Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses.

Introduction: Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. Methods: A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. Results: This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/µL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. Discussion: The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.

Immune cells within tertiary lymphoid structures are associated with progression-free survival in patients with locoregional recurrent breast cancer.

INTRODUCTION: Locoregional recurrent breast cancers have a poor prognosis. Little is known about the prognostic impact of immune microenvironment, and tertiary lymphoid structures (TLSs) in particular have not been reported. Thus, we aimed to characterize the immune microenvironment in locoregional recurrent breast tumors and to investigate its relationship with prognosis. METHODS: We retrospectively included 112 patients with locoregional recurrent breast cancer, and hematoxylin-eosin staining and immunohistochemical staining (CD3, CD4, CD8, CD19, CD38, and CD68) were performed on locoregional recurrent tumor samples. The association of immune cells and TLSs with progression-free survival (PFS) were analyzed by survival analysis. RESULTS: We found more immune cells in the peritumor than stroma. After grouping according to estrogen receptor (ER) status, a low level of peritumoral CD3+ cells in ER+ subgroup (p = 0.015) and a low level of stromal CD68+ cells in ER- subgroup (p = 0.047) were both associated with longer PFS. TLSs were present in 68% of recurrent tumors, and CD68+ cells within TLSs were significantly associated with PFS as an independent prognostic factor (p = 0.035). TLSs and immune cells (CD3, CD38, and CD68) within TLSs were associated with longer PFS in ER- recurrent tumors (p = 0.044, p = 0.012, p = 0.050, p < 0.001, respectively), whereas CD38+ cells within TLSs were associated with shorter PFS in ER+ recurrent tumors (p = 0.037). CONCLUSION: Our study proposes potential predictors for the clinical prognosis of patients with locoregional recurrent breast cancer, emphasizing the prognostic value of immune cells within TLSs, especially CD68+ cells.

Characterization of two chicken origin highly pathogenic H7N9 viruses isolated in northern China.

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs.

The Epidemiological Analysis of Pseudorabies Virus and Pathogenicity of the Variant Strain in Shandong Province.

Pseudorabies (PR) is a disease that is seriously endangering the pig industry in China. To understand the current prevalence of pseudorabies virus (PRV) in Shandong Province, China, 19,292 serum samples were collected from 16 locations in Shandong from 2018 to 2020. The gE antibody was detected by enzyme-linked immunosorbent assay. Ninety-seven suspected cases of PRV infection were collected from sick pigs vaccinated with Bartha-K61 to isolate PRV. The results showed that the average positive rate of the PRV gE antibody decreased from 38.20% in 2018 to 18.12% in 2020, but there was a high positive rate in sows. The isolation rate of PRV was 13.40% (13/97), and four strains were purified through plaque assay (named PRV-SD1, PRV-SD2, PRV-SD3, and PRV-SD4). The homology and genetic evolution of four PRV strains based on gE, gC, gI, and TK genes were analyzed and showed that these four strains shared more than 99.0% nucleotide homology with the variant PRV XJ5 strain, and they clustered in the same sub-branch with the domestic variant PRV strains, including JS-2012 and XJ5. Furthermore, the pathogenicity of the isolated variant strain was assessed by intranasal infection of 16-week-old pigs with 1 mL PRV-SD1 strain. The results of the animal experiment demonstrated that the PRV-SD1-infected pigs exhibited obvious clinical symptoms as early as 2 days post inoculation (dpi), and all infected pigs died within 1 week. The severe hyperemia of meninges and swelling of lungs and tonsils were observed. Histopathology analysis showed the obvious lymphocytes necrosis of tonsils, interstitial pneumonia, and viral encephalitis. Many positive staining cells were observed in tonsils and brains through immunohistochemistry staining assay. Viral shedding in oropharyngeal and rectal swabs were detected at 2 dpi, reached a peak at 3 dpi, and then gradually decreased. The detection of viral loads in the tissues showed that tonsils had the highest virus titer, further proving it may be the target organ of variant PRV infection. In conclusion, variant PRV strains were still highly prevalent in Shandong Province, and they had a strong pathogenicity in pigs.

Emergence of a novel reassortant avian influenza virus (H10N3) in Eastern China with high pathogenicity and respiratory droplet transmissibility to mammals.

Decades have passed since the first discovery of H10-subtype avian influenza virus (AIV) in chickens in 1949, and it has been detected in many species including mammals such as minks, pigs, seals and humans. Cases of human infections with H10N8 viruses identified in China in 2013 have raised widespread attention. Two novel reassortant H10N3 viruses were isolated from chickens in December 2019 in eastern China during routine surveillance for AIVs. The internal genes of these viruses were derived from genotype S (G57) H9N2 and were consistent with H5N6, H7N9 and H10N8, which cause fatal infections in humans. Their viral pathogenicity and transmissibility were further studied in different animal models. The two H10N3 isolates had low pathogenicity in chickens and were transmitted between chickens via direct contact. These viruses were highly pathogenic in mice and could be transmitted between guinea pigs via direct contact and respiratory droplets. More importantly, these viruses can bind to both human-type SAα-2,6-Gal receptors and avian-type SAα-2,3-Gal receptors. Asymptomatic shedding in chickens and good adaptability to mammals of these H10N3 isolates would make it easier to transmit to humans and pose a threat to public health.

Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Genetic and antigenic diversity of H7N9 highly pathogenic avian influenza virus in China.

Avian influenza virus (AIV) H7N9 that emerged in 2013 in eastern China is a novel zoonotic agent mainly circulating in poultry without clinical signs but causing severe disease with high fatality in humans in more than 5 waves. Since the emergence of highly pathogenic (HP) H7N9 variants in 2016, it has induced heavy losses in the poultry industry leading to the implementation of an intensive nationwide vaccination program at the end of wave 5 (September 2017). To characterize the ongoing evolution of H7N9 AIV, we conducted analyses of H7N9 glycoprotein genes obtained from 2013 to 2019. Bayesian analyses revealed a decreasing population size of HP H7N9 variants post wave 5. Phylogenetic topologies revealed that two novel small subclades were formed and carried several fixed amino acid mutations that were along HA and NA phylogenetic trees since wave 5. Some of the mutations were located at antigenic sites or receptor binding sites. The antigenic analysis may reveal a significant antigenic drift evaluated by hemagglutinin inhibition (HI) assay and the antigenicity of H7N9 AIV might evolute in large leaps in wave 7. Molecular simulations found that the mutations (V135T, S145P, and L226Q) around the HA receptor pocket increased the affinity to α2,3-linked sialic acid (SIA) while decreased to α2,6-linked SIA. Altered affinity may suggest that HP H7N9 variations aggravate the pathogenicity to poultry but lessen the threat to public health. Selection analyses showed that the HP H7N9 AIV experienced an increasing selection pressure since wave 5, and the national implementation of vaccination might intensify the role of natural selection during the evolution waves 6 and 7. In summary, our data provide important insights about the genetic and antigenic diversity of circulating HP H7N9 viruses from 2017 to 2019. Enhanced surveillance is urgently warranted to understand the current situation of HP H7N9 AIV.

Identification of Isoflavonoid Biosynthesis-Related R2R3-MYB Transcription Factors in Callerya speciosa (Champ. ex Benth.) Schot Using Transcriptome-Based Gene Coexpression Analysis.

The R2R3-MYB family is one of the largest plant transcription factor (TF) families playing vital roles in defense, plant growth, and secondary metabolism biosynthesis. Although this gene family has been studied in many species, isoflavonoid biosynthesis-related R2R3-MYB TFs in Callerya speciosa (Champ. ex Benth.) Schot, a traditional Chinese medicinal herb, are poorly understood. Here, a total of 101 R2R3-MYB TFs were identified from C. speciosa transcriptome dataset. 25 clades divided into five functional groups were clustered based on the sequence similarity and phylogenetic tree. Conserved motifs and domain distribution, expression patterns, and coexpression networks were also employed to identify the potential R2R3-MYB TFs in the regulation of isoflavonoid biosynthesis. In silico evaluation showed that the deduced R2R3-CsMYB proteins contain highly conserved R2R3 repeat domain at the N-terminal region, that is the signature motif of R2R3-type MYB TFs. Eight potential TFs (CsMYB17, CsMYB36, CsMYB41, CsMYB44, CsMYB45, CsMYB46, CsMYB72, and CsMYB81) had high degrees of coexpression with four key isoflavonoid biosynthetic genes (CsIFS, CsCHS7, CsHID-1, and CsCHI3), in which CsMYB36 as a potential regulator possessed the highest degree. HPLC analysis showed that formononetin and maackiain contents were significantly increased during the development of tuberous roots, which might be controlled by both related R2R3-CsMYBs and structural genes involved in the isoflavonoid biosynthesis pathway. The transcriptome data were further validated by reverse transcription real-time PCR (RT-qPCR) analysis, and similar expression profiles between TFs and key structural genes were identified. This study was the first step toward the understanding of the R2R3-MYB TFs regulating isoflavonoid biosynthesis in C. speciosa. The results will provide information for further functional analysis and quality improvement through genetic manipulation of these potential R2R3-CsMYB genes in C. speciosa.

Hormonal and transcriptional analyses provides new insights into the molecular mechanisms underlying root thickening and isoflavonoid biosynthesis in Callerya speciosa (Champ. ex Benth.) Schot.

Callerya speciosa (Champ. ex Benth.) Schot is a traditional Chinese medicine characterized by tuberous roots as the main organ of isoflavonoid accumulation. Root thickening and isoflavonoid accumulation are two major factors for yield and quality of C. speciosa. However, the underlying mechanisms of root thickening and isoflavonoid biosynthesis have not yet been elucidated. Here, integrated morphological, hormonal and transcriptomic analyses of C. speciosa tuberous roots at four different ages (6, 12, 18, 30 months after germination) were performed. The growth cycle of C. speciosa could be divided into three stages: initiation, rapid-thickening and stable-thickening stage, which cued by the activity of vascular cambia. Endogenous changes in phytohormones were associated with developmental changes during root thickening. Jasmonic acid might be linked to the initial development of tuberous roots. Abscisic acid seemed to be essential for tuber maturation, whereas IAA, cis-zeatin and gibberellin 3 were considered essential for rapid thickening of tuberous roots. A total of 4337 differentially expressed genes (DEGs) were identified during root thickening, including 15 DEGs participated in isoflavonoid biosynthesis, and 153 DEGs involved in starch/sucrose metabolism, hormonal signaling, transcriptional regulation and cell wall metabolism. A hypothetical model of genetic regulation associated with root thickening and isoflavonoid biosynthesis in C. speciosa is proposed, which will help in understanding the underlying mechanisms of tuberous root formation and isoflavonoid biosynthesis.

Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination.

Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China.