Small interfering RNA (siRNA)-based therapeutic applications against viruses: principles, potential, and challenges.

RNA has emerged as a revolutionary and important tool in the battle against emerging infectious diseases, with roles extending beyond its applications in vaccines, in which it is used in the response to the COVID-19 pandemic. Since their development in the 1990s, RNA interference (RNAi) therapeutics have demonstrated potential in reducing the expression of disease-associated genes. Nucleic acid-based therapeutics, including RNAi therapies, that degrade viral genomes and rapidly adapt to viral mutations, have emerged as alternative treatments. RNAi is a robust technique frequently employed to selectively suppress gene expression in a sequence-specific manner. The swift adaptability of nucleic acid-based therapeutics such as RNAi therapies endows them with a significant advantage over other antiviral medications. For example, small interfering RNAs (siRNAs) are produced on the basis of sequence complementarity to target and degrade viral RNA, a novel approach to combat viral infections. The precision of siRNAs in targeting and degrading viral RNA has led to the development of siRNA-based treatments for diverse diseases. However, despite the promising therapeutic benefits of siRNAs, several problems, including impaired long-term protein expression, siRNA instability, off-target effects, immunological responses, and drug resistance, have been considerable obstacles to the use of siRNA-based antiviral therapies. This review provides an encompassing summary of the siRNA-based therapeutic approaches against viruses while also addressing the obstacles that need to be overcome for their effective application. Furthermore, we present potential solutions to mitigate major challenges.

Hymenobacter canadensis sp. nov., isolated from freshwater of the pond in Cambridge Bay, Canada.

A Gram-stain-negative, aerobic, reddish-coloured, rod-shaped and non-motile strain PAMC 29467T, was isolated from freshwater of the pond in Cambridge Bay, Canada. Strain PAMC 29467T was closely related to Hymenobacter yonginensis (98.1 % 16S rRNA gene similarity). Genomic relatedness analyses showed that strain PAMC 29467T is distinguishable from H. yonginensis based on average nucleotide identity (91.3 %) and digital DNA-DNA hybridization values (39.3 %). The major fatty acids (>10 %) of strain PAMC 29467T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C15 : 0 iso, C16 : 1 ω5c and summed feature 4 (C17 : 1 iso l and/or anteiso B). The major respiratory quinone was menaquinone-7. The genomic DNA G+C content was 61.5 mol%. Strain PAMC 29467T was separated from the type species in the genus Hymenobacter by its distinct phylogenetic position and some physiological characteristics. As a result, a novel species is proposed, with the name Hymenobacter canadensis sp. nov. (type strain, PAMC 29467T=KCTC 92787T=JCM 35843T).

Factors associated with seropositivity to Aino virus among sheep and goats in South Korea.

Aino virus infection is responsible for epizootic and/or sporadic outbreaks of abortions, stillbirths and premature delivery among pregnant ruminants. The epizootiology of Aino virus infection is poorly defined in South Korea, therefore our aim was to assess its seroprevalence among sheep and goats. We also wanted to evaluate management and regional risk factors that might influence the frequency of infection. Between 2012 and 2013, 26 of 331 flocks (7.9%) and 139 of 915 heads (15.2%) were found serologically positive. In 2018, when samples were again collected in the same regions, 35 of 308 flocks (11.4%) and 89 of 735 heads (12.1%) showed serum-neutralising antibodies against Aino virus. Our results revealed that the age class and history of reproductive problems in the flocks are connected to an increased risk of being positive. The management risk factor attributes showed that preventive measures, such as the routine application of insecticide in farms, decreased the odds for seropositivity to Aino virus (OR = 0.453, P = 0.001). We observed a significant difference in the individual likelihood of being positive in the southern and western provinces with respect to that in the northern and eastern provinces, respectively (OR = 2.199, P < 0.001 and OR = 2.177, P < 0.001). The results of this study may serve as a basis for future epizootic studies on Aino virus infection in South Korea.

Radicicol Inhibits Chikungunya Virus Replication by Targeting Nonstructural Protein 2.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a debilitating febrile illness characterized by persistent muscle and joint pain. The widespread distribution of transmission-competent vectors, Aedes species mosquitoes, indicates the potential risk of large-scale epidemics with high attack rates that can severely impact public health globally. Despite this, currently, there are no antivirals available for the treatment of CHIKV infections. Thus, we aimed to identify potential drug candidates by screening a chemical library using a cytopathic effect-based high-throughput screening assay. As a result, we identified radicicol, a heat shock protein 90 (Hsp90) inhibitor that effectively suppressed CHIKV replication by blocking the synthesis of both positive- and negative-strand viral RNA as well as expression of viral proteins. Interestingly, selection for viral drug-resistant variants and mutational studies revealed nonstructural protein 2 (nsP2) as a putative molecular target of radicicol. Moreover, coimmunoprecipitation and in silico modeling analyses determined that G641D mutation in the methyltransferase (MT)-like domain of nsP2 is essential for its interaction with cytoplasmic Hsp90ß chaperone. Our findings collectively support the potential application of radicicol as an anti-CHIKV agent. The detailed study of the underlying mechanism of action further contributes to our understanding of virus-host interactions for novel therapeutics against CHIKV infection.

Adiponectin Controls Nutrient Availability in Hypothalamic Astrocytes.

Adiponectin, an adipose tissue-derived hormone, plays integral roles in lipid and glucose metabolism in peripheral tissues, such as the skeletal muscle, adipose tissue, and liver. Moreover, it has also been shown to have an impact on metabolic processes in the central nervous system. Astrocytes comprise the most abundant cell type in the central nervous system and actively participate in metabolic processes between blood vessels and neurons. However, the ability of adiponectin to control nutrient metabolism in astrocytes has not yet been fully elucidated. In this study, we investigated the effects of adiponectin on multiple metabolic processes in hypothalamic astrocytes. Adiponectin enhanced glucose uptake, glycolytic processes and fatty acid oxidation in cultured primary hypothalamic astrocytes. In line with these findings, we also found that adiponectin treatment effectively enhanced synthesis and release of monocarboxylates. Overall, these data suggested that adiponectin triggers catabolic processes in astrocytes, thereby enhancing nutrient availability in the hypothalamus.

Serological evidence of bluetongue virus infection and serotype distribution in dairy cattle in South Korea.

BACKGROUND: Bluetongue is a vector-borne viral disease, and bluetongue virus (BTV) outbreaks can cause substantial economic losses. Even subclinical infection may carry significant associated costs, including a loss of condition, reduced milk yield, and infertility and abortion, and indirect costs, largely due to the export restrictions and surveillance requirements imposed to limit the spread of the virus. However, the BTV epidemiology in the Far East remains incompletely understood, especially in the cattle population in South Korea. In this study, the seroprevalence of BTV antibodies and distribution of BTV serotypes in dairy cattle in South Korea were evaluated to improve the understanding of the BTV epidemiological situation in the Asia-Pacific region. RESULTS: Between 2012 and 2013, a total of 37 out of 171 dairy cattle herds (21.6%) and 85 out of 466 dairy cattle heads (18.2%) showed antibodies against BTV. Neutralizing antibodies to BTV-1, - 2, - 3, - 4, - 7, - 15, and - 16 serotypes were identified, and the RNAs of the BTV-1, - 2, - 3, - 15, and - 16 serotypes were detected, indicating that BTV was circulating in the dairy cattle population in South Korea. CONCLUSIONS: These findings indicate that BTV is widespread and has circulated in dairy cattle in South Korea. This is the first report presenting evidence of circulating antibodies against BTV and the serotype distribution in bovine populations in South Korea.

Nioella aestuarii sp. nov., of the family Rhodobacteraceae, isolated from tidal flat.

A bacterium, designated strain MME-018T, was isolated from a tidal flat of the Muui-do in the Republic of Korea and identified within the family Rhodobacteraceae. The 16S rRNA gene sequence of the isolate showed the highest similarity to that of Nioella sediminis JS7-11T (98.9 %), followed by Nioella nitratireducens SSW136T (97.1 %). In phylogenetic analyses, these taxa formed a clade at neighbour-joining, maximum-likelihood, and maximum-parsimony algorithms, in which it was separated from other genus belonging to the family Rhodobacteraceae. Ubiquinone-10 (Q-10) was the major respiratory quinone. Major polar lipids included phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, and an unidentified lipid. Major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) C16 : 0, cyclo C19 : 0ω8c, and 11-methyl C18 : 1ω7c. Genomic DNA G+C content was 61 mol%. Cells were Gram-stain negative, non-motile, aerobic, and rod-shaped. This strain grew in 1‒4 % (w/v) NaCl, at 4-40 °C and pH 6.0‒8.0, with optimal growth in 2 % (w/v) NaCl, at 25‒30 °C and pH 7.0. DNA-DNA hybridization values between strain MME-018T and Nioella sediminis KCTC 42144T and Nioella nitratireducens KCTC 32417T were 17±3 and 13±1 %, respectively. On the basis of polyphasic taxonomic analysis, strain MME-018T is proposed to represent a novel species of the genus Nioella, for which the name Nioellaaestuarii sp. nov. The type strain of Nioellaaestuarii is MME-018T (=KCCM 43135T=JCM 30752T).

Lawsonia intracellularis in the feces of wild rodents and stray cats captured around equine farms.

BACKGROUND: Proliferative enteropathy is a global enteric disease of particular importance in pigs. The causative bacterium, Lawsonia intracellularis, has a wide range of susceptible host species. Recently, L. intracellularis has been recognized as an etiologic agent of an emerging enteric disease in foals called equine proliferative enteropathy (EPE). The presence of L. intracellularis in nonruminant wildlife has raised questions regarding the role of these species in EPE transmission. RESULTS: This study investigated exposure to L. intracellularis in wild rodents and feral cats from eight farms with confirmed EPE. Serum (42) and fecal (40) samples from resident foals and fecal samples (131), intestinal mucosa tissues (14), and mesenteric lymph nodes (14) from wild and feral animals were collected for the evaluation of the farm status and the molecular detection of L. intracellularis following the diagnosis of EPE in index cases. Fresh feces from wild rodents and feral cats were collected from the ground while walking the premises or after trapping the animals using live traps. A total of 3 brown rats, 7 house mice, 1 striped field mouse, 2 grey red-backed voles, and 3 feral cats showed evidence of prior exposure to L. intracellularis. CONCLUSIONS: Our data add to increasing evidence demonstrating the potential for L. intracellularis transmission and infection in wild rodents and feral cats and provide possible evidence of interspecies transmission. The exposure of wild rodents and feral cats provides potential evidence for the spillover of L. intracellularis to wildlife species and raises the question of spillback to horses. Additionally, these animals may represent an indicator of environmental exposure or may be actively involved in the transmission of L. intracellularis to foals by acting as potential reservoir/amplifier hosts. This study is the first to demonstrate the magnitude of L. intracellularis shedding in the feces of wild rodents and feral cats and to indicate the significant infection risk that wild rodents and feral cats pose to naïve horses in South Korea.

Suppression of lytic replication of Kaposi's sarcoma-associated herpesvirus by autophagy during initial infection in NIH 3T3 fibroblasts.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the angioproliferative neoplasm Kaposi's sarcoma (KS). We first confirmed the susceptibility of NIH 3T3 fibroblasts to KSHV by infecting them with BCP-1-derived KSHV. Lytic replication of KSHV was confirmed by PCR amplification of viral DNA isolated from culture supernatants of KSHV-infected cells. The template from KSHV-infected NIH 3T3 cells resulted in an intense viral DNA PCR product. A time course experiment revealed the disappearance of KSHV-specific DNA in culture supernatant of NIH 3T3 cells during a period between 48 h and 72 h postinfection. Furthermore, 3 days postinfection, infected NIH 3T3 cells showed no evidence of latent or lytic transcripts, including LANA, vFLIP, vCyclin, and vIL-6. These results imply that KSHV infection in NIH 3T3 cells is unstable and is rapidly lost on subsequent culturing. Additionally, we detected an enhancement of autophagy early in infection with KSHV. More interestingly, inhibition of autophagy by Beclin 1 siRNA or 3-methyladenine significantly increased the amount of KSHV-specific DNA in the culture supernatant of NIH 3T3 cells when compared to the group treated with KSHV infection alone, implying that autophagy prevents lytic replication of KSHV. Taken together, our data suggest that autophagy could be one of the cellular mechanisms utilized by host cells to promote viral clearance.

First report of Bluetongue virus isolation in the Republic of Korea and analysis of the complete coding sequence of the segment 2 gene.

This study investigated the possible presence of the Bluetongue virus (BTV) in the Republic of Korea (ROK). Cell cultures were used to test blood samples collected from abattoirs throughout the country. Testing identified a single BTV isolate, which was characterized as BTV serotype 1 based on a nucleotide sequence analysis of the segment 2 gene. This report therefore indicates that BTV serotype 1 is present in the ROK. The potential importance of BTV in the ROK has been overlooked because cattle are mostly unaffected by the virus and because sheep, the most severely infected hosts, are uncommon in the ROK. However, as recent BTV serotype 8 outbreaks in Europe have demonstrated, certain BTV strains have the potential to cause severe disease in cattle. Additionally, with climate change continuously expanding the regions in which Culicoides vectors are able to survive, there is an increased need to study BTV in the Far East and ROK. To better prepare for future outbreaks of BTV, a sustained and effective level of surveillance for BTV in livestock will need to be established.

Serological surveillance studies confirm the Rift Valley fever virus free status in South Korea.

Rift Valley fever is a mosquito-borne zoonotic disease of domestic ruminants. This disease causes abortions in pregnant animals, and it has a high mortality rate in newborn animals. Recently, a Rift Valley fever virus (RVFV) outbreak in the Arabian Peninsula increased its potential spread to new regions worldwide. In non-endemic or disease-free countries, early detection and surveillance are important for preventing the introduction of RVFV. In this study, a serological surveillance was conducted to detect antibodies against RVFV. A total of 2382 serum samples from goats and cattle were randomly collected from nine areas in South Korea from 2011 to 2013. These samples were tested for antibodies against RVFV, using commercial ELISA kits. None of the goats and cattle were positive for antibodies against RVFV. This finding suggests that this disease is not present in South Korea, and furthermore presents the evidence of the RVFV-free status of this country.

Seroprevalence of porcine proliferative enteropathy among wild boars in the Republic of Korea.

BACKGROUND: The importance of the wild boar as a reservoir of Lawsonia intracellularis was assessed by investigating the seroprevalence of this pathogen among wild boars in the Republic of Korea. The extent of exposure to L. intracellularis among wild boars (Sus scrofa coreanus) was monitored by a country-wide serological survey using an immunoperoxidase monolayer assay. RESULTS: In this study, antibodies to L. intracellularis were observed in 165 of 716 clinically healthy wild boars tested. The overall apparent prevalence calculated directly from the sample and the true prevalence calculated based on the accuracy of the test method were 23.0% (95% confidence interval: 20.0-26.3%) and 25.6% (95% confidence interval: 23.9-27.2%), respectively. Serologically positive animals were found in all the tested provinces. CONCLUSIONS: Our results confirm that L. intracellularis is present in the wild boar population worldwide, even in Far East Asia. Despite the high seroprevalence shown in wild boars, further studies are warranted to evaluate their potential as a reservoir species because seroprevalence does not prove ongoing infection nor shedding of the bacteria in amounts sufficient to infect other animals. It should also be determined whether the wild boar, like the domestic pig, is a natural host of L. intracellularis.

Coding-complete genome sequence and phylogenetic analysis of Coxsackievirus B3 isolated from South Korea.

Whole-genome sequencing of a Coxsackievirus B3 strain isolated from the stool of a febrile patient with aseptic meningoencephalitis, South Korea, in 2002 was performed. This strain exhibits a high nucleotide sequence identity with various strains circulating in China from 2001 to 2019.

Risk factors associated with Coxiella burnetii in wild boars: A study in South Korea.

Coxiella burnetii is a Gram-negative bacterium that causes the zoonotic disease Q fever. Wild boars serve as reservoirs for C. burnetii. This study aimed to identify the risk factors associated with C. burnetii infection in wild boars. We analyzed the data from 975 wild boar samples collected from June to November 2021 in South Korea. We utilized the indirect ELISA to detect antibodies against C. burnetii. A sample optical density to positive-control optical density value exceeding 50% was classified as positive. We gathered data on the forestation, terrain, weather, agriculture, and animal density of the region where the samples were collected. Continuous variables were categorized into tertiles. We performed a univariate logistic regression analysis and included variables with a p-value < 0.2 in the final multivariable logistic regression model. In our multivariable logistic regression analysis to identify risk factors for C. burnetii infection in wild boars, we used a forward selection method to enter variables based on the order of their significance. We performed the final multivariable logistic regression analyses using either continuous variables or variables categorized into tertiles. The prevalence of C. burnetii was 14.6% (n=142). Locations with the highest maximum wind speeds (3.92-8.24 m/s) showed a 59% increase in infection odds compared to locations with the lowest speeds (1.45-3.25 m/s)(p=0.044). For each 1 m/s increase in maximum wind speed, infection odds increased by 24.1% (p=0.037). Regions with the highest percentage of paddy fields per area (8.3-45%) showed a 76% increase in infection odds compared to regions with the lowest percentage (0-1.5%)(p=0.011). For each 1% increase in the proportion of paddy fields per area, infection odds increased by 3.3% (p=0.003). High maximum wind speed and a high percentage of paddy field were identified as significant risk factors for C. burnetii infection in wild boars.

Control of foot-and-mouth disease during 2010-2011 epidemic, South Korea.

An outbreak of foot-and-mouth disease caused by serotype O virus occurred in cattle and pigs in South Korea during November 2010-April 2011. The highest rates of case and virus detection were observed 44 days after the first case was detected. Detection rates declined rapidly after culling and completion of a national vaccination program.

Complete genome analysis of three live attenuated Rinderpest virus vaccine strains derived through serial passages in different culture systems.

The genomes of three South Korean Rinderpest virus vaccine strains (L72, LA77, and LA96) were analyzed in order to investigate their genetic variability. These three vaccine strains were all derived from the same virus strain origin (Fusan) through repeated passages in different culture systems. The full genome length of the three strains was 15,882 nucleotides, and the sequence similarity between the three South Korean RPV strains at the nucleotide level was 98.1 to 98.9%. The genetic distance between Nakamura III, L72, LA77, LA96, and LATC06 and the Kabete strain was greater than that between the Fusan and Kabete strains for the P, V, and C genes. The difference in pathogenicity among these strains might be due to the V gene, which has a positive (>1) selection ratio based on the analysis of synonymous (dS) and nonsynonymous (dN) substitution rates (dN/dS ratio [ω]).

Antibodies to H5 subtype avian influenza virus and Japanese encephalitis virus in northern pintails (Anas acuta) sampled in Japan.

Blood samples from 105 northern pintails (Anas acuta) captured on Hokkaido, Japan were tested for antibodies to avian influenza virus (AIV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) to assess possible involvement of this species in the spread of economically important and potentially zoonotic pathogens. Antibodies to AIV were detected in 64 of 105 samples (61%). Of the 64 positives, 95% and 81% inhibited agglutination of two different H5 AIV antigens (H5N1 and H5N9), respectively. Antibodies to JEV and WNV were detected in five (5%) and none of the samples, respectively. Results provide evidence for prior exposure of migrating northern pintails to H5 AIV which couldhave implications for viral shedding and disease occurrence. Results also provide evidence for limited involvement of this species in the transmission and spread of flaviviruses during spring migration.

Prevalence and associated risk factors for Lawsonia intracellularis infection in farmed rabbits: A serological and molecular cross-sectional study in South Korea.

Lawsonia intracellularis is the etiological agent of proliferative enteropathy, which is globally considered an important enteric disease in pigs and horses. Experimental studies suggest that the organism spreads by subclinical infection of many animals, including rabbits. Despite the importance of rabbits in the epidemiology of L. intracellularis, the extent of exposure to L. intracellularis in the rabbit population is poorly defined and remains unclear. The objective of this cross-sectional study was to investigate the seroprevalence and shedding of L. intracellularis in farmed rabbits. Furthermore, we aimed to identify risk factors associated with seropositivity. Sera from the rabbits were used to measure L. intracellularis-specific antibodies by immunoperoxidase monolayer assay, and rectal swabs were used to detect L. intracellularis DNA using a real-time PCR assay. Antibodies against L. intracellularis were detected in 12.3% of farms (20/163) and 6.3% of rabbits (49/774). Lawsonia intracellularis DNA in rectal swabs was detected in 3.8% of farms (6/156) and 1.2% of rabbits (8/667). The risk factor analysis showed that the presence of pigs or horses on the farm or the neighboring farm was associated with an increase in the risk of seropositivity (p < 0.05). We observed significantly increased odds of positivity for L. intracellularis in rabbits with a history of digestive trouble (diarrhea) on the farm during the 3 months before the samples were obtained (p < 0.05). Collectively, these findings demonstrated that L. intracellularis infection was evident among farmed rabbits and that rabbits might serve as an important reservoir for L. intracellularis epidemiology.

Epidemiology of Chuzan Virus Infection in Free-Ranging and Farmed Cervids in South Korea: A Seroprevalence Survey.

Chuzan virus is teratogenic in cattle and causes congenital abnormalities, such as hydranencephaly and cerebellar hypoplasia, in calves. Chuzan virus seroprevalence among free-ranging and farmed cervids in South Korea was found to be 4.4% (38/873), confirming exposure to this virus in cervids in South Korea.

Role of cervids in the epidemiology of bovine ephemeral fever virus infection in the Republic of Korea: A cross-sectional retrospective study.

BACKGROUND: Bovine ephemeral fever (BEF) is a viral disease in cattle and buffaloes, with subclinical involvement in various ruminant species. OBJECTIVES: This study aimed to investigate bovine ephemeral fever virus (BEFV) transmission in deer in the Republic of Korea (ROK) and the potential risk factors associated with seropositivity. METHODS: We conducted a retrospective cross-sectional serological survey of neutralising antibodies against BEFV in cervid sera collected from the ROK. RESULTS: The seroprevalence of BEFV was estimated to be 10.8% (95% confidence interval [CI] = 8.5-14.1), demonstrating that exposure to this virus is prevalent among farmed and free-ranging cervids in the ROK. The results revealed that age class and geographic location affected seroprevalence. Older age and the presence of neighbouring ruminant farms were significant risk factors (odds ratio [OR] = 2.394, 95% CI = 1.195-4.796) and (OR = 1.533, 95% CI = 1.026-2.288), respectively. We also observed that the individual likelihood of positivity in the southern provinces was significantly higher than that in the northern provinces (OR = 1.744, 95% CI = 1.001-3.037). There were also significant differences in the seroprevalence of cervids between the western and eastern provinces (OR = 2.021, 95% CI = 1.047-3.900). Factors that were not significantly associated with BEFV antibody prevalence included herd size and species (p > 0.05). CONCLUSIONS: These results suggest that cervid species may serve as important reservoirs for the transmission of BEFV, highlighting the need for closer monitoring of BEFV infections in cervids in the ROK.