A class of independently evolved transcriptional repressors in plant RNA viruses facilitates viral infection and vector feeding.

Plant viruses employ diverse virulence strategies to achieve successful infection, but there are few known general strategies of viral pathogenicity and transmission used by widely different plant viruses. Here, we report a class of independently evolved virulence factors in different plant RNA viruses which possess active transcriptional repressor activity. Rice viruses in the genera Fijivirus, Tenuivirus, and Cytorhabdovirus all have transcriptional repressors that interact in plants with the key components of jasmonic acid (JA) signaling, namely mediator subunit OsMED25, OsJAZ proteins, and OsMYC transcription factors. These transcriptional repressors can directly disassociate the OsMED25-OsMYC complex, inhibit the transcriptional activation of OsMYC, and then combine with OsJAZ proteins to cooperatively attenuate the JA pathway in a way that benefits viral infection. At the same time, these transcriptional repressors efficiently enhanced feeding by the virus insect vectors by repressing JA signaling. Our findings reveal a common strategy in unrelated plant viruses in which viral transcriptional repressors hijack and repress the JA pathway in favor of both viral pathogenicity and vector transmission.

Distinct modes of manipulation of rice auxin response factor OsARF17 by different plant RNA viruses for infection.

Plant auxin response factor (ARF) transcription factors are an important class of key transcriptional modulators in auxin signaling. Despite the well-studied roles of ARF transcription factors in plant growth and development, it is largely unknown whether, and how, ARF transcription factors may be involved in plant resistance to pathogens. We show here that two fijiviruses (double-stranded RNA viruses) utilize their proteins to disturb the dimerization of OsARF17 and repress its transcriptional activation ability, while a tenuivirus (negative-sense single-stranded RNA virus) directly interferes with the DNA binding activity of OsARF17. These interactions impair OsARF17-mediated antiviral defense. OsARF17 also confers resistance to a cytorhabdovirus and was directly targeted by one of the viral proteins. Thus, OsARF17 is the common target of several very different viruses. This suggests that OsARF17 plays a crucial role in plant defense against different types of plant viruses, and that these viruses use independently evolved viral proteins to target this key component of auxin signaling and facilitate infection.

A rice LRR receptor-like protein associates with its adaptor kinase OsSOBIR1 to mediate plant immunity against viral infection.

Plants sense pathogen attacks using a variety of receptors at the cell surface. The LRR receptor-like proteins (RLP) and receptor-like kinases (RLK) are widely reported to participate in plant defence against bacterial and fungal pathogen invasion. However, the role of RLP and RLK in plant antiviral defence has rarely been reported. We employed a high-throughput-sequencing approach, transgenic rice plants and viral inoculation assays to investigate the role of OsRLP1 and OsSOBIR1 proteins in rice immunity against virus infection. The transcript of a rice LRR receptor-like protein, OsRLP1, was markedly up-regulated following infection by RBSDV, a devastating pathogen of rice and maize. Viral inoculation on various OsRLP1 mutants demonstrated that OsRLP1 modulates rice resistance against RBSDV infection. It was also shown that OsRLP1 is involved in the RBSDV-induced defence response by positively regulating the activation of MAPKs and PTI-related gene expression. OsRLP1 interacted with a receptor-like kinase OsSOBIR1, which was shown to regulate the PTI response and rice antiviral defence. Our results offer a novel insight into how a virus-induced receptor-like protein and its adaptor kinase activate the PTI response and antiviral defence in rice.

Spexin inhibits excessive autophagy-induced ferroptosis to alleviate doxorubicin-induced cardiotoxicity by upregulating Beclin 1.

BACKGROUND AND PURPOSE: Doxorubicin is widely used in the treatment of malignant tumours, but doxorubicin-induced cardiotoxicity severely limits its clinical application. Spexin is a neuropeptide that acts as a novel biomarker in cardiovascular disease. However, the effects of spexin on doxorubicin-induced cardiotoxicity is unclear. EXPERIMENTAL APPROACH: We established a model of doxorubicin-induced cardiotoxicity both in vivo and in vitro. Levels of cardiac damage in mice was assessed through cardiac function assessment, determination of serum cardiac troponin T and CKMB levels and histological examination. CCK8 and PI staining were used to assess the doxorubicin-induced toxicity in cultures of cardiomyocytes in vitro. Ferroptosis was assessed using FerroOrange staining, determination of MDA and 4-HNE content and ferroptosis-associated proteins SLC7A11 and GPX4. Mitochondrial membrane potential and lipid peroxidation levels were measured using TMRE and C11-BODIPY 581/591 probes, respectively. Myocardial autophagy was assessed by expression of P62 and Beclin1. KEY RESULTS: Spexin treatment improved heart function of mice with doxorubicin-induced cardiotoxicity, and attenuated doxorubicin-induced cardiotoxicity by decreasing iron accumulation, abnormal lipid metabolism and inhibiting ferroptosis. Interestingly, doxorubicin caused excessive autophagy in cardiomyocyte in culture, which could be alleviated by treatment with spexin. Knockdown of Beclin 1 eliminated the protective effects of spexin in mice with DIC. CONCLUSION AND IMPLICATIONS: Spexin ameliorated doxorubicin-induced cardiotoxicity by inhibiting excessive autophagy-induced ferroptosis, suggesting that spexin could be a drug candidate against doxorubicin-induced cardiotoxicity. Beclin 1 might be critical in mediating the protective effect of spexin against doxorubicin-induced cardiotoxicity.

Development of a Duplex Insulated Isothermal PCR Assay for Rapid On-Site Detection and Differentiation of Genotypes 1 and 2 of African Swine Fever Virus.

Genotype II African swine fever virus (ASFV) has been plaguing Asian pig industry since 2018. Recently, genotype I ASFV was reported for the first time in China. Since there is no commercial vaccine available against ASFV, early onsite detection and quick culling procedures are commonly used by many countries all over the world. It is important that the above two genotypes of ASFV could be quickly differentiated during onsite detection at the same time. In this study, we established a sensitive and simple Fluorescent Probe Hydrolysis-Insulated isothermal PCR (iiPCR) that can detect and differentiate two genotypes of ASFV within 40 minutes. The positive or negative results of tested samples were displayed on the screen of the device automatically after PCR amplification was complete. The detection limit of the iiPCR was tested to be 20 copies for both genotype I and genotype II ASFVs. There was no cross-reactivity with other swine viruses by using the established iiPCR. Fifty-eight ASFV positive samples confirmed by National ASF Reference Laboratory were subjected to the established duplex iiPCR for genotype differentiation. The results showed that all these ASFV-positive samples belong to genotype II. At last, we found serum samples could be directly used as the templates for iiPCR without comprising sensitivity and specificity. Therefore, the duplex iiPCR established in study provide a useful tool for ASFV onsite detection and genotype differentiation.

Development and application of a duplex real-time PCR assay for differentiation of genotypes I and II African swine fever viruses.

Genotype II African swine fever virus (ASFV) has been plaguing Chinese pig industry and caused severe morbidity and mortality of pigs resulting in huge economic losses since its first report in August 2018. Most recently, two genotype I ASFVs with low virulence but efficient transmissibility in pigs were reported in China, which makes the diagnosis and control of this lethal disease more challenging. Therefore, it is prerequisite and important to differentiate genotype I from genotype II upon ASFV outbreaks before making any stringent control procedures. In this study, a duplex real-time PCR assay based on ASFV E296R gene was established which could simultaneously detect genotypes I and II ASFVs with two pairs of primers and two probes. Plasmid containing ASFV genes was used to test the sensitivity, repeatability, and reproducibility. DNA or cDNA samples of ASFV and other swine viruses were used to test the specificity. The results showed that the established duplex real-time PCR assay has satisfied specificity, sensitivity, repeatability, and reproducibility. In addition, the assay was applied to differentiate 84 ASFV positive clinical samples including lymph nodes, spleen, kidney, lung, liver, blood, nasal swab, and environmental swab samples which were sent to National ASF Reference Laboratory from April 2020 to September 2021. The results showed that all these ASFV positive samples belong to genotype II ASFV. The established duplex real-time PCR in this study provides a powerful tool for rapid detection and differentiation between genotypes I and II ASFVs and will facilitate efficient control of ASFV in China.

Same Dosages of rPRV/XJ5-gI-/gE-/TK- Prototype Vaccine or Bartha-K61 Vaccine Similarly Protects Growing Pigs Against Lethal Challenge of Emerging vPRV/XJ-5 Strain.

Variant pseudorabies viruses (vPRV) have constantly emerged in China since late 2011. In the present study, a 1 × 106.0 TCID50 per-animal dosage of a commercially available Bartha-K61 vaccine and an rPRV/XJ5-gI-/gE-/TK- prototype vaccine freshly extracted from the vPRV/XJ-5 at the same dose were administered to evaluate the immune effectiveness thereof on growing pigs to prevent lethal strikes caused by vPRV/XJ-5. The results suggest that the Bartha-K61 vaccine at a dose of 1 × 106.0 TCID50 per animal and the same dosage of the rPRV/XJ5-gI-/gE-/TK- prototype vaccine protected growing pigs against the lethal challenge of vPRV/XJ-5 strain with 100% survive rate. Furthermore, the outcome of the clinical score, virus shedding, weight gain, and viral loads in different pig tissues in these two groups demonstrates that either the Bartha-K61 vaccine or the rPRV/XJ5-gI-/gE-/TK- prototype vaccine at the same dose exhibited parallel efficacy in pigs against the lethal challenge with the XJ-5 strain of vPRV.

Genome-Wide Analysis of the RAV Transcription Factor Genes in Rice Reveals Their Response Patterns to Hormones and Virus Infection.

The RAV family is part of the B3 superfamily and is one of the most abundant transcription factor families in plants. Members have highly conserved B3 or AP2 DNA binding domains. Although the RAV family genes of several species have been systematically identified from genome-wide studies, there has been no comprehensive study to identify rice RAV family genes. Here, we identified 15 genes of the RAV family in the rice genome and analyzed their phylogenetic relationships, gene structure, conserved domains, and chromosomal distribution. Based on domain similarity and phylogenetic topology, rice RAV transcription factors were phylogenetically clustered into four groups. qRT-PCR analyses showed that expression of these RAV genes was significantly up-regulated or down-regulated by plant hormone treatments, including BL, NAA, IAA, MeJA, and SA. Most of the rice RAV genes were dramatically down-regulated in response to rice stripe virus (RSV) and mostly up-regulated in response to Southern rice black-streaked dwarf virus (SRBSDV). These results suggest that the rice RAV genes are involved in diverse signaling pathways and in varied responses to virus infection.

Development and application of an indirect ELISA for the detection of antibody to porcine circovirus 4 in pigs.

Porcine circovirus type 4 (PCV4) was first reported in 2019 in China. So far, the viral DNA was detected from both healthy and diseased pigs in China and South Korea by using molecular techniques including PCR and real-time PCR. In contrast, a serological survey regarding the presence of PCV4 antibodies in the pig population was seldomly reported. In the present study, we describe the development of an indirect enzyme-linked immunosorbent assay (ELISA) based on capsid protein for the detection of PCV4 antibodies in swine sera. After validating the specificity and sensitivity, the ELISA was used in a retrospective serological survey for PCV4 antibodies in pig sera from Jiangsu Province of China. Note that 3.44% of analyzed samples collected between 2018 and 2021 were tested positive for PCV4 antibodies. However, PCV4 genome was absent in all ELISA-positive serum samples. Therefore, the dynamic of viremia and antibody response to PCV4 infection in pigs warrant further investigation.

Colonisation of mice and pigs by a chimeric porcine circovirus 1-2 prototype vaccine strain and a PCV2 isolate originating in China and their induction of cytokines.

A chimeric porcine circovirus (PCV) 1-2b vaccine strain and its parental wild-type PCV2b strain from China (PCV2-J) were used separately to vaccinate BALB/c mice and tissue and serum samples were collected from the mice to investigate whether the replication properties of the viruses differed. The spleen lymphocytes from the infected mice were cultured in vitro; the amounts of interferon-γ-secreting cells (IFN-γ-SCs) and levels of interleukin (IL) 2, IL-4 and IL-10 in the culture fluids were monitored. The results showed that PCV1-2b induced higher levels of antibody production in the infected mice than the PCV2b-J isolate. Viremia declined gradually in both infection groups and the DNA copy numbers were nearly equal in both groups of mouse tissues tested. The IFN-γ-SC levels were clearly up-regulated in both the PCV1-2b- and PCV2b-J-infected mice. In both mouse groups, IL-2 was up-regulated, and IL-10 was detected at low levels, while IL-4 was always below the limit of detection. Similar experiments were performed in pigs and the results showed that when infected with either PCV1-2b or PCV2b-J the pigs experienced high-level antibody responses, with no significant differences between the infection groups. In the pig model, the development of IFN-γ-SCs in response to PCV1-2b and PCV2b-J infections was detected. However, the PCV1-2b strain tended to elicit more IFN-γ-SCs in the peripheral blood mononuclear cell population of the infected pigs from 21 to 28 days post infection than the PCV2b-J isolate did. The concentrations of IL-2 were transiently different between the PCV1-2b and PCV2b-J infected pigs, while those of IL-10 and IL-2 were similar in both groups, but were lower than those elicited in mice. These results indicated that BALB/c mouse could be used as an alternate model for evaluating the efficacy of attenuated PCV1-2b vaccines.

Characterization and evolution of the coronavirus porcine epidemic diarrhoea virus HLJBY isolated in China.

A strain of porcine epidemic diarrhoea virus (PEDV), namely HLJBY, was isolated in Heilongjiang province, China. To provide insight into the understanding of the phylogenetic and the current epidemiological status of PEDV, PEDV HLJBY was compared with CV777 and other PEDV strains deposited in the GenBank. The homology between the entire genomic nucleotide sequences of PEDV HLJBY and CV777 was 97.7%. The homology of M gene was the highest (99.0%). However, the homology of ORF3 gene was 97.7%, and protein of ORF3 was 90.1%. In addition, HLJBY showed the highest nucleotide identity (99.9%) with PEDV-SX/China/2017 strain and lowest similarity (91.2%) to PEDV/Belgorod/dom/2008 strain. We analysed the changes in S gene and its protein of PEDV HLJBY with 65 historic PEDV strains. The highest nucleotide identity was 99.9% compared with PEDV-SX/China/2017 strain, and the lowest nucleotide identity was 60.0% compared with PEDV/Belgorod/dom/2008 strain. The length of deduced amino acid sequences of S proteins varied from 1,372 to 1,390 amino acids (aa). Compared with most aa sequences of S proteins, HLJBY exhibited 5 aa deletions (position 55, 59-61, 144). Analysis and comparison of open reading frame 3 (ORF3) proteins between HLJBY strain and other PEDV strains were also focused in this study. We revealed that the length of deduced amino acid sequences of ORF3 proteins was 80-224 aa among tested strains and the identity of HLJBY ORF3 amino acids with other PEDV strains was 71.4%-98.9%. ORF3 protein of both HLJBY strain and PEDV-SX/China/2017 strain consists of 91 aa, with 133 aa deletions at their C' end in relation to the other tested PEDV strains. The phylogenetic tree based on different proteins or genes resulted in different phylogenetic groups. For pathogenicity evaluation of PEDV HLJBY strain, colostrum deprivation piglets were challenged with PEDV HLJBY, and PEDV reference strain CV777 as a control, the results showed that animals challenged with either of these PEDV strains developed diarrhoea, and histopathological examination of small intestines of challenged animals showed acute viral enteritis with villous atrophy in either PEDV HLJBY-P10 or PEDV CV777-P8 inoculated piglets.

Efficacy of the Bartha-K61 vaccine and a gE-/gI-/TK- prototype vaccine against variant porcine pseudorabies virus (vPRV) in piglets with sublethal challenge of vPRV.

Pseudorabies has caused huge economic losses in China's pig industry and recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Therefore, the prevention and control of this recurrence of pseudorabies in China has been given priority. In a previous study, we showed that a suitable dose [1 × 106.3 50% tissue culture infectious dose (TCID50) per animal] of commercial Bartha-K61 vaccine protects growing pigs against lethal challenge by the emerging vPRV strain XJ5. In this study, different doses of the Bartha-K61 vaccine and our newly developed rPRV-gI-/gE-/TK- prototype vaccine derived from the vPRV strain XJ5 were used to evaluate immune protection against sublethal challenge by the vPRV strain XJ5. Pigs vaccinated with high doses of the Bartha-K61 vaccine or rPRV-gI-/gE-/TK- prototype vaccine showed no differences in their humoral immune responses, clinical symptoms, body weight gains, viral shedding, or gross and histological lesions after sublethal challenge by the vPRV strain XJ5. Therefore, we concluded that the Bartha-K61 vaccine at a dose of 1 × 105 TCID50 per animal protects pigs against sublethal challenge by the vPRV strain XJ5 and performs equally well as the same dose of the rPRV-gI-/gE-/TK- vaccine, whereas lower doses of the Bartha-K61 vaccine alone do not protect pigs from this challenge. These findings provide useful information for vaccination interventions and the ultimate eradication of pseudorabies caused by vPRV strains emerging in China.

The Influence of Spraying Strategy on the Dynamic Response of Polyurea-Coated Metal Plates to Localized Air Blast Loading: Experimental Investigations.

Polyurea has attracted considerable attention owing to its potential applications in protective fields to improve the resistant performance of structures subjected to damage loads resulting from intentional or accidental explosions. However, different spraying strategies of polyurea may lead to significant differences in overall resistance performance of polyurea-coated structures, and the underlying mechanisms have not been clear until now. This study aims to elucidate the influence of spraying strategy, i.e., spraying area, spraying thickness, and spraying interface condition, on the dynamic response of polyurea-coated steel plates under localized air blast loading. Three types of plates manufactured using different spraying strategies were adopted to evaluate their blast-resistant performance. The spraying strategies used were (i) whole-area spraying, (ii) partial-area spraying, and (iii) in-contact backing of polyurea on the rear surfaces of steel plates. In addition, the influence of spraying thickness of polyurea for whole-area sprayed plates was evaluated. The energy absorbing mechanisms of polyurea backing layers were highlighted. The energy absorption of plates was quantitatively analyzed. The results show that the air blast resistances of whole-area sprayed and in-contact backed plates are both superior to, whereas that of partial-area sprayed plates is inferior to, bare steel counterparts. A suitable spraying thickness of polyurea can significantly reduce the damage of the front steel layer, whereas excessive spraying thickness decreases the overall air blast resistance of plates. The polyurea backing layer exhibits favorable performance in absorbing energy under a whole-area spraying condition. This study provides useful guidance for the design of polyurea-coated metal plates in engineering applications.

Detection of Avian H7N9 Influenza A Viruses in the Yangtze Delta Region of China During Early H7N9 Outbreaks.

Since the first H7N9 human case in Shanghai, February 19, 2013, the emerging avian-origin H7N9 influenza A virus has become an epizootic virus in China, posing a potential pandemic threat to public health. From April 2 to April 28, 2013, some 422 oral-pharyngeal and cloacal swabs were collected from birds and environmental surfaces at five live poultry markets (LPMs) and 13 backyard poultry farms (BPFs) across three cities, Wuxi, Suzhou, and Nanjing, in the Yangtze Delta region. In total 22 isolates were recovered, and six were subtyped as H7N9, nine as H9N2, four as H7N9/H9N2, and three unsubtyped influenza A viruses. Genomic sequences showed that the HA and NA genes of the H7N9 viruses were similar to those of the H7N9 human isolates, as well as other avian-origin H7N9 isolates in the region, but the PB1, PA, NP, and MP genes of the sequenced viruses were more diverse. Among the four H7N9/H9N2 mixed infections, three were from LPM, whereas the other one was from the ducks at one BPF, which were H7N9 negative in serologic analyses. A survey of the bird trading records of the LPMs and BPFs indicates that trading was a likely route for virus transmission across these regions. Our results suggested that better biosecurity and more effective vaccination should be implemented in backyard farms, in addition to biosecurity management in LPMs.