Simultaneous detection and phylogenetic analysis of porcine epidemic diarrhea virus and porcine circovirus 4 in Henan province, China.

Porcine circovirus 4 (PCV4) is a recently discovered circovirus that was first reported in 2019 in several pigs in Hunan province of China and has also been identified in pigs infected with porcine epidemic diarrhea virus (PEDV). To further investigate the coinfection and genetic diversity of these two viruses, 65 clinical samples (including feces and intestinal tissues) were collected from diseased piglets on 19 large-scale pig farms in Henan province of China, and a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (qPCR) assay was developed for detecting PEDV and PCV4 simultaneously. The results showed that the limit of detection was 55.2 copies/µL and 44.1 copies/µL for PEDV and PCV4, respectively. The detection rate for PEDV and PCV4 was 40% (26/65) and 38% (25/65), respectively, and the coinfection rate for the two viruses was 34% (22/65). Subsequently, the full-length spike (S) gene of eight PEDV strains and a portion of the genome containing the capsid (Cap) gene of three PCV4 strains were sequenced and analyzed. Phylogenetic analysis showed that all of the PEDV strains from the present study clustered in the G2a subgroup and were closely related to most of the PEDV reference strains from China from 2011 to 2021, but they differed genetically from a vaccine strain (CV777), a Korean strain (virulent DR1), and two Chinese strains (SD-M and LZC). It is noteworthy that two PEDV strains (HEXX-24 and HNXX-24XIA) were identified in one sample, and the HNXX-24XIA strain had a large deletion at amino acids 31-229 of the S protein. Moreover, a recombination event was observed in strain HEXX-24. Phylogenetic analysis based on the amino acid sequence of the PCV4 Cap protein revealed that PCV4 strains were divided into three genotypes: PCV4a1, PCV4a2, and PCV4b. Three strains in the present study belonged to PCV4a1, and they had a high degree of sequence similarity (>98% identity) to other PCV4 reference strains. This study not only provides technical support for field investigation of PEDV and PCV4 coinfection but also provides data for their prevention and control.

[Chemical Constituents of Ethyl Acetate Fraction of Suaeda glauca].

OBJECTIVE: To study the chemical constituents of Suaeda glauca. METHODS: The chemical constituents were isolated and purified with several separation and purification techniques. Their structures were identified by physicochemical properties and various spectroscopic methods. RESULTS: Ten compounds were isolated from the ethyl acetate fraction as lignoceric acid (1), ß-amyrin-n-nonyl ether(2), ß-sitosterol(3), ß-daucosterol(4), quercetin(5), luteolin(6), luteolin-7-O-ß-D-glucoside(7), isorhamnetin(8), scopoletin (9) and stigmasterol(10). CONCLUSION: Compounds 1, 2, 6, 7, 8, 9 and 10 are isolated from Suaeda genus for the first time and compounds 3 - 5 are isolated from this plant for the first time.

[Study on Chemical Constituents from Roots of Lonicera macranthoides].

OBJECTIVE: To study chemical constituents of the roots of Lonicera macranthoides. METHODS: The chemical constituents were isolated and purified by means of several chromatographic techniques and their structures were elucidated by spectroscopic methods. RESULTS: Seven compounds were isolated and identified as ribenol (1), excoecarin C (2), 18-hydroxy-13-epi-manoyloxide (3), asiatic acid (4), oleanolic acid (5), ß-sitosterol (6) and ß-daucosterol (7). CONCLUSION: Compounds 1-4 are obtained from this genus for the first time. Compound 5 is obtained from this plant for the first time. All the compounds are found from the roots of Lonicera mac- ranthoides for the first time.

Integrated ribosome and proteome analyses reveal insights into sevoflurane-induced long-term social behavior and cognitive dysfunctions through ADNP inhibition in neonatal mice.

A growing number of studies have demonstrated that repeated exposure to sevoflurane during development results in persistent social abnormalities and cognitive impairment. Davunetide, an active fragment of the activity-dependent neuroprotective protein (ADNP), has been implicated in social and cognitive protection. However, the potential of davunetide to attenuate social deficits following sevoflurane exposure and the underlying developmental mechanisms remain poorly understood. In this study, ribosome and proteome profiles were analyzed to investigate the molecular basis of sevoflurane-induced social deficits in neonatal mice. The neuropathological basis was also explored using Golgi staining, morphological analysis, western blotting, electrophysiological analysis, and behavioral analysis. Results indicated that ADNP was significantly down-regulated following developmental exposure to sevoflurane. In adulthood, anterior cingulate cortex (ACC) neurons exposed to sevoflurane exhibited a decrease in dendrite number, total dendrite length, and spine density. Furthermore, the expression levels of Homer, PSD95, synaptophysin, and vglut2 were significantly reduced in the sevoflurane group. Patch-clamp recordings indicated reductions in both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). Notably, davunetide significantly ameliorated the synaptic defects, social behavior deficits, and cognitive impairments induced by sevoflurane. Mechanistic analysis revealed that loss of ADNP led to dysregulation of Ca 2+ activity via the Wnt/ß-catenin signaling, resulting in decreased expression of synaptic proteins. Suppression of Wnt signaling was restored in the davunetide-treated group. Thus, ADNP was identified as a promising therapeutic target for the prevention and treatment of neurodevelopmental toxicity caused by general anesthetics. This study provides important insights into the mechanisms underlying social and cognitive disturbances caused by sevoflurane exposure in neonatal mice and elucidates the regulatory pathways involved.

First Molecular Detection and Genetic Analysis of a Novel Porcine Circovirus (Porcine Circovirus 4) in Dogs in the World.

A novel circovirus species was identified in farmed pigs and designated porcine circovirus 4 (PCV4); it has recently been proved to be pathogenic to piglets. However, little is known about its cross-species transmission, and there is no evidence of PCV4 in dogs. A total of 217 fecal samples were collected from diarrheal dogs in Henan Province, China, and tested for the presence of PCV4 using a real-time PCR assay. Among the 217 samples, the total positivity rate for PCV4 was 5.99% (13/217 samples), with rates of 7.44% and 4.17% in 2020 and 2021, respectively. PCV4 was detected in dogs in 6 of 10 cities, demonstrating that PCV4 could be detected in dogs in Henan Province, China. One PCV4 strain (HN-Dog) was sequenced in this study and shared high levels of identity (97.9% to 99.6%) with reference strains at the genome level. Phylogenetic analysis based on complete genome sequences of HN-Dog and 42 reference strains showed that the HN-Dog strain was closely related to 3 PCV4 reference strains (from pig, raccoon dog, and fox) but differed genetically from other viruses in the genus Circovirus. Three genotypes, i.e., PCV4a, PCV4b, and PCV4c, were confirmed by phylogenetic analysis of complete genome sequences of 42 PCV4 strains, and one amino acid variation in Rep protein (V239L) and three amino acid variations in Cap protein (N27S, R28G, and M212L) were considered conserved genotype-specific molecular markers. In conclusion, the present study is the first to report the discovery of the PCV4 genome in dogs, and the association between PCV4 infection and diarrhea warrants further study. IMPORTANCE This study is the first to report the presence of PCV4 in dogs worldwide, and the first complete genome sequence was obtained from a dog affected with diarrhea. Three genotypes of PCV4 strains (PCV4a, PCV4b, and PCV4c) were determined, as supported by specific amino acid markers (V239L for open reading frame 1 [ORF1] and N27S R28G and M212L for ORF2). These findings help us understand the current status of intestinal infections in pet dogs in Henan Province, China, and also prompted us to accelerate research on the pathogenesis, epidemiology, and cross-species transmission of PCV4.

Molecular detection and genetic characteristics of a novel porcine circovirus (porcine circovirus 4) and porcine reproductive and respiratory syndrome virus in Shaanxi and Henan Provinces of China.

Porcine circovirus 4 (PCV4) is a recently discovered circovirus that was first reported in 2019 in several pigs with severe clinical disease in Hunan province of China, and also identified in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). To further investigate the epidemic profile and genetic characteristics of the two viruses, 150 clinical samples were collected from 9 swine farms in Shaanxi and Henan provinces of China, and a SYBR Green I-based duplex quantitative real-time polymerase chain reaction (qPCR) was developed for detecting PCV4 and PRRSV simultaneously. The results showed the limits of detection were 41.1 copies/µL and 81.5 copies/µL for PCV4 and PRRSV, respectively. The detection rates of PCV4 and PRRSV were 8.00% (12/150) and 12.00% (18/150) respectively, and a case of co-infection with PCV4 and PRRSV was found in the lung tissue of a suckling pig with respiratory symptom. Subsequently, the complete genomic sequences of five PCV4 strains were obtained, of which one PCV4 strain (SX-ZX) was from Shaanxi province, and these strains were 1770 nucleotides in length and had 97.7%-99.4% genomic identity with 59 PCV4 reference strains. The genome characteristic of the SX-ZX strain was evaluated from three aspects, a "stem-loop" structure, ORF1 and ORF2. As essential elements for the replication, the 17-bp iterative sequence was predicted as the stem structure, in which three non-tandem hexamers were found at downstream with H1/H2 (12-CGGCACACTTCGGCAC-27) as the minimal binding site. Three of the five PCV4 strains were clustered into PCV4b, which was composed of Suidae, fox, dairy cow, dog and raccoon dog. Phylogenetic analysis revealed that seven PRRSV strains from the present study were clustered into the PRRSV-2 genotype. Collectively, these data extend our understanding of the genome characteristic of PCV4 as well as the molecular epidemiology and the genetic profile of PCV4 and PRRSV.

[Water-soluble chemical constituents in flower buds of Lonicera macranthoides].

OBJECTIVE: To study the water-soluble chemical constituents of the flower buds of Lonicera macranthoides. METHODS: The chemical constituents were isolated and purified by means of chromatographic techniques and their structures were elucidated by spectroscopic methods. RESULTS: Six compounds were isolated and identified as trans-linalool-3,7-oxide-6-O-beta-D-glucopyranoside(1), secol oganoside(2), secoxyloganin(3),chlorogenic acid(4), caffeic acid(5), sucrose(6). CONCLUSION: Compound 1 is isolated from nature as a single compound for the first time, compounds 2-3 are isolated from this plant for the first time.

Genetic Characteristics and Pathogenicity of a Novel Porcine Epidemic Diarrhea Virus with a Naturally Occurring Truncated ORF3 Gene.

Porcine epidemic diarrhea virus (PEDV) is the major pathogen that causes diarrhea and high mortality in newborn piglets, with devastating impact on the pig industry. To further understand the molecular epidemiology and genetic diversity of PEDV field strains, in this study the complete genomes of four PEDV variants (HN2021, CH-HNYY-2018, CH-SXWS-2018, and CH-HNKF-2016) obtained from immunized pig farms in central China between 2016 to 2021 were characterized and analyzed. Phylogenetic analysis of the genome and S gene showed that the four strains identified in the present study had evolved into the subgroup G2a, but were distant from the vaccine strain CV777. Additionally, it was noteworthy that a new PEDV strain (named HN2021) belonging to the G2a PEDV subgroup was successfully isolated in vitro and it was further confirmed by RT-PCR that this isolate had a large natural deletion at 207-373 nt of the ORF3 gene, which has never been reported before. Particularly, in terms of pathogenicity evaluation, colostrum deprivation piglets challenged with PEDV HN2021 showed severe diarrhea and high mortality, confirming that PEDV HN2021 was a virulent strain. Hence, PEDV strain HN2021 of subgroup G2a presents a promising vaccine candidate for the control of recurring porcine epidemic diarrhea (PED) in China. This study lays the foundation for better understanding of the genetic evolution and molecular pathogenesis of PEDV.

[Study on chemical constituents of the bulbs of Lycoris longituba].

OBJECTIVE: To study the chemical constituents of the Bulbs of Lycoris longituba. METHODS: The chemical constituents were isolated and purified by means of chromatographic techniques and their structures were elucidated by spectroscopic methods. RESULTS: Six compounds were isolated and identified as Crinine (1), Narciclasine (2), Squamigine (3), 11-Hydroxyvittatine (4), Pancracine (5), Lycorine (6). CONCLUSION: Compounds 1 - 6 are isolated from this plant for the first time.

[Studies on the chemical constituents of Salicornia europaea].

OBJECTIVE: To study the chemical constituents of Salicornia europaea. METHODS: The chemical constituents were isolated and purified by means of several chromatographic techniques and their structures were elucidated by spectroscopic methods. RESULTS: Five compounds were isolated and identified as syringaresinol 4-O-beta-D-glucopyranoside(1), icariside B2(2), erythro-1-(4-O-beta-D-glucopyranosyl-3,5-dimethyoxyphenyl)-2-syringaresinoxyl-propane-1,3-diol(3), longifloroside B(4), 7-O-beta-D-glucopyranosyl-6-methoxychromone(5). CONCLUSION: Compounds 1-4 are obtained from this genus for the first time.

[Studies on the chemical constituents of the aerial part of Bupleurum chinense (II)].

OBJECTIVE: To study the chemical constituents of the aerial part of Bupleurum chinense. METHODS: Dried aerial parts of Bupleurum chinense were extracted with EtOH, the chemical constituents in the extract were isolated and their structures were identified by physical and chemical properties and spectral analysis. RESULTS: Five compounds were obtained and identified as 1-O-caffeoylglycerol (1), esculetin (2), scopoletin (3), kaempferol-3-O-beta-D-rutinoside (4) and alpha-spinastero (5). CONCLUSION: Compounds 1-3 are obtained from Bupleurum chinense for the first time.