Erratum: MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene : Erratum.

[This corrects the article DOI: 10.7150/ijbs.25106.].

Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II.

IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.

Glycyrrhizic Acid-Based Carbonized Dots Boost Antiviral Activity against Influenza A Virus via Multisite Inhibition Mechanisms.

Influenza A virus (IVA) has been continuously causing pandemics in several animal hosts and has become a worldwide public health threat. Currently, antiviral drugs have become associated with a lot of side effects and limited activity against emerging drug-resistant influenza viruses. Therefore, the development of novel antiviral drugs is of great importance. In this study, we synthesized a kind of carbon dots (CDs) with high dispersibility from glycyrrhizic acid (GA) using a simple dry heating method. Compared with glycyrrhizic acid alone, GA-CDs exhibit superior solubility and significantly improve the antiviral property against IVA. Investigation of the mechanism revealed that GA-CDs act against IVA mainly by inhibiting viral internalization, replication of the viral genome, neuraminidase activity, and host inflammatory responses. More importantly, in a mouse model, GA-CDs can significantly alleviate the clinical symptoms and decrease mortality and lung viral titers. In vitro and in vivo experiments demonstrate that GA-CDs possess extraordinary therapeutic effects; therefore, we propose that GA-CDs may be a promising alternative therapy for IVA infection.

In vitro and in vivo effects of 3-indoleacetonitrile-A potential new broad-spectrum therapeutic agent for SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has resulted in significant global morbidity, mortality, and societal disruption. Currently, effective antiviral drugs for the treatment of SARS-CoV-2 infection are limited. Therefore, safe and effective antiviral drugs to combat COVID-19 are urgently required. In previous studies, we showed that 3-indoleacetonitrile, a plant growth hormone produced by cruciferous (Brassica) vegetables, is effective in treating influenza A virus infection. However, the molecular mechanisms underlying these effects remain unclear. Herein, we demonstrated that 3-indoleacetonitrile exhibits broad-spectrum antiviral activity and is effective against HSV-1 and VSV infections in vitro. This phenomenon prompted us to study its role in the anti-SARS-CoV-2 process. Interestingly, 3-indoleacetonitrile exhibited antiviral activity against SARS-CoV-2 in vitro. Importantly, tail vein injection of 3-indoleacetonitrile resulted in good antiviral activity in mouse models infected with WBP-1 (a mouse adaptation of the SARS-CoV-2 strain). Mechanistically, 3-indoleacetonitrile promoted the host interferon signalling pathway response and inhibited autophagic flux. Furthermore, we demonstrated that 3-indoleacetonitrile induced an increase in mitochondrial antiviral-signalling (MAVS) protein levels, which might be attributed to its inhibition of the interaction between MAVS and the selective autophagy receptor SQSTM1. Overall, our results demonstrate that 3-indoleacetonitrile is potently active against SARS-CoV-2 in vitro and in vivo, which may provide a foundation for further clinical testing for the treatment of COVID-19. In addition, considering its broad-spectrum antiviral effect, it should be explored whether it also has an effect on other viruses that threaten human health.

Infection Characteristics and Transcriptomics of African Swine Fever Virus in Bama Minipigs.

Animal experiments on African swine fever virus (ASFV) are vital to the study of ASFV; however, ASFV can only infect pigs, and animal experiments need to be performed in animal biosafety level 3 (ABSL-3) laboratories, meaning that many small ABSL-3 laboratories are unable to carry out in vivo ASFV experiments. Therefore, miniaturized experimental animals for ASFV infection are urgently needed. Here, we successfully isolated genotype II of ASFV SY-1 from wild boars and evaluated ASFV-infected Bama minipigs in a negative-pressure isolator of a small ABSL-3 laboratory. The pathological changes of ASFV-infected Bama minipigs were consistent with characteristic lesions of ASFV-infected domestic pigs and wild boars. All pigs died 5 to 14 days postinfection (dpi) through intramuscular injection. Viral genomic DNA from nasal, oral, and rectal swab samples was first detectable at 2 to 4 dpi. The common differentially expressed genes were clustered in the immune-related, metabolic, and inflammatory response pathways from the spleen and inguinal lymph node samples comparing infected to mock. In summary, these results demonstrated that the Bama minipig was an appropriate model for ASFV infection in small ABSL-3 laboratories that can accelerate the research of vaccines and antiviral drugs and uncover pathogenic mechanisms of ASFV infection. IMPORTANCE African swine fever virus (ASFV) can only infect pigs rather than other animals. However, the domestic pigs cannot be kept in small ABSL-3 laboratories for a long time due to the characteristics of rapid growth and large size, which hinder ASFV research, including research of vaccines, antiviral drugs, and mechanisms. In contrast, Bama minipigs have unique advantages consisting of low growth and small size. In the research, Bama minipigs were used to evaluate the characteristics of ASFV infection in small ABSL-3 laboratories. The pathological changes, viral shedding, and gene regulation were consistent with those of domestic pigs infected with ASFV. Therefore, Bama minipigs can be a suitable model for ASFV infection in small ABSL-3 laboratories.

African swine fever virus infection activates inflammatory responses through downregulation of the anti-inflammatory molecule C1QTNF3.

African swine fever (ASF) is the most dangerous pig disease, and causes enormous economic losses in the global pig industry. However, the mechanisms of ASF virus (ASFV) infection remains largely unclear. Hence, this study investigated the host response mechanisms to ASFV infection. We analyzed the differentially expressed proteins (DEPs) between serum samples from ASFV-infected and uninfected pigs using quantitative proteomics. Setting the p-value < 0.05 and |log2 (fold change)| > 1.5, we identified 173 DEPs, comprising 57 upregulated and 116 downregulated proteins, which belonged to various biological processes and pathways based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The enriched pathways include immune responses, metabolism, and inflammation signaling pathways. Western blot analysis validated the DEPs identified using quantitative proteomics. Furthermore, our proteomics data showed that C1QTNF3 regulated the inflammatory signaling pathway. C1QTNF3 knockdown led to the upregulation of pro-inflammatory factors IL-1ß, IL-8, and IL-6, thus inhibiting ASFV replication. These results indicated that C1QTNF3 was critical for ASFV infection. In conclusion, this study revealed the molecular mechanisms underlying the host-ASFV interaction, which may contribute to the development of novel antiviral strategies against ASFV infection in the future.

One-Step Rapid and Sensitive ASFV p30 Antibody Detection via Nanoplasmonic Biosensors.

African swine fever (ASF) is one of the most serious transnational swine diseases in the world. The case fatality rate of susceptible pigs is up to 100%. Currently, no commercial vaccine is available, so the prevention and control of ASF mainly relies on early diagnosis and culling of infected pigs. As the ASF virus continues to evolve, develop, and diversify, nucleic acid testing becomes less efficient. Here, we developed a method for the rapid and direct optical measurement of African swine fever virus (ASFV) antibody in vitro. This one-step procedure requires nearly no sample preparation and involves p30 protein-specific label-free integration into standard 96-well plates. Using a nanoplasmonic biosensor with extraordinary optical transmission (EOT) effect, one-step sample addition, ASFV antibody was detected within 20 min. The positive antibody showed a satisfactory sensitivity and linear relationship in the dilution ratio of 1:100-1:16000. It was used for the detection of clinical serum samples with a coincidence rate of 96.6%. The measurement results can be automatically analyzed and displayed on a conventional microplate meter computer and connected device. Our detection method can be widely applied in point-of-care testing (POCT) of ASFV antibody in pig farms. IMPORTANCE African swine fever (ASF) is a serious transnational disease caused by the African swine fever virus (ASFV), which is highly contagious in wild boars and domestic pigs. There is currently no available vaccine for ASF; therefore, development efforts are a key priority as ASFV continues to evolve and diversify. The ASF antibody rapid detection platform comprising the nanoplasmonic biosensor with extraordinary optical transmission effect can greatly reduce the detection time and improve detection flux while maintaining detection sensitivity and specificity. The one-step sample addition can effectively avoid cross contamination of samples in the detection process. The detection method provides a solution for the rapid and accurate real-time monitoring of ASF in pig farms.

Flow cytometry-based multiplexing antibody detection for diagnosis of African swine fever virus.

African swine fever (ASF) is an infectious disease that has a mortality rate of nearly 100% in domestic pigs. To date, no vaccine or effective treatment for ASF is available, necessitating the development of an accurate and sensitive diagnostic method to monitor ASF virus (ASFV) antibodies for prevention and control. Herein, a reliable and sensitive suspension microarray technology-based multiplexing method was developed for ASFV antibody detection using recombinant CD2v, p30, p54, and p22 antigen protein coated size-encoded microbeads as probes to capture the target antibody. Compared to commercial ELISA kits, the newly developed method showed a 16-fold improvement in detection sensitivity. Differential diagnosis of CD2v-unpressed low-virulence mutant (genotype II) and wild-type ASFV (genotype II) was readily achieved by fluorescence signal analysis of the CD2v-coated probe in the microbead mixture solution. In addition, the real serum assay revealed a 97% consistency rate between the novel method and commercial ELISA kits, demonstrating excellent potential for ASF epidemic surveillance and control.

A serological survey of severe acute respiratory syndrome coronavirus 2 in dogs in Wuhan.

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis and economic losses. Although several cases of cats and dogs infected with SARS-CoV-2 have been reported during this outbreak, the prevalence of SARS-CoV-2 in dog and its transmission among other companion animals are still unknown. Here, we report an extensive serological study of SARS-CoV-2 infection in dogs in Wuhan and analyse the infection rates at different stages of the pandemic outbreak. A total of 946 dogs serum samples were collected from Wuhan, of which 36 samples were obtained prior to the pandemic outbreak. Indirect enzyme-linked immunosorbent assay (ELISA) showed that 16 sera collected during the outbreak were detected as positive through the receptor-binding domain (RBD) of SARS-CoV-2. Of these 16 sera, 10 exhibited measurable SARS-CoV-2-specific neutralizing antibodies whose titres ranged from 1/20 to 1/180. No serological cross-reactivity was detected between SARS-CoV-2 and canine coronavirus (CCV). Furthermore, with the effective control of the outbreak, a decrease in the SARS-CoV-2 seropositive dog number was observed. Our results suggest that SARS-CoV-2 has infected companion dogs during the outbreak, and that COVID-19 patient families have a higher risk of dog infection. Our findings deepen our understanding of the infection of SARS-CoV-2 in dogs and provide an important reference for prevention of COVID-19.

Characterization of Erysipelothrix rhusiopathiae Isolates from Diseased Pigs in 15 Chinese Provinces from 2012 to 2018.

Erysipelothrix rhusiopathiae can cause erysipelas in animals and erysipeloid in humans. Since its recurrence in 2012, swine erysipelas has caused serious losses within the pig industry in China. The aim of this study was to perform multilocus sequence typing and understand the virulence and antimicrobial susceptibility of E. rhusiopathiae isolates in China. Multilocus sequence typing (MLST) of a total of 120 strains was performed, and as a result, three different sequence types were identified, of which ST48 was the main one. Five isolates of each MLST type were randomly selected to be used to challenge mice. ST48 was associated with a higher virulence. Antimicrobial susceptibility was tested using a microdilution technique and, to analyze the resistance mechanism, six strains were selected for genome sequencing. A comparison of the six genomes indicated the presence of a suspected macrolide resistance gene, namely, Erm(A)-like, in erythromycin-resistant strains, which increased the minimum inhibitory concentration (MIC) of erythromycin against E. coli C600 at least four-fold. In addition, three mutations (gyrA86T-I, gyrA90D-N, and parC81S-I) were observed in the quinolone resistance-determining regions (QRDRs) of gyrA and parC in quinolone-resistant strains. After the gyrA gene with the 86T-I mutation or the parC gene with the 81S-I mutation was transfected into E. coli C600, the MIC of enrofloxacin against this strain increased at least two-fold. Our findings provide a theoretical basis for developing antibacterial drugs and may contribute to the clinical prevention and control of E. rhusiopathiae.

Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV.

African swine fever virus (ASFV) is an important viral pathogen infecting pigs worldwide throughout the pig industry. CD2v (an outer-membrane glycosylated protein of ASFV)-unexpressed lower-virulence mutants have appeared in China and other countries in recent years. Using OIE-recommended quantitative PCR and ELISA methods, people can accurately judge whether pigs are infected with wild-type ASFV. However, the strategy has failed to distinguish ΔCD2v lower-virulence mutants and wild-type ASFV infection. Here, we expressed and purified the CD2v and p30 proteins via CHO cells and successfully established a dual enzyme-linked immunosorbent assay (ELISA), which can be used to differentiate pigs infected with wild-type ASFV or with CD2v-unexpressed lower-virulence mutants. The dual ELISA showed excellent specificity without cross-reactions with antibodies of PRRSV, CSFV, JEV, PRV, or PPV. The dual ELISA could detect ASFV-infected positive serum samples up to dilutions of 5120 times, possessing high sensitivity. Therefore, the application of this dual ELISA approach can play an important role in ASFV epidemiology study and fill the gaps in differential diagnosis.

Development of a chemiluminescence immunoassay to accurately detect African swine fever virus antibodies in serum.

The outbreak of African swine fever (ASF) has caused significant economic losses to animal husbandry worldwide. Currently, there is no effective vaccine or treatment available to control the disease, and therefore, efficient disease control is dependent on early detection and diagnosis of ASF virus (ASFV). In this study, a chemiluminescent immunoassay (CLIA) was developed using the ASFV protein p54 as a serum diagnostic antigen and an anti-p54 monoclonal antibody. After optimizing the working parameters of the CLIA, the sensitivity of the established CLIA was 1:128, ASFV-specific serum antibody was identified, and there was no cross-reaction with other swine virus antibodies. After testing 49 clinical serum samples, the consistency rate between the CLIA and the World Organization for Animal Health (OIE) recommended commercial kit was 100 %. Thus, this CLIA had a high degree of specificity, sensitivity, and reliability, and could be used as a rapid detection method for epidemiological investigations of ASFV infection.

SARS-CoV-2 rapidly adapts in aged BALB/c mice and induces typical pneumonia.

Age is a risk factor for coronavirus disease 2019 (COVID-19) associated morbidity and mortality in humans; hence, in this study, we compared the course of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in young and aged BALB/c mice. We found that SARS-CoV-2 isolates replicated in the respiratory tracts of 12-month-old (aged) mice and caused pathological features of pneumonia upon intranasal infection. In contrast, rapid viral clearance was observed 5 days following infection in 2-month-old (young) mice with no evidence of pathological changes in the lungs. Infection with SARS-CoV-2 elicited significantly upregulated production of cytokines, especially interleukin 6 and interferon gamma, in aged mice; whereas this response was much weaker in young mice. Subsequent challenge of infected aged BALB/c mice with SARS-CoV-2 resulted in neutralized antibody responses, a significantly reduced viral burden in the lungs, and inflammation mitigation. Deep sequencing showed a panel of mutations potentially associated with the enhanced infection in aged BALB/c mice, such as the Q498H mutations which are located at the receptor binding domain (RBD) of the spike (S) protein. We further found that the isolates can not only multiply in the respiratory tract of mice but also cause disease in aged mice. Overall, viral replication and rapid adaption in aged BALB/c mice were associated with pneumonia, confirming that the age-related susceptibility to SARS-CoV-2 in mice resembled that in humans.ImportanceAged BALB/c model are in use as a model of disease caused by SARS-CoV-2. Our research demonstrated SARS-CoV-2 can rapidly adapt in aged BALB/c mice through causing mutations at the RBD of the S protein. Moreover, SARS-CoV-2-infected aged BALB/c mice indicated that alveolar damage, interstitial pneumonia, and inflammatory immune responses were similar to the clinical manifestations of human infections. Therefore, our aged BALB/c challenge model will be useful for further understanding the pathogenesis of SARS-CoV-2 and for testing vaccines and antiviral agents.

C1QBP inhibits proliferation of porcine circovirus type 2 by restricting nuclear import of the capsid protein.

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.

Ivermectin treatment inhibits the replication of Porcine circovirus 2 (PCV2) in vitro and mitigates the impact of viral infection in piglets.

Porcine circovirus 2 (PCV2) capsid protein (Cap) has a nuclear localization signal (NLS) and can enter the nucleus. In this study, ivermectin, a small-molecule nuclear import inhibitor of proteins was used to determine the role of nuclear localization of Cap on PCV2 replication. Observation by fluorescence microscopy of the intracellular localization of Cap and Cap NLS in cells cultured with ivermectin (50 µg/mL) determined that Cap and Cap NLS were located in the cytoplasm; in contrast, for cells cultured without ivermectin, they accumulated in the cell nucleus. Ivermectin treatment also reduced nuclear transport of Cap derived from PCV2 infection as well as PCV2 replication in PK-15 cells. In addition, lower levels of PCV2 in tissues and sera of piglets treated with ivermectin were detected by qPCR. These results established for the first time that ivermectin has potent antiviral activity towards PCV2 both in vitro and vivo.

MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene.

MicroRNAs regulate post-transcriptional gene expression via either translational repression or mRNA degradation. They have important roles in both viral infection and host anti-infection processes. We discovered that the miR-375 is significantly upregulated in Newcastle disease virus (NDV)-infected chicken embryonic visceral tissues using a small RNA sequencing approach. Further research revealed that the overexpression of miR-375 markedly decreases the replication of the velogenic NDV F48E9 and the lentogenic NDV La Sota by targeting the M gene of NDV in DF-1 cells. Interestingly, miR-375 has another target, ELAVL4, which regulates chicken fibrocyte cell cycle progression and decreases NDV proliferation. In addition, miR-375 can influence bystander cells by its secretion in culture medium. Our results indicated that miR-375 is an inhibitor of NDV, but can also enhance NDV growth by reducing the expression of its target ELAVL4. These results emphasize the complex roles of microRNAs in the regulation of viral infections.

Ivermectin inhibits DNA polymerase UL42 of pseudorabies virus entrance into the nucleus and proliferation of the virus in vitro and vivo.

Pseudorabies virus (PRV) is an important viral pathogen of pigs that causes huge losses in pig herds worldwide. Ivermectin is a specific inhibitor of importin-α/ß-dependent nuclear transport and shows antiviral potential against several RNA viruses by blocking the nuclear localization of viral proteins. Since the replication of DNA viruses is in the nucleus, ivermectin may be functional against DNA virus infections if the DNA polymerase or other important viral proteins enter the nucleus via the importin-α/ß-mediated pathway. Here, we determined whether ivermectin suppresses PRV replication in hamster kidney BHK-21 cells and investigated the effect of ivermectin on the subcellular localization of the PRV UL42 protein, the accessory subunit of PRV DNA polymerase. Also, an in vivo anti-PRV assay was conducted in mice. Our data demonstrate that ivermectin treatment inhibits PRV infection in cells in a dose-dependent manner. Treatment of PRV-infected cells with ivermectin significantly suppressed viral DNA synthesis and progeny virus production. Ivermectin disrupted the nuclear localization of UL42 by targeting the nuclear localization signal of the protein in transfected cells. Ivermectin treatment increased the survival rates of mice infected with PRV and relieved infection as indicated by lower clinical scores and fewer gross lesions in the brain. Together, our results suggest that ivermectin may be a therapeutic or preventative agent against PRV infection.

Common microRNA⁻mRNA Interactions in Different Newcastle Disease Virus-Infected Chicken Embryonic Visceral Tissues.

To investigate the roles and explore the altered expression of microRNAs (miRNAs) and mRNAs in chicken embryos in response to Newcastle disease virus (NDV) infection, deep sequencing was performed. Then, a conjoint analysis of small RNA-seq and mRNA-seq was performed to screen interactional miRNA⁻mRNA pairs during NDV infection. In total, 15 and 17 up- and downregulated miRNAs were identified that potentially targeted 4279 and 6080 mRNAs in NDV-infected chicken embryonic tissues, respectively; in addition, 595 upregulated and 480 downregulated mRNAs were identified. The conjoint analysis of the obtained data identified 1069 miRNA⁻mRNA pairs. Among these pairs, 130 pairs were related to immune or inflammatory responses. The relationship between gga-miR-203a and its target transglutaminase 2 (TGM2) was confirmed using a dual-luciferase reporter system and a real time quantitative polymerase chain reaction (RT-qPCR) assay. Overall, the discovery of miRNAs, mRNAs, and their potential pairing relationships, which may be involved in the regulation of NDV infection, will facilitate our understanding of the complex regulatory relationship between the host and the virus.