NiV attachment glycoprotein (G) to detect NiV-specific immunoglobulin G by expressing a fusion protein of nanoluciferase (NanoLuc) and the target antigen. Sensitivity analysis was performed and compared to an indirect enzyme-linked immunosorbent assay (ELISA), and specificity and cross-reactivity assessments were performed using NiV-positive horse serum and Ebola virus-, Crimean-Congo hemorrhagic fever virus-, and West Nile virus-positive horse sera. The optimal structural domain for NiV detection was located within amino acids 176-602 of the NiV G protein head domain. Moreover, the LISA showed at least fourfold more sensitivity than the indirect ELISA, and the cross-reactivity results suggested that the LISA had good specificity and was capable of detecting NiV-specific immunoglobulin G in both mouse and horse serum. In conclusion, the establishment of a rapid, simple NiV LISA using the G protein head domain provides a resource for NiV monitoring.

Blowflies are potential vector for avian influenza virus at enzootic area in Japan

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

Novel Genotype of HA Clade 2.3.4.4b H5N8 Subtype High Pathogenicity Avian Influenza Virus Emerged at a Wintering Site of Migratory Birds in Japan, 2021/22 Winter.

Surveillance of avian influenza virus (AIV) was conducted in the 2021-2022 winter season at a wintering site of migratory Anatidae in Japan. An H5N8 subtype high pathogenicity AIV (HPAIV) with a unique gene constellation and four low pathogenicity AIVs (LPAIVs) were isolated from environmental samples. The genetic origin of the HPAIV (NK1201) was determined with whole-genome sequencing and phylogenetic analyses. Six of NK1201's eight genes were closely related to HA clade 2.3.4.4b H5N8 subtype HPAIVs, belonging to the G2a group, which was responsible for outbreaks in poultry farms in November 2021 in Japan. However, the remaining two genes, PB1 and NP, most closely matched those of the LPAIVs H7N7 and H1N8, which were isolated at the same place in the same 2021-2022 winter. No virus of the NK1201 genotype had been detected prior to the 2021-2022 winter, indicating that it emerged via genetic reassortment among HPAIV and LPAIVs, which were prevalent at the same wintering site. In addition, experimental infection in chickens indicated that NK1201 had slightly different infectivity compared to the reported infectivity of the representative G2a group H5N8 HPAIV, suggesting that the PB1 and NP genes derived from LPAIVs might have affected the pathogenicity of the virus in chickens. Our results directly demonstrate the emergence of a novel genotype of H5N8 HPAIV through gene reassortment at a wintering site. Analyses of AIVs at wintering sites can help to identify the emergence of novel HPAIVs, which pose risks to poultry, livestock, and humans.

Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

A multi-species, multi-pathogen avian viral disease outbreak event: Investigating potential for virus transmission at the wild bird – poultry interface

ABSTRACT A free-range organic broiler (Gallus gallus domesticus) premises in Staffordshire was infected by high pathogenicity avian influenza virus (HPAIV) H5N8 during the 2020–2021 epizootic in the United Kingdom (UK). Following initial confirmation of the infection in poultry, multiple wild bird species were seen scavenging on chicken carcasses. Detected dead wild birds were subsequently demonstrated to have been infected and succumbed to HPAIV H5N8. Initially, scavenging species, magpie (Pica pica) and raven (Corvus corax) were found dead on the premises but over the following days, buzzards (Buteo buteo) were also found dead within the local area with positive detection of HPAIV in submitted carcasses. The subacute nature of microscopic lesions within a buzzard was consistent with the timeframe of infection. Finally, a considerable number of free-living pheasants (Phasianus colchicus) were also found dead in the surrounding area, with carcasses having higher viral antigen loads compared to infected chickens. Limited virus dissemination was observed in the carcasses of the magpie, raven, and buzzard. Further, an avirulent avian paramyxovirus type 1 (APMV-1) was detected within poultry samples as well as in the viscera of a magpie infected with HPAIV. Immunohistochemistry did not reveal colocalization of avian paramyxovirus antigens with lesions, supporting an avirulent APMV-1 infection. Overall, this case highlights scenarios in which bi-directional transmission of avian viral diseases between commercial and wild bird species may occur. It also underlines the importance of bio separation and reduced access when infection pressure from HPAIV is high.

EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.