Genetic variation and recombination analysis of the GP5 gene of the porcine reproductive and respiratory syndrome virus in Thailand.

Introduction: Porcine reproductive and respiratory syndrome (PRRS) is a significant threat to the global swine industry, and its prevalence in Thailand spans over two decades. Methods: To understand the genetic variation and recombination of the PRRS virus (PRRSV) GP5 gene in Thailand, we retrieved 726 GP5 gene sequences from the NCBI database. Phylogenetic trees were constructed using the neighbor-joining (NJ) and maximum likelihood (ML) methods, and recombination analysis was performed. Results: Homology analysis was conducted on 83 PRRSV-1 and 83 PRRSV-2 strains. Phylogenetic analysis revealed the prevalence of both PRRSV-1 and PRRSV-2 strains in Thailand, with the latter exhibiting wider distribution. PRRSV-1 strains clustered into clades A, D, and H, while PRRSV-2 strains grouped into lineages 1, 5, and sublineage 8.7, further divided into 8.7/HP and 8.7/NA sublineages. Sublineage 8.7/NA strains accounted for a significant proportion of circulating PRRSV-2 strains. Homology analysis showed nucleotide and amino acid similarities ranging from 75.4 to 100.0% and 41.3 to 100.0% for PRRSV-1, and 78.6 to 100.0% and 70.8 to 100.0% for PRRSV-2 strains. Amino acid sequence alignments revealed mutations, insertions, and deletions in PRRSV-1 GP5, and key residue mutations in PRRSV-2 GP5 associated with biological functions. Recombination analysis identified two recombination events within PRRSV-2 sublineage 8.7 strains. Discussion: These findings confirm the variability of the GP5 protein. This study enhances our understanding of PRRSV prevalence and genetic variation in Thailand, contributing valuable insights for PRRS prevention and control.

Molecular and serological investigations of Batai virus in cattle and goats in the border area of Yunnan, China (2021-2022).

Introduction: Batai virus (BATV), a zoonotic pathogen transmitted by mosquitoes, infects vertebrates, including livestock, birds, and humans. Although BATV has been detected and isolated in mosquitoes in Yunnan Province, China, there have been no reports of livestock infection. Thus, we conducted a molecular and serological investigation of BATV in cattle and goat sera collected in spring and autumn from 2021 to 2022 in Honghe Prefecture, Yunnan Province, on the China-Vietnam border. Methods: We used indirect enzyme-linked immunosorbent assays and reverse transcription real-time PCR (RT-qPCR) to test 929 cattle and 973 goat serum samples. Results: BATV antibodies were detected in 262/929 (28.2%) cattle and 263/973 (27.0%) goat serum samples. RT-qPCR did not detect BATV RNA. Discussion: The positive rate of BATV serum antibodies in cattle and goats in Luxi County was higher compared with other areas, and it was also higher in autumn compared with spring, which may be related to climate, temperature, and mosquito density. Although our findings indicated the presence of BATV infection in livestock in the region, RT-qPCR did not detect BATV RNA. Therefore, BATV monitoring in cattle and goats should be heightened in autumn, and the scope of host monitoring should be expanded to clarify the hosts and vectors of BATV infection.

Epidemiological and evolutionary analysis of canine circovirus from 1996 to 2023.

BACKGROUND: Canine circovirus (CanineCV), a non-enveloped virus with a circular DNA genome, has been identified in various avian and mammalian species, including domestic and wild canids. This study aimed to comprehensively analyze the prevalence of CanineCV across diverse animal species in 11 provinces of China. RESULTS: A total of 1,666 serum samples were collected, revealing a 5.82% prevalence of CanineCV in dogs, with the highest rates being observed in southern and eastern China. Phylogenetic analysis of 266 global CanineCV genomes sourced from the NCBI identified six distinct genotypes, elucidating the complex dynamics of their evolution. Evidence suggested a potential bat origin for CanineCV, with positive selection and high rates of evolution being observed. Recombination analysis revealed dynamic genetic exchange, highlighting the intricate nature of CanineCV evolution. Mutational analysis identified key amino acid substitutions likely to influence the virus's adaptation. Additionally, glycosylation, palmitoylation, and SUMOylation sites were predicted, shedding light on crucial functional properties of the virus. CONCLUSIONS: This study provides a global perspective on the origin, genetic diversity, and evolutionary dynamics of CanineCV. Understanding these factors is crucial for elucidating its epidemiology and potential health risks.

The Hanks-like serine/threonine protein kinase YihE is crucial for Aeromonas veronii virulence and adhesion.

Aeromonas veronii is an important pathogen found in various aquatic environments and products, posing a threat to public health. The Hanks-like serine/threonine protein kinase is closely linked to the pathogenesis of pathogenic bacteria, but the exact role of YihE in A. veronii remains still unknown. To study the specific function of the YihE kinase, we constructed a knockout mutant of the yihE gene in A. veronii. The deletion of the yihE gene resulted in changes to the metabolism of L-arginine-AMC and acetic acid, as well as enhanced resistance to ampicillin and kanamycin in A. veronii. Additionally, the ΔyihE strain demonstrated a 1.4-fold increase in biofilm formation ability and a 1.8-fold decrease in adhesion and invasion to EPCs when compared to the wild-type strain. A significant decrease in cytotoxicity was observed at 2 and 3 h post-infection with EPCs compared to the wild-type strain. Additionally, the deletion of the yihE gene was associated with a significant decrease in motility of the strain. Furthermore, the deletion of the yihE gene resulted in a 1.44-fold increase in the LD50 of A. veronii in zebrafish. These findings offer valuable insights into the pathogenic mechanisms of A. veronii.

NSP4 promotes replication of porcine reproductive and respiratory syndrome virus-2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3 C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24 h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.

The peptidoglycan-associated lipoprotein gene mutant elicits robust immunological defense in mice against Salmonella enteritidis.

Background: Salmonella enteritidis (S. enteritidis), a zoonotic pathogen with a broad host range, presents a substantial threat to global public health safety. Vaccination stands as an effective strategy for the prevention and control of S. enteritidis infection, highlighting an immediate clinical need for the creation of safe and efficient attenuated live vaccines. Methods: In this study, a S. enteritidis peptidoglycan-associated lipoprotein (pal) gene deletion strain (Δpal), was constructed. To assess its virulence, we conducted experiments on biofilm formation capability, motility, as well as cell and mouse infection. Subsequently, we evaluated the immune-protective effect of Δpal. Results: It was discovered that deletion of the pal gene reduced the biofilm formation capability and motility of S. enteritidis. Cell infection experiments revealed that the Δpal strain exhibited significantly decreased abilities in invasion, adhesion, and intracellular survival, with downregulation of virulence gene expression, including mgtC, invH, spvB, sipA, sipB, ssaV, csgA, and pipB. Mouse infection experiments showed that the LD50 of Δpal increased by 104 times, and its colonization ability in mouse tissue organs was significantly reduced. The results indicated that the pal gene severely affected the virulence of S. enteritidis. Further, immunogenicity evaluation of Δpal showed a significant enhancement in the lymphocyte transformation proliferation capability of immunized mice, producing high titers of specific IgG and IgA, suggesting that Δpal possesses good immunogenicity. Challenge protection tests demonstrated that the strain could provide 100% immune protection against wild-type strains in mice. Discussion: This study proves that the pal gene influences the virulence of S. enteritidis, and Δpal could serve as a candidate strain for attenuated live vaccines, laying the foundation for the development of attenuated live vaccines against Salmonella.

The H4 subtype of avian influenza virus: a review of its historical evolution, global distribution, adaptive mutations and receptor binding properties.

The H4 subtype of avian influenza virus (AIV) exhibits a wide host range and is commonly found in migratory waterfowl. Recent studies have revealed that the H4N6 AIV can infect guinea pigs via aerosol transmission without prior adaptation. Additionally, the Q226L/G228S substitutions in the receptor-binding site have led to structural changes in globular head of H4 AIV, resulting in a configuration similar to that of pandemic H2N2 and H3N2 human influenza viruses. This article provides an updated review of the historical evolution, global distribution, adaptive mutations, receptor-binding preferences, and host range of H4 AIV. The insights presented herein will help in assessing the potential risk of future H4 AIV epidemics.

Establishment of a chip digital PCR detection method for canine circovirus.

Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/µL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Genetic and Evolutionary Analysis of Porcine Deltacoronavirus in Guangxi Province, Southern China, from 2020 to 2023.

Porcine deltacoronavirus (PDCoV) has shown large-scale global spread since its discovery in Hong Kong in 2012. In this study, a total of 4897 diarrheal fecal samples were collected from the Guangxi province of China from 2020 to 2023 and tested using RT-qPCR. In total, 362 (362/4897, 7.39%) of samples were positive for PDCoV. The S, M, and N gene sequences were obtained from 34 positive samples after amplification and sequencing. These PDCoV gene sequences, together with other PDCoV S gene reference sequences from China and other countries, were analyzed. Phylogenetic analysis revealed that the Chinese PDCoV strains have diverged in recent years. Bayesian analysis revealed that the new China 1.3 lineage began to diverge in 2012. Comparing the amino acids of the China 1.3 lineage with those of other lineages, the China 1.3 lineage showed variations of mutations, deletions, and insertions, and some variations demonstrated the same as or similar to those of the China 1.2 lineage. In addition, recombination analysis revealed interlineage recombination in CHGX-MT505459-2019 and CHGX-MT505449-2017 strains from Guangxi province. In summary, the results provide new information on the prevalence and evolution of PDCoV in Guangxi province in southern China, which will facilitate better comprehension and prevention of PDCoV.

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3.

Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

Learn Zero-Constraint-Violation Safe Policy in Model-Free Constrained Reinforcement Learning.

We focus on learning the zero-constraint-violation safe policy in model-free reinforcement learning (RL). Existing model-free RL studies mostly use the posterior penalty to penalize dangerous actions, which means they must experience the danger to learn from the danger. Therefore, they cannot learn a zero-violation safe policy even after convergence. To handle this problem, we leverage the safety-oriented energy functions to learn zero-constraint-violation safe policies and propose the safe set actor-critic (SSAC) algorithm. The energy function is designed to increase rapidly for potentially dangerous actions, locating the safe set on the action space. Therefore, we can identify the dangerous actions prior to taking them and achieve zero-constraint violation. Our major contributions are twofold. First, we use the data-driven methods to learn the energy function, which releases the requirement of known dynamics. Second, we formulate a constrained RL problem to solve the zero-violation policies. We prove that our Lagrangian-based constrained RL solutions converge to the constrained optimal zero-violation policies theoretically. The proposed algorithm is evaluated on the complex simulation environments and a hardware-in-loop (HIL) experiment with a real autonomous vehicle controller. Experimental results suggest that the converged policies in all environments achieve zero-constraint violation and comparable performance with model-based baseline.

Strain-restricted transfer of ferromagnetic electrodes for constructing reproducibly superior-quality spintronic devices.

Spintronic device is the fundamental platform for spin-related academic and practical studies. However, conventional techniques with energetic deposition or boorish transfer of ferromagnetic metal inevitably introduce uncontrollable damage and undesired contamination in various spin-transport-channel materials, leading to partially attenuated and widely distributed spintronic device performances. These issues will eventually confuse the conclusions of academic studies and limit the practical applications of spintronics. Here we propose a polymer-assistant strain-restricted transfer technique that allows perfectly transferring the pre-patterned ferromagnetic electrodes onto channel materials without any damage and change on the properties of magnetism, interface, and channel. This technique is found productive for pursuing superior-quality spintronic devices with high controllability and reproducibility. It can also apply to various-kind (organic, inorganic, organic-inorganic hybrid, or carbon-based) and diverse-morphology (smooth, rough, even discontinuous) channel materials. This technique can be very useful for reliable device construction and will facilitate the technological transition of spintronic study.

Application and Research Progress of Ultrasound-Guided Brachial Plexus Block Through Costoclavicular Space Approach in Upper Limb Surgery.

Objective: Exploring newer approaches to brachial plexus block is crucial for improving surgical outcomes and patient comfort. This study aims to review the application and research progress of ultrasound-guided brachial plexus block via the costoclavicular space approach in upper limb surgery. Methods: This study provides a comprehensive review of existing literature, studies, and clinical cases related to the costoclavicular approach. The advantages and disadvantages of conventional approaches for brachial plexus block, including the intermuscular groove method, supraclavicular method, and axillary approach, are discussed. The anatomical characteristics of the costoclavicular space are examined, and the methods of brachial plexus nerve block using ultrasound-guided costoclavicular space approach are described. It holds great promise for enhancing patient care and increasing the overall success rate of surgical procedures. Results: The costoclavicular space approach for brachial plexus block offers several advantages, including stable anatomical structure, low nerve variation rate, and clear visualization of each nerve bundle under ultrasound imaging. Compared to traditional approaches, ultrasound-guided brachial plexus block via the costoclavicular space approach has a high success rate, rapid onset of anesthesia, and high safety. Conclusion: Ultrasound-guided brachial plexus block via the costoclavicular space approach is effective and safe in upper limb surgery. It provides good anesthesia and postoperative analgesia, making it a valuable technique for various upper limb surgeries. The potential clinical significance of our findings lies in the possibility that ultrasound-guided costoclavicular space approach, with its enhanced precision and patient outcomes, could play a pivotal role in improving upper limb surgical procedures.

A Case of Subglottic Marginal B-Cell Lymphoma With Laryngeal Amyloidosis.

Laryngeal mucosa-associated lymphoid tissue (MALT) is an extra-nodal margin zone B-cell lymphoma (MALT lymphoma) and a low-grade malignant lymphoma with a low incidence, the etiology of the condition remains obscure, and the process of differential diagnosis poses a significant challenge, so it is easy to miss diagnosis and misdiagnosis clinically. The present article presents a clinical case study of a patient who was diagnosed with subglottic MALT lymphoma, which was associated with laryngeal amyloidosis. The patient underwent a successful treatment regimen comprising carbon dioxide laser and radiotherapy. In addition, the article provides an overview of relevant literature that can aid in the diagnosis and management of this rare disease. The study is expected to contribute to the existing body of knowledge on the treatment of subglottic MALT lymphoma and laryngeal amyloidosis.

Outcome and associated factors of high-risk human papillomavirus infection without cervical lesions.

OBJECTIVE: To study the outcome of human papillomavirus (HPV) infection in women with cervical pathology results of non-cervical intraepithelial neoplasia (CIN) or cervical cancer and positive high-risk HPV test, as well as analyze the associated risk factors affecting the outcome of infection. METHODS: To investigate the outcome of high-risk (HR)-HPV infection in the female genital tract and analyze the associated risk factors affecting their outcome, a total of 196 women with positive HR-HPV test results and non-CIN or cervical cancer cervical pathology results were selected for follow-up at the Cervical Disease Clinic of the Obstetrics and Gynecology Hospital, Zhejiang University School of Medicine from January 2017 to March 2020. The follow-up interval was every 6 months, and both cervical cytology (TCT) and HR-HPV testing were performed at each follow-up visit. If the cervical cytology results were normal upon recheck and the HR-HPV test was negative, the woman was considered to be cleared of the HPV infection and was entered into the routine cervical screening population. When the repeat HR-HPV test remained positive after 6 months, the woman was defined as having a persistent HR-HPV infection. If HR-HPV persisted but the TCT results were normal, follow-up was continued. If HR-HPV persisted and the TCT results were abnormal, a colposcopy-guided biopsy was performed immediately. In this situation, if the histological results were still non-CIN or cervical cancer, the follow-up was continued. If the histological results confirmed the development of CIN or invasive cancer, then enter another study follow-up to further track its development and outcome, and the woman commenced the treatment process. The HPV infection clearance time was analyzed by the Kaplan-Meier method, and the comparison of the HPV clearance rate and infection clearance time between each of the different groups was performed using aχ2 test or Fisher's exact test, as appropriate. After the univariate analysis, several significant factors were included in the Cox model and independent risk factors were analyzed. RESULTS: A total of 163 women were enrolled in this study. The median age was 40.0 years (22-67 years) and the median follow-up time was 11.5 months (6-31 months). The spontaneous clearance rate of HR-HPV infection was 51.5%, and the median time to viral clearance was 14.5 months. Age and the initial viral load were high risk factors affecting the spontaneous clearance of HR-HPV infection. The factors significantly associated with HPV clearance rate and time to HPV clearance consisted of menopause and full-term delivery (P < 0.05). CONCLUSIONS: In women with normal or low-grade lesions on the cell smear, the spontaneous clearance rate of HR-HPV infection was 51.5% and the time to clearance was 14.5 months. Age and the initial viral load were independent associated factors affecting the spontaneous clearance of HR-HPV infection in the female genital tract. These findings suggest that non-young women or those with high viral loads have a higher rate of persistent HR-HPV infection. Thus, intensive screening should be recommended.

Seneca Valley virus 3Cpro antagonizes host innate immune responses and programmed cell death.

Seneca Valley virus (SVV), a member of the Picornaviridae family, may cause serious water blister diseases in pregnant sows and acute death in newborn piglets, which have resulted in economic losses in pig production. The 3C protease is a vital enzyme for SVV maturation and is capable of regulating protein cleavage and RNA replication of the virus. Additionally, this protease can impede the host's innate immune response by targeting the interferon pathway's principal factor and enhance virus replication by modulating the host's RNA metabolism while simultaneously triggering programmed cell death. This article reviews recent studies on SVV 3C functions, which include viral replication promotion, cell apoptosis modulation and host immune response evasion, and provides a theoretical basis for research on preventing and controlling SVV infection.

Development of a MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although cSCC contributes to substantial morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. Our systematic computational drug repurposing screen predicted that selumetinib, a MAPK (mitogen-activated protein kinase) kinase inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development, consistent with our genomic analysis implicating MEK as a chemoprevention target. Although systemic MEKi suppresses the formation of cSCC in mice, systemic MEKi can cause severe adverse effects. Here, we report the development of a metabolically labile MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin before rapid metabolism in the systemic circulation. NFX-179 was identified on the basis of its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our ultraviolet-induced cSCC mouse model, topical application of NFX-179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. We further confirmed the localized nature of these effects in an additional split-mouse randomized controlled study where suppression of cSCC was observed only in drug-treated areas. No toxicities were observed. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner, and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together, our data provide a compelling rationale for using topical MEK inhibition through the application of NFX-179 gel as an effective strategy for cSCC chemoprevention.