In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro. METHODS: The effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected. RESULTS: Compared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control. CONCLUSION: Taken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.

Molecular epidemiology of pseudorabies virus in Yunnan and the sequence analysis of its gD gene.

Outbreaks of pseudorabies (PRs) have occurred in Yunnan, China, which caused significant economic loss. To determine the prevalence and origin of PR in Yunnan, especially among vaccinated pigs, overall 791 samples of blood, tissue, semen, and sera were analyzed by serological methods, PCR, and sequence analysis of gD gene. Detection with viral gI antibody or PCR showed that the yearly positive rates of PR virus (PRV) in Yunnan from 2010 to 2014 were 48.15, 21.26, 2.17, 5.22, and 0.35%, respectively, with an average of 15.43%. In general, the incidence declined through the period of 2010-2014 probably due to the application of PRV eradication strategies. A phylogenetic tree was constructed based on the complete sequence of gD gene, with all strains clustered into two independent clades, i.e., Asian and European-American clades. The virus isolates from Henan, Tianjin, Heilongjiang, Sichuan, Shandong, Fujian, Xinjiang, Hubei, Guangdong, and Yunnan fell into Asian group, which harbored South Korea isolate. Four Yunnan virus isolates together with South Korean Namyangju fell into in the European-American clade. It showed that PR was pandemic as there was not a clear clue about the geographical origin of the PRV isolates in China since 2010.

Genetic heterogeneity of swine hepatitis E virus isolates from Yunnan province, China in 2011-2012.

BACKGROUND: Hepatitis E is a disease of major public-health concern mainly in developing countries. Although molecular and sero-epidemiological investigations of HEV have been performed in many provinces in China, the epidemiological data from Yunnan Province are limited and genotypes are not be fully characterized. In this study the prevalence and characteristics of hepatitis E virus (HEV) detected in pigs from Yunnan province, China was evaluated. RESULTS: A total of 13 out of 187 pig fecal samples collected in 2011 revealed HEV positive results; likewise, 7 out of 69 samples collected in 2012 exhibited positive results. These findings indicated a total prevalence of 7.8% (20/256). Phylogenetic and molecular evolutionary analysis results revealed that nine strains were found in the samples obtained in 2011, in which 87.1% to 99.4% nucleotide sequence identity was shared among these strains; and 77.0% to 81.9%, 52.2% to 53.6%, 77.0% to 88.2% and 77.9% to 96.8% nucleotide sequence identities were shared with strains representing genotypes 1, 2, 3, and 4. Five strains were detected in the samples obtained in 2012, in which 94.2% to 99.3% nucleotide sequence identity was shared among the strains, and 81.0% to 82.5%, 81.8% to 83.2%, 81.0% to 92.7% and 81.0% to 97.8% nucleotide sequence identities were shared with strains representing the genotypes 1, 2, 3, and 4. CONCLUSIONS: Analysis of fourteen detected HEV strains revealed that three of them were subtype 4d, two were subtype 4b; the nine remaining isolated strains were subtype 4 h. These results indicated that the prevalence of HEV in the swine herds of Yunnan was quite high, additional public-health concerns should focus on pork safety.

Therapeutic and immune-regulation effects of Scutellaria baicalensis Georgi polysaccharide on pseudorabies in piglets.

Pseudorabies virus (PRV) can cause fatal encephalitis in newborn pigs and escape the immune system. While there is currently no effective treatment for PRV, Scutellaria baicalensis Georgi polysaccharides (SGP) and Rodgersia sambucifolia Hemsl flavonoids (RHF) are traditional Chinese herbal medicines with potential preventive and therapeutic effects against PRV infection. In order to explore which one is more effective in the prevention and treatment of PRV infection in piglets. We investigate the therapeutic effects of RHF and SGP in PRV-infected piglets using clinical symptom and pathological injury scoring systems. The immune regulatory effects of RHF and SGP on T lymphocyte transformation rate, cytokines, T cells, and Toll-like receptors were also measured to examine the molecular mechanisms of these effects. The results showed that SGP significantly reduced clinical symptoms and pathological damage in the lungs, liver, spleen, and kidneys in PRV-infected piglets and the T lymphocyte conversion rate in the SGP group was significantly higher than that in the other treatment groups, this potential dose-dependent effect of SGP on T lymphocyte conversation. Serum immunoglobulin and cytokine levels in the SGP group fluctuated during the treatment period, with SGP treatment showing better therapeutic and immunomodulatory effects in PRV-infected piglets than RHF or the combined SGP + RHF treatment. In conclusion, RHF and SGP treatments alleviate the clinical symptoms of PRV infection in piglets, and the immunomodulatory effect of SGP treatment was better than that of the RHF and a combination of both treatments. This study provides evidence for SGP in controlling PRV infection in piglets.

Isolation and Characterization of Yunnan Variants of the Pseudorabies Virus and Their Pathogenicity in Rats.

Porcine pseudorabies has long existed in China and is a serious threat to the Chinese farming industry. To understand the prevalence and genetic variation of the porcine pseudorabies virus (PRV) and its pathogenicity in Yunnan Province, China, we collected 560 serum samples across seven Yunnan Province regions from 2020 to 2021 and detected anti-gE antibodies in these samples. Sixty-one clinical tissue samples were also collected from pigs with suspected PRV that were vaccinated with Bartha-K61. PRV-gE antibodies were found in 29.6% (166/560) of the serum samples. The PRV positivity rate in clinical tissue samples was 13.1% (8/61). Two isolates, PRV-KM and PRV-QJ, were obtained. The identity of the gB, gD, and gE genes between these isolates and the Chinese mutants exceeded 99.5%. These isolates and the classical Fa strain were used to infect 4-week-old rats intranasally to assess their pathogenicity. All infected rats showed the typical clinical and pathological features of PRV two days post-infection. The viral loads in the organs differed significantly among the infected groups. Viruses were detected in the saliva and feces at 12 h. Significant dynamic changes in total white blood cell counts (WBC), lymphocyte counts (Lym), and neutrophil counts (Gran) occurred in the blood of the infected groups at 24 and 48 h. These results show that mutant PRV strains are prevalent in Bartha-K61-vaccinated pigs in Yunnan Province, China. Moreover, rats shed PRV in their saliva and feces during early infection, indicating the need for rodent control in combatting PRV infections in Yunnan Province, China.

A study on the treatment effects of Crataegus pinnatifida polysaccharide on non-alcoholic fatty liver in mice by modulating gut microbiota.

The objective of this study was to investigate the protective effect of Crataegus pinnatifida polysaccharide (CPP) on non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet (HFD) in mice. The findings demonstrated that CPP improved free fatty acid (FFA)-induced lipid accumulation in HepG2 cells and effectively reduced liver steatosis and epididymal fat weight in NAFLD mice, as well as decreased serum levels of TG, TC, AST, ALT, and LDL-C. Furthermore, CPP exhibited inhibitory effects on the expression of fatty acid synthesis genes FASN and ACC while activating the expression of fatty acid oxidation genes CPT1A and PPARα. Additionally, CPP reversed disturbances in intestinal microbiota composition caused by HFD consumption. CPP decreased the firmicutes/Bacteroidetes ratio, increased Akkermansia abundance, and elevated levels of total short-chain fatty acid (SCFA) content specifically butyric acid and acetic acid. Our results concluded that CPP may intervene in the development of NAFLD by regulating of intes-tinal microbiota imbalance and SCFAs production. Our study highlights that CPP has a potential to modulate lipid-related pathways via alterations to gut microbiome composition thereby ex-erting inhibitory effects on obesity and NAFLD development.

Host cellular factors involved in pseudorabies virus attachment and entry: a mini review.

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily and serves as an exceptional animal model for investigating the infection mechanism of Herpes simplex virus type 1. Notably, PRV has the capability to infect a wide range of mammals, including humans, highlighting its potential as an overlooked zoonotic pathogen. The attachment and entry steps of PRV into host cells are crucial to accomplish its life cycle, which involve numerous cellular factors. In this mini review, we offer a comprehensive summary of current researches pertaining to the role of cellular factors in PRV attachment and entry stages, with the overarching goal of advancing the development of novel antiviral agents against this pathogen.

Epidemiological investigation and phylogenetic analysis of Classical Swine Fever virus in Yunnan province from 2015 to 2021.

BACKGROUND: Classical swine fever virus (CSFV), the causative agent of classical swine fever (CFS), is a highly contagious disease that poses a serious threat to Chinese pig populations. OBJECTIVES: Many provinces of China, such as Shandong, Henan, Hebei, Heilongjiang, and Liaoning provinces, have reported epidemics of CSFV, while the references to the epidemic of CSFV in Yunnan province are rare. This study examined the epidemic characteristics of the CSFV in Yunnan province. METHODS: In this study, 326 tissue samples were collected from different regions in Yunnan province from 2015 to 2021. A reverse transcription-polymerase chain reaction (RT-PCR), sequences analysis, and phylogenetic analysis were performed for the pathogenic detection and analysis of these 326 clinical specimens. RESULTS: Approximately 3.37% (11/326) of specimens tested positive for the CSFV by RT-PCR, which is lower than that of other regions of China. Sequence analysis of the partial E2 sequences of eleven CSFV strains showed that they shared 89.0-100.0% nucleotide (nt) and 95.0-100.0% amino acid (aa) homology, respectively. Phylogenetic analysis showed that these novel isolates belonged to the subgenotypes 2.1c and 2.1d, with subgenotype 2.1c being predominant. CONCLUSIONS: The CSFV was sporadic in China's Yunnan province from 2015 to 2021. Both 2.1c and 2.1d subgenotypes were found in this region, but 2.1c was dominant.

Emergence of a Novel Recombinant Pseudorabies Virus Derived From the Field Virus and Its Attenuated Vaccine in China.

The occurrence of pseudorabies (PR) caused by the PR virus (PRV) causes huge economic losses to the pig industry in China. Moreover, the potential threat of PRV to humans' health has received wide attention recently. The prevalence of two PRV genotypes and the application of their corresponding live attenuated vaccines increase the recombination possibility. In the present study, a novel recombinant PRV strain designed as HN-2019 was isolated from one sick piglet in Hunan province, China, its genetic features and pathogenicity were further investigated. The results showed that the glycoprotein E (gE) and gG genes of the HN-2019 strain displayed higher nucleotide homology with PRV classical strains (such as Ea and Fa) compared to others. However, its TK gene with continuous nucleotide deletions shared 100% nucleotide identity with the HB-98 vaccine strain, which was derived from the Ea strain. Moreover, the HN-2019 strain exhibited similar growth characteristics to that of the Ea strain, but its pathogenicity in mice was significantly lower than the latter one. The results above suggested that a naturally recombinant event might occur in the genome of the HN-2019 strain between the PRV classical strain and the HB-98 vaccine strain, which will provide useful guidelines for PRV vaccine design in the future.

Homologous recombination technology generated recombinant pseudorabies virus expressing EGFP facilitates to evaluate its susceptibility to different cells and screen antiviral compounds.

Pseudorabies virus (PRV) is considered as an emerging zoonotic pathogen since its isolation from a human case. Meanwhile, the disease caused by PRV infection has led huge economic losses to Chinese pig industry since 2011. In this study, we constructed a recombinant PRV stably expressing the enhanced green fluorescent protein (EGFP) by homologous recombination technology for evaluating its susceptibility to different human cell lines and screening antiviral compounds. Stably expressed EGFP by this designed rPRVHuN-EGFP virus was confirmed in the infected cells, moreover, the growth kinetics of which was similar to that of wild type strain. Importantly, the application of this rPRV allowed us to easily verify its infectivity in all tested human cell lines, although the infection efficiencies were lower than that in PK15 cells. Meanwhile, the antiviral activities of harmine and PHA767491 were also conveniently validated in vitro, as directly reflected by the reduced EGFP signals. These results demonstrate that this recombinant PRV virus should be a useful tool for basic virology researches and antiviral agent screening.

Epidemiological Investigation and Genetic Analysis of Pseudorabies Virus in Yunnan Province of China from 2017 to 2021.

In recent years, the prevalence of pseudorabies virus (PRV) has caused huge economic losses to the Chinese pig industry. Meanwhile, PRV infection in humans also sounded the alarm about its cross-species transmission from pigs to humans. To study the regional PRV epidemic, serological and epidemiological investigations of PRV in pig populations from Yunnan Province during 2017-2021 were performed. The results showed that 31.37% (6324/20,158, 95% CI 30.73-32.01) of serum samples were positive for PRV glycoprotein E (gE)-specific antibodies via enzyme-linked immunosorbent assay (ELISA). The risk factors, including the breeding scale and development stage, were significantly associated with PRV seroprevalence among pigs in Yunnan Province. Of the 416 tissue samples collected from PRV-suspected pigs in Yunnan Province, 43 (10.33%, 95% CI 7.41-13.26) samples were positive for PRV-gE nucleic acid in which 15 novel PRV strains from these PRV-positive samples were isolated, whose gC and gE sequences were analyzed. Phylogenetic analysis showed that all 15 isolates obtained in this study belonged to the genotype II. Additionally, the gC gene of one isolate (YuN-YL-2017) was genetically closer to variant PRV strains compared with others, while the gE gene was in the same clade with other classical PRV strains, indicating that this isolate might be a recombinant strain generated from the classical and variant strains. The results revealed the severe PRV epidemic in Yunnan Province and indicated that PRV variants are the major genotypes threatening the pig industry development.

Embryonic organizer formation disorder leads to multiorgan dysplasia in Down syndrome.

Despite the high prevalence of Down syndrome (DS) and early identification of the cause (trisomy 21), its molecular pathogenesis has been poorly understood and specific treatments have consequently been practically unavailable. A number of medical conditions throughout the body associated with DS have prompted us to investigate its molecular etiology from the viewpoint of the embryonic organizer, which can steer the development of surrounding cells into specific organs and tissues. We established a DS zebrafish model by overexpressing the human DYRK1A gene, a highly haploinsufficient gene located at the "critical region" within 21q22. We found that both embryonic organizer and body axis were significantly impaired during early embryogenesis, producing abnormalities of the nervous, heart, visceral, and blood systems, similar to those observed with DS. Quantitative phosphoproteome analysis and related assays demonstrated that the DYRK1A-overexpressed zebrafish embryos had anomalous phosphorylation of ß-catenin and Hsp90ab1, resulting in Wnt signaling enhancement and TGF-ß inhibition. We found an uncovered ectopic molecular mechanism present in amniocytes from fetuses diagnosed with DS and isolated hematopoietic stem cells (HSCs) of DS patients. Importantly, the abnormal proliferation of DS HSCs could be recovered by switching the balance between Wnt and TGF-ß signaling in vitro. Our findings provide a novel molecular pathogenic mechanism in which ectopic Wnt and TGF-ß lead to DS physical dysplasia, suggesting potential targeted therapies for DS.

Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China.

In this study, the prevalence and characteristics of hepatitis E virus (HEV) in pigs and the general population in the Yunnan province, China, were evaluated. Nine hundred sixty sera, 95 liver and 60 feces samples were randomly collected from pig farms and abattoirs, in addition 173 human sera were sampled in the provincial capital city for a serological survey and an RT-nPCR assay. The screening results showed that among 621 samples collected from five pig farms, the HEV-specific IgG positive rate ranged from 73.2% to 83.5%, and the overall seroprevalence was 78.9% (490/621). A further analysis revealed that the seroprevalence increased with age. The positive rate of human serum samples was 39.9% (69/173). HEV RNA was detected in five swine feces, six swine liver and one anti-HEV-IgM-positive human serum sample by RT-nPCR. Sequence and alignment of the 348-nt PCR-amplified products of 12 HEV strains identified nine distinct nucleotide sequences. Phylogenetic and molecular evolutionary analysis revealed that these nine sequences shared 84.2% to 100.0% nucleotide sequence identity with each other, with all isolates belonging to genotype 4 HEV and clustering with other Chinese swine and human HEV sequences determined earlier. This study results suggest that the prevalence of genotype 4 HEV is serious, both in pig herds and in the human population, and authorities should pay more attention to the prevalence of HEV in southwest China.

Histopathology of brain functional areas in pigs infected by porcine pseudorabies virus.

Porcine pseudorabies virus (PRV) infection is a major disease in swine. It is challenging to eradicate the virus entirely after it has invaded Chinese farms, resulting in significant economic losses. This study aimed to explore the histopathological correlation of brain regions in PRV-infected pigs. Twenty pigs were randomly divided into two experimental groups (the PRV-infected and sham-inoculated groups; n = 10 per group). The pigs were then observed for clinical signs at specified time points. Brain tissue samples were collected for histopathological examination on days 3, 10, and 14. The correlation analysis was based on clinical observation, lesion characterization, and pathogen location. Clinical observation showed that the severity of clinical neurological signs increased with time. Pathological dissection and microscopic observation revealed gross pathological changes such as degeneration and necrosis of nerve cells, increase in microglia, eosinophilic inclusion body, lymphocyte infiltration, and loose cortical tissue structure. Immunohistochemistry showed that the virus was mainly localized in neurons, microglia, nerve fibers, cerebellar granular layer, and Purkinje cell layer. The virus invasion route was from the cerebrum to the cerebellum and eventually to the brainstem, and the severity of brain damage increased with time. The route of virus infection was from the olfactory bulb to the hippocampus and eventually to the medulla oblongata, and the viral expression increased with time. Of note, brain injury, viral expression, and clinical neurological signs were positively correlated with the infection period; similarly, the severity and degrees of their changes were positively correlated.

Effect of porcine circovirus type 2 on the severity of lung and brain damage in piglets infected with porcine pseudorabies virus.

Porcine circovirus type 2 (PCV2) is widespread throughout Chinese farms, and the infection rate of porcine pseudorabies virus (PRV) is very high. The emergence of mixed infection involving PCV2 and PRV has been difficult to prevent and control and has caused considerable economic loss. The present study investigated lung and brain damage caused by PRV in piglets with PCV2 infection. Twenty piglets were divided randomly into two experiment groups (PRV group and PRV + PCV2 group; n = 10 per group). The pigs were observed for clinical signs at specified times. At necropsy, lung and brain tissue samples were collected for histopathological examination, and tissue virus load was determined using quantitative polymerase chain reaction. Severe pathogenicity due to PRV was evident in two-month-old piglets. PCV2 and PRV co-infection led to more severe neurological and respiratory symptoms and a higher mortality rate in the piglets. In addition, the pathological damage to the lung and brain was also aggravated. The co-infection was associated with a significant increase in the content of PRV in the brain and lung tissue. In conclusion, PCV2 and PRV co-infection could cause severe and irreversible damage to piglets.

Isolation, sequence identification and tissue expression profiles of 3 novel porcine genes: ASPA, NAGA, and HEXA.

The complete coding sequences of 3 porcine genes - ASPA, NAGA, and HEXA - were amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referenced pig ESTs. These 3 novel porcine genes were then deposited in the NCBI database and assigned GeneIDs: 100142661, 100142664 and 100142667. The phylogenetic tree analysis revealed that the porcine ASPA, NAGA, and HEXA all have closer genetic relationships with the ASPA, NAGA, and HEXA of cattle. Tissue expression profile analysis was also carried out and results revealed that swine ASPA, NAGA, and HEXA genes were differentially expressed in various organs, including skeletal muscle, the heart, liver, fat, kidney, lung, and small and large intestines. Our experiment is the first one to establish the foundation for further research on these 3 swine genes.

Association between SLC11A1 (NRAMP1) polymorphisms and susceptibility to tuberculosis in Chinese Holstein cattle.

We investigated the associations between SLC11A1 polymorphisms and susceptibility to tuberculosis (TB) in Chinese Holstein cattle, using a case-control study of 136 animals that had positive reactions to TB tests and showed symptoms and 96 animals that had negative reactions to tests and showed no symptoms. Polymerase chain reaction (PCR) sequencing and the restriction fragment length polymorphism (RFLP) technique were used to detect and determine SLC11A1 polymorphisms. Association analysis identified significant correlations between SLC11A1 polymorphisms and susceptibility/resistance to TB, and two genetic markers for SLC11A1 were established using PCR-RFLP. Sequence alignment of SLC11A1 revealed seven single-nucleotide polymorphisms (SNPs). This is the first report of MaeII PCR-RFLP markers for the SLC11A1-SNP3 site and PstI PCR-RFLP markers for the SLC11A1-SNP5 and SLC11A1-SNP6 sites in Chinese Holstein cattle. Logistic regression analysis indicated that SLC11A1-SNP1, SLC11A1-SNP3, and SLC11A1-SNP5 were significantly associated with susceptibility/resistance to TB. Two genotypes of SLC11A1-SNP3 were susceptible to TB, whereas one genotype of SLC11A1-SNP1 and two genotypes of SLC11A1-SNP5 were resistant. Haplotype analysis showed that nine haplotypes were potentially resistant to TB. After Bonferroni correction, three of the haplotypes remained significantly associated with TB resistance. SLC11A1 is a useful candidate gene related to TB in Chinese Holstein cattle.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in southwest China from 2007 to 2009.

To gain insight into the molecular epidemiology and possible mechanisms of genetic variation of porcine reproductive and respiratory syndrome (PRRS) in Yunnan Province of China, the ORF5 gene of 32 PRRSV isolates from clinical samples collected from 2007 to 2009 were sequenced and analyzed. Nucleotide and amino acid analyses were carried out on 32 isolates and representative strains of the North American genotype, European genotype and two representative Chinese isolates. Results revealed that these isolates share 86.9-99.0% nucleotide and 87.5-98.0% amino acid identity with VR-2332 the prototypical North American PRRSV, 61.7-62.9% and 54.3-57.8% with Lelystad virus (LV) the representative strain of European genotype, 91.2-95.4% and 90.0-94.5% with CH-1a that was isolated in mainland China in 1996, 88.1-99.3% and 85.5-99.0% with JX-A1 the representative strain of High pathogenic PRRSV in China, and 86.2-99.8% and 85.5-100.0% between isolated strains of different years, respectively. Phylogenetic analysis revealed that all 32 PRRSV isolates belonged to the North American genotype and were further divided into two different subgenotypes. Subgenotype 1 comprised twenty two Yunnan isolates which divided into two branches. Subgenotype 2 comprised ten isolates which closely related to the RespPRRS vaccine and its parent strain VR-2332. The functional domains of GP5 such as the signal peptide, ectodomain, transmembrane regions and endodomain were identified and some motifs in GP5 with known functions, such as primary neutralizing epitope (PNE) and decoy epitope were also further analyzed. Our study shown the great genetic diversity of PRRSV in southwest China, rendering the guide for control and prevention of this disease.