Interpretatively automated identification of circulating tumor cells from human peripheral blood with high performance.

The detection and analysis of circulating tumor cells (CTCs) would be of aid in a precise cancer diagnosis and an efficient prognosis assessment. However, traditional methods that rely heavily on the isolation of CTCs based on their physical or biological features suffer from intensive labor, thus being unsuitable for rapid detection. Furthermore, currently available intelligent methods are short of interpretability, which creates a lot of uncertainty during diagnosis. Therefore, we propose here an automated method that takes advantage of bright-field microscopic images with high resolution, so as to take an insight into cell patterns. Specifically, the precise identification of CTCs was achieved by using an optimized single-shot multi-box detector (SSD)-based neural network with integrated attention mechanism and feature fusion modules. Compared to the conventional SSD system, our method exhibited a superior detection performance with the recall rate of 92.2%, and the maximum average precision (AP) value of 97.9%. To note, the optimal SSD-based neural network was combined with advanced visualization technology, i.e., the gradient-weighted class activation mapping (Grad-CAM) for model interpretation, and the t-distributed stochastic neighbor embedding (T-SNE) for data visualization. Our work demonstrates for the first time the outstanding performance of SSD-based neural network for CTCs identification in human peripheral blood environment, showing great potential for the early detection and continuous monitoring of cancer progression.

Correction: A molecularly imprinted antibiotic receptor on magnetic nanotubes for the detection and removal of environmental oxytetracycline.

Correction for 'A molecularly imprinted antibiotic receptor on magnetic nanotubes for the detection and removal of environmental oxytetracycline' by Jixiang Wang et al., J. Mater. Chem. B, 2022, 10, 6777-6783, https://doi.org/10.1039/D2TB00497F.

CD163-Expressing Porcine Macrophages Support NADC30-like and NADC34-like PRRSV Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a strict cell tropism. In addition to the primary alveolar macrophages, PRRSV is strictly cytotropic to African green monkey kidney cells, such as MARC-145 cells; however, MARC-145 cells are not infected by most NADC30-like and NADC34-like PRRSV strains. The essential scavenger receptor CD163 has been proved to mediate productive infection of PRRSV in various non-permissive cell lines. In this study, we systematically tested the porcine CD163 stably expressing 3D4/21 cells for infections with various PRRSV strains. The results showed that the porcine CD163-expressing macrophages support the infections of PRRSV2 of lineages 1, 5, and 8, as evidenced by Western blotting, immunofluorescence assay, quantitative PCR, and virus titration assay. Considering the current prevalence of NADC30-like and NADC34-like PRRSV2 of lineage 1 in China, the CD163-expressing macrophages are very useful for PRRSV research and disease management.

A molecularly imprinted antibiotic receptor on magnetic nanotubes for the detection and removal of environmental oxytetracycline.

The detection and elimination of antibiotic contaminants, such as oxytetracycline (OTC), a broad-spectrum tetracycline antibiotic, would be of help in efficient environmental monitoring, agriculture and food safety tests. Nevertheless, currently available methodologies, which mostly rely on the chromatographic separation of OTC, suffer from low sensitivity and complicated processes. Thus, we report here on the design and synthesis of a fluorescent sensor based on molecularly imprinted magnetic halloysite nanotubes (referred to as MHNTs@FMIPs) for the effective detection and purification of OTC in actual environmental samples. The fluorescence of the MHNTs@FMIPs was quenched obviously upon loading with OTC, covering a linear concentration range of 10-300 nM with a limit of detection (LOD) as low as 8.1 nM. The imprinting factor is 4.47, indicating an excellent specificity. Furthermore, the MHNTs@FMIPs can be applied to the quantitative detection of OTC (5 cycles of 300 nM) in aquaculture wastewater and Yangtze River water, demonstrating their immense application potential.

Systemic Homologous Neutralizing Antibodies Are Inadequate for the Evaluation of Vaccine Protective Efficacy against Coinfection by High Virulent PEDV and PRRSV.

G2 porcine epidemic diarrhea virus (G2 PEDV) and highly pathogenic porcine reproductive and respiratory syndrome virus 2 (HP-PRRSV2) are two of the most prevalent swine pathogens in China's swine herds, and their coinfection occurs commonly. Several PED and PRRS vaccines have been utilized in China for decades, and systemic homologous neutralizing antibodies (shnAbs) in serum are frequently used to evaluate the protective efficacy of PED and PRRS vaccines. To develop a vaccine candidate against G2 PEDV and HP-PRRSV2 coinfection, in this study, we generated a chimeric virus (rJSTZ1712-12-S) expressing S protein of G2 PEDV using an avirulent HP-PRRSV2 rJSTZ1712-12 infectious clone as the viral vector. The rJSTZ1712-12-S strain has similar replication efficacies as the parental rJSTZ1712-12 virus. In addition, animal inoculation indicated that rJSTZ1712-12-S is not pathogenic to piglets and can induce shnAbs against both G2 PEDV and HP-PRRSV2 isolates after prime-boost immunization. However, passive transfer study in neonatal piglets deprived of sow colostrum showed that rJSTZ1712-12-S-induced shnAbs may only decrease PEDV and PRRSV viremia but cannot confer sufficient protection against dual challenge of high virulent G2 PEDV XJ1904-34 strain and HP-PRRSV2 XJ17-5 isolate. Overall, this study provides the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for the evaluation of protective efficacy of PED and PRRS bivalent vaccine (especially for the PED vaccine). IMPORTANCE Porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) coinfection occurs commonly and can synergistically reduce feed intake and pig growth. Vaccination is an effective strategy utilized for PED and PRRS control, and systemic homologous neutralizing antibodies (shnAbs) in serum are commonly used for protective efficacy evaluation of PED and PRRS vaccines. Currently, no commercial vaccine is available against PEDV and PRRSV coinfection. This study generated a chimeric vaccine candidate against the coinfection of prevalent PEDV and PRRSV in China. The chimeric strain can induce satisfied shnAbs against both PEDV and PRRSV after prime-boost inoculation in pigs. But the shnAbs cannot confer sufficient protection against PEDV and PRRSV coinfection in neonatal piglets. To the best of our knowledge, these findings provide the first evidence that shnAbs confer insufficient protection against PEDV and PRRSV coinfection and are inadequate for evaluating PED and PRRS bivalent vaccine protective efficacy.

Screening of Porcine Innate Immune Adaptor Signaling Revealed Several Anti-PRRSV Signaling Pathways.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The current strategies for controlling PRRSV are limited and complete understanding of anti-PRRSV innate immunity is needed. Here, we utilized nine porcine innate immune signaling adaptors which represent all currently known innate immune receptor signaling pathways for screening of anti-PRRSV activity. The analysis of PRRSV N gene transcription and protein expression both suggested that the multiple ectopic adaptors exhibited varying degrees of anti-PRRSV activities, with TRIF and MAVS most effective. To better quantify the PRRSV replication, the GFP signal of PRRSV from reverse genetics were measured by flow cytometry and similarly varying anti-PRRSV activities by different signaling adaptors were observed. Based on the screening data, and considering the importance of viral nucleic acid in innate immune response, endogenous TRIF, MAVS and STING were selected for further examination of anti-PRRSV activity. Agonist stimulation assay showed that MAVS and STING signaling possessed significant anti-PRRSV activities, whereas siRNA knockdown assay showed that TRIF, MAVS and STING are all involved in anti-PRRSV activity, with TLR3-TRIF displaying discrepancy in anti-PRRSV infection. Nevertheless, our work suggests that multiple pattern recognition receptor (PRR) signaling pathways are involved in anti-PRRSV innate immunity, which may have implications for the development of future antiviral strategies.

The infectious cDNA clone of commercial HP-PRRS JXA1-R-attenuated vaccine can be a potential effective live vaccine vector.

Multiple commercial porcine reproductive and respiratory syndrome (PRRS) modified live vaccines are currently utilized in Chinese swine herds due to the limited cross-protection of vaccines and coexistence of different PRRS viruses. In this study, an infectious cDNA clone of the highly pathogenic PRRS (HP-PRRS) vaccine JXA1-R strain was generated. We successfully rescued the virus from direct in vitro DNA transfection of rJXA1-R clone, which has similar growth kinetics to the parental JXA1-R virus in Marc-145 cells. To further evaluate the potential use of the cloned rJXA1-R virus as a live vector for foreign gene expression, the enhanced green fluorescent protein (EGFP) was inserted between non-structural and structural genes. Our results showed that the dynamic expression of EGFP can be visualized by live cell imaging system during the infection in Marc-145 cells. The availability of our cloned JXA1-R viruses provides a crucial platform to study the fundamental biology of HP-PRRS virus vaccine and also serves as a potential effective vector for developing live vector vaccines against swine pathogens.

High genetic diversity of Chinese porcine reproductive and respiratory syndrome viruses from 2016 to 2019.

High genetic diversity and limited cross-protection are two major reasons for ineffective control of porcine reproductive and respiratory syndrome virus (PRRSV) infection. Therefore, it's important to dynamically monitor the prevalence of PRRSV for adopting appropriate control strategy. In this study, we analyzed PRRSV infection by detecting 712 clinical samples collected from 2016 to 2019 in China. Totally 100 samples were detected as PRRSV positive, including 2 and 98 samples were infected with PRRSV1 and PRRSV2, respectively. In addition, two out of the 98 PRRSV2 positive samples were co-infected with two distinct viruses. ORF5-based phylogenetic analysis showed that JXA1-like HP-PRRSV2 (lineage 8) and NADC30-like PRRSV2 (lineage 1) isolates are currently predominant, but QYYZ-like PRRSV2, CH-1a-like PRRSV2 and PRRSV1 isolates also co-exist in Chinese swine herds. In addition, two commercial MLV-derived viruses (TJM-F92-like and JXA1-R-like) were frequently detected. GP5 alignment also detected insertion and deletion in the extravirion domain. Our study presents the up-to-date PRRSV infection status and highlights the high genetic diversity of PRRSV currently circulating in China.

Identification of Two Porcine Reproductive and Respiratory Syndrome Virus Variants Sharing High Genomic Homology but with Distinct Virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to the global swine industry. Even though several control strategies have been applied, PRRS is still not effectively controlled due to the continuous emergence of new variants and limited cross-protection by current vaccines. During the routine epidemiological investigation in 2017, two PRRSV variants were identified from a severe abortion farm and a clinically healthy farm, respectively. The viruses were isolated and denominated as XJ17-5 and JSTZ1712-12. Genomic sequencing indicated that their genomes are both 14,960 bp in length sharing 99.45% nucleotide identity. Sequence alignments identified a discontinuous 30-amino-acid deletion and a continuous 120-amino-acid deletion in nsp2 of both isolates. Genome-based phylogenetic analysis confirmed that XJ17-5 and JSTZ1712-12 belong to the HP-PRRSV subtype but form a new branch with other isolates containing the same 150-amino-acid deletion in nsp2. Pathogenic analysis showed that XJ17-5 is highly virulent causing 60% mortality, while JSTZ1712-12 is avirulent for piglets. Furthermore, fragment comparisons identified 34-amino-acid differences between XJ17-5 and JSTZ1712-12 that might be associated with the distinct virulence. The identification of highly homologous HP-PRRSV variants with new genetic feature and distinct virulence contributes to further analyze the pathogenesis and evolution of PRRSV in the field.

Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA-PRRSV2), highly pathogenic (HP-PRRSV2) and NADC30-like (NL-PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real-time RT-PCR assays using TaqMan-MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real-time RT-PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV-positive, including 2 samples that were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay. ORF5 sequencing confirmed the real-time RT-PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA-PRRSV2, NL-PRRSV2 and HP-PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.

A novel NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) plays a limited role in the pathogenicity of porcine circoviruses (PCV2 and PCV3) and PRRSV co-infection.

Co-infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co-infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17-36) collected from a diseased pig was co-infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17-36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30-like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17-36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30-like PRRSV SD17-36 isolate is low pathogenic. Our results indicate that the co-infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co-infection.

Co-infection status of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018.

Classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) are economically important swine viruses that cause reproductive failure and/or respiratory symptoms in pigs. However, the co-infection status of these viruses in Chinese swine herds is not well clarified. In this study, we evaluated the co-infection of these four viruses in 159 pigs collected from 63 herds in eight regions of China from 2016 to 2018. CSFV, PRRSV, PCV2 and PCV3 were detected in 14, 56, 43 and 4 of the pigs, respectively. The percentage of singular infections was 32.71%, while the percentages of dual infections and multiple infections were 15.72% and 3.15%, respectively. The E2 of CSFV, ORF5 of PRRSV, ORF2s of PCV2 and PCV3 from all positive samples were determined and used for phylogenetic analyses. E2-based phylogenetic tree showed that all 14 CSFVs identified in this study belong to 2.1b subtype. ORF5-based phylogenetic tree showed that PRRSV2 is predominant in China while PRRSV1 can also be detected. In addition, 35, 16, 4 and 1 of our PRRSVs are clustered with highly pathogenic PRRSV2, NADC30-like PRRSV2, classical PRRSV2 and PRRSV1, respectively. ORF2-based phylogenetic trees showed that our PCVs are grouped with 2 PCV2 subtypes (PCV2d and PCV2b) and 3 PCV3 subtypes (PCV3a, PCV3b and PCV3c), respectively. Our results provide the latest co-infection status and the diversity of four important swine viruses in Chinese swine herds, which is beneficial for understanding the epidemiology of these viruses.

Emergence of a novel highly pathogenic recombinant virus from three lineages of porcine reproductive and respiratory syndrome virus 2 in China 2017.

A novel porcine reproductive and respiratory syndrome virus 2 (PRRSV2) was isolated from diseased piglets in Shandong, China in 2017 and denominated as SD17-38. ORF5 sequencing showed that SD17-38 contains a unique serine/asparagine deletion at position 33 and an asparagine insertion at position 60 of GP5, which has never been described. The SD17-38 complete genome was then determined, and genome-based phylogenetic analysis showed that SD17-38 is clustered with NADC30-like isolates. Sequence alignment and recombination analyses by RDP4 and SimPlot all indicated that SD17-38 is a recombinant virus from NADC30 (lineage 1), BJ-4 (lineage 5) and TJ (lineage 8) isolates. Animal challenge study in 4-week piglets showed that SD17-38 causes high fever (≥41°C), 100% morbidity and 40% mortality. In addition, significantly lower weight gain and severe histopathological lung lesions could be observed in SD17-38-infected pigs. In particular, the unique deletion and insertion in GP5 were stable during the challenge study. This study provides direct evidence for the natural occurrence of recombination events among three lineages of PRRSV2 in Chinese swine herds, resulting in the emergence of novel PRRSV variant with unique genetic property and high pathogenicity.

Two novel porcine epidemic diarrhea virus (PEDV) recombinants from a natural recombinant and distinct subtypes of PEDV variants.

Porcine epidemic diarrhea virus (PEDV) causes devastating impact on global pig-breeding industry and current vaccines have become not effective against the circulating PEDV variants since 2011. During the up-to-date investigation of PEDV prevalence in Fujian China 2016, PEDV was identified in vaccinated pig farms suffering severe diarrhea while other common diarrhea-associated pathogens were not detected. Complete genomes of two PEDV representatives (XM1-2 and XM2-4) were determined. Genomic comparison showed that these two viruses share the highest nucleotide identities (99.10% and 98.79%) with the 2011 ZMDZY strain, but only 96.65% and 96.50% nucleotide identities with the attenuated CV777 strain. Amino acid alignment of spike (S) proteins indicated that they have the similar mutation, insertion and deletion pattern as other Chinese PEDV variants but also contain several unique substitutions. Phylogenetic analysis showed that 2016 PEDV variants belong to the cluster of recombination strains but form a new branch. Recombination detection suggested that both XM1-2 and XM2-4 are inter-subgroup recombinants with breakpoints within ORF1b. Remarkably, the natural recombinant HNQX-3 isolate serves as a parental virus for both natural recombinants identified in this study. This up-to-date investigation provides the direct evidence that natural recombinants may serve as parental viruses to generate recombined PEDV progenies that are probably associated with the vaccination failure.