Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a.

Largemouth bass ranavirus (LMBV) is an epidemic disease that seriously jeopardizes the culture of largemouth bass（Micropterus salmoides）, and it has a very high incidence in largemouth bass. Once an outbreak occurs, it may directly lead to the failure of the culture, resulting in substantial economic losses, but there is no effective vaccine or special effective drug yet. Consequently, it is important to establish an accurate, sensitive, convenient and specific detection approach for preventing LMBV infection. The recombinant enzyme-assisted amplification (RAA) technology was used in combination with clustered regularly interspaced short palindromic repeats (CRISPR), and associated protein 13a (CRISPR/Cas13a) to detect LMBV. We designed RAA primers and CRISPR RNA (crRNA) that targeted the conserved region in the LMBV main capsid protein (MCP) gene, amplified sample nucleic acids using the RAA technology, performed CRISPR/Cas13a fluorescence detection and evaluated the sensitivity and specificity of the established method with qPCR as a control method. This technique was able to determine the results by collecting fluorescence signals, visualizing fluorescence by UV excitation and combining with lateral flow strips (LFS). The sensitivity and specificity of the established method were consistent with the qPCR method. Besides, it was performed at a constant temperature of 37 °C and the sensitivity of the reaction system was 3.1 × 101 copies/µL, with no cross-reactivity with other common aquatic pathogens. Further, the positive detection rate of the proposed method in 32 clinical samples was consistent with that of qPCR. In conclusion, our established RAA-CRISPR/Cas13 method for detecting LMBV is sensitive, simple and specific, which is applicable in the rapid on-site detection and epidemiological monitoring of LMBV.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.

Multiple Roles of TRIM21 in Virus Infection.

The tripartite motif protein 21 (TRIM21) belongs to the TRIM family, possessing an E3 ubiquitin ligase activity. Similar to other TRIMs, TRIM21 also contains three domains (named RBCC), including the Really Interesting New Gene (RING) domain, one or two B-Box domains (B-Box), and one PRY/SPRY domain. Notably, we found that the RING and B-Box domains are relatively more conservative than the PRY/SPRY domain, suggesting that TRIM21 of different species had similar functions. Recent results showed that TRIM21 participates in virus infection by directly interacting with viral proteins or modulating immune and inflammatory responses. TRIM21 also acts as a cytosol high-affinity antibody Fc receptor, binding to the antibody-virus complex and triggering an indirect antiviral antibody-dependent intracellular neutralization (ADIN). This paper focuses on the recent progress in the mechanism of TRIM21 during virus infection and the application prospects of TRIM21 on virus infection.

Exosome-Autophagy Crosstalk in Enveloped Virus Infection.

Exosomes, which are extracellular vesicles (EVs) predominantly present in bodily fluids, participate in various physiological processes. Autophagy, an intracellular degradation mechanism, eliminates proteins and damaged organelles by forming double-membrane autophagosomes. These autophagosomes subsequently merge with lysosomes for target degradation. The interaction between autophagy and endosomal/exosomal pathways can occur at different stages, exerting significant influences on normal physiology and human diseases. The interplay between exosomes and the autophagy pathway is intricate. Exosomes exhibit a cytoprotective role by inducing intracellular autophagy, while autophagy modulates the biogenesis and degradation of exosomes. Research indicates that exosomes and autophagy contribute to the infection process of numerous enveloped viruses. Enveloped viruses, comprising viral nucleic acid, proteins, or virions, can be encapsulated within exosomes and transferred between cells via exosomal transport. Consequently, exosomes play a crucial role in the infection of certain viral diseases. This review presents recent findings on the interplay between exosomes and autophagy, as well as their implications in the infection of enveloped viruses, thereby offering valuable insights into the pathogenesis and vaccine research of enveloped virus infection.

Non-structural proteins of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) belongs to the family Flaviviridae genus pestivirus. The viral genome is a single-stranded, positive-sense RNA that encodes four structural proteins (i.e., C, Erns, E1, and E2) and eight non-structural proteins (NSPs) (i.e., Npro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Cattle infected with BVDV exhibit a number of different clinical signs including diarrhea, abortion, and other reproductive disorders which have a serious impact on the cattle industry worldwide. Research on BVDV mainly focuses on its structural protein, however, progress in understanding the functions of the NSPs of BVDV has also been made in recent decades. The knowledge gained on the BVDV non-structural proteins is helpful to more fully understand the viral replication process and the molecular mechanism of viral persistent infection. This review focuses on the functions of BVDV NSPs and provides references for the identification of BVDV, the diagnosis and prevention of Bovine viral diarrhea mucosal disease (BVD-MD), and the development of vaccines.

PCV2 and PRV Coinfection Induces Endoplasmic Reticulum Stress via PERK-eIF2α-ATF4-CHOP and IRE1-XBP1-EDEM Pathways.

Porcine circovirus 2 (PCV2) and pseudorabies virus (PRV) are two important pathogens in the pig industry. PCV2 or PRV infection can induce endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). However, the effect of PCV2 and PRV coinfection on the ERS and UPR pathways remains unclear. In this study, we found that PRV inhibited the proliferation of PCV2 mainly at 36 to 72 hpi, while PCV2 enhanced the proliferation of PRV in the middle stage of the infection. Notably, PRV is the main factor during coinfection. The results of the transcriptomic analysis showed that coinfection with PCV2 and PRV activated cellular ERS, and upregulated expressions of the ERS pathway-related proteins, including GRP78, eIF2α, and ATF4. Further research indicated that PRV played a dominant role in the sequential infection and coinfection of PCV2 and PRV. PCV2 and PRV coinfection induced the ERS activation via the PERK-eIF2α-ATF4-CHOP axis and IRE1-XBP1-EDEM pathway, and thus may enhance cell apoptosis and exacerbate the diseases.

Coinfection of Porcine Circovirus 2 and Pseudorabies Virus Enhances Immunosuppression and Inflammation through NF-κB, JAK/STAT, MAPK, and NLRP3 Pathways.

Porcine circovirus 2 (PCV2) and pseudorabies virus (PRV) are economically important pathogens in swine. PCV2 and PRV coinfection can cause more severe neurological and respiratory symptoms and higher mortality of piglets. However, the exact mechanism involved in the coinfection of PRV and PCV2 and its pathogenesis remain unknown. Here, porcine kidney cells (PK-15) were infected with PCV2 and/or PRV, and then the activation of immune and inflammatory pathways was evaluated to clarify the influence of the coinfection on immune and inflammatory responses. We found that the coinfection of PCV2 and PRV can promote the activation of nuclear factor-κB (NF-κB), c-Jun N-terminal protein kinases (JNK), p38, and nod-like receptor protein 3 (NLRP3) pathways, thus enhancing the expression of interferon-γ (IFN-γ), interferon-λ1 (IFN-λ1), interferon-stimulated gene (ISG15), interleukin 6 (IL6), and interleukin 1ß (IL1ß). Meanwhile, PCV2 and PRV also inhibit the expression and signal transduction of IFN-ß, tumor necrosis factor α (TNFα), and the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway. In addition, PCV2 and PRV infection can also weaken extracellular-signal-regulated kinase (ERK) activity. These results indicate that the regulations of cellular antiviral immune responses and inflammatory responses mediated by NF-κB, JAK/STAT, mitogen-activated protein kinase (MAPK), and NLRP3 pathways, contribute to immune escape of PCV2 and PRV and host antiviral responses.

Porcine Circovirus Type 4 Strains Circulating in China Are Relatively Stable and Have Higher Homology with Mink Circovirus than Other Porcine Circovirus Types.

Porcine circovirus type 4 (PCV4) is a newly identified porcine circovirus (PCV) belonging to the Circovirus genus Circoviridae family. Although several groups have conducted epidemiological investigations on PCV4 and found that PCV4 also exists widely in pigs, there are few reports on the origin and evolution of PCV4. In this study, the genetic relationship between PCV4, mink circovirus (MiCV), bat circovirus (BtCV), PCV1, PCV2, and PCV3 was analyzed, and the consistency of viral proteins in three-dimensional (3D) structure and epitopes was predicted. We found that the genome and protein structure of PCV4 was relatively stable among current circulating PCV4 strains. Furthermore, PCV4 was more similar to MiCV in terms of its genome, protein structure, and epitope levels than other PCVs and BtCVs, suggesting that PCV4 may be derived from MiCV or have a common origin with MiCV, or mink may be an intermediate host of PCV4, which may pose a great threat to other animals and/or even human beings. Therefore, it is necessary to continuously monitor the infection and variation of PCV4, analyze the host spectrum of PCV4, and establish the prevention and treatment methods of PCV4 infection in advance.

Mutation and Interaction Analysis of the Glycoprotein D and L and Thymidine Kinase of Pseudorabies Virus.

Pseudorabies (also called Aujeszky's disease) is a highly infectious viral disease caused by the pseudorabies virus (PRV, or Suid herpesvirus 1). Although the disease has been controlled by immunization with the PRV-attenuated vaccine, the emerging PRV variants can escape the immune surveillance in the vaccinated pig, resulting in recent outbreaks. Furthermore, the virus has been detected in other animals and humans, indicating cross-transmission of PRV. However, the mechanism of PRV cross-species transmission needs further study. In this study, we compared the amino acid sequences of glycoproteins (gD), gL, and thymidine kinase (TK) of PRV strains, human PRV hSD-1 2019 strain, and the attenuated strain Bartha-K61, followed by predication of their spatial conformation. In addition, the interactions between the viral gD protein and host nectin-1, nectin-2, and HS were also evaluated via molecular docking. The results showed that the amino acid sequence homology of the gD, gL, and TK proteins of hSD-1 2019 and JL-CC was 97.5%, 94.4%, and 99.1%, respectively. Moreover, there were mutations in the amino acid sequences of gD, gL, and TK proteins of hSD-1 2019 and JL-CC compared with the corresponding reference sequences of the Bartha strain. The mutations of gD, gL, and TK might not affect the spatial conformation of the protein domain but may affect the recognition of antibodies and antigen epitopes. Moreover, the gD protein of JL-CC, isolated previously, can bind to human nectin-1, nectin-2, and HS, suggesting the virus may be highly infectious and pathogenic to human beings.

Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO.

BACKGROUND: UTP-glucose-1-phosphoryl transferase (UGPase) catalyzes the synthesis of UDP-glucose, which is essential for generating the glycogen needed for the synthesis of bacterial lipopolysaccharide (LPS) and capsular polysaccharide, which play important roles in bacterial virulence. However, the molecular function of UGPase in Brucella is still unknown. RESULTS: In this study, the ubiquitination modification of host immune-related protein in cells infected with UGPase-deleted or wild-type Brucella was analyzed using ubiquitination proteomics technology. The ubiquitination modification level and type of NF-κB Essential Modulator (NEMO or Ikbkg), a molecule necessary for NF-κB signal activation, was evaluated using Coimmunoprecipitation, Western blot, and dual-Luciferase Assay. We found 80 ubiquitin proteins were upregulated and 203 ubiquitin proteins were downregulated in cells infected with B. melitensis 16 M compared with those of B. melitensis UGPase-deleted strain (16 M-UGPase-). Moreover, the ubiquitin-modified proteins were mostly enriched in the categories of regulation of kinase/NF-κB signaling and response to a bacterium, suggesting Brucella UGPase inhibits ubiquitin modification of related proteins in the host NF-κB signaling pathway. Further analysis showed that the ubiquitination levels of NEMO K63 (K63-Ub) and Met1 (Met1-Ub) were significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M-infected cells, further confirming that the ubiquitination levels of NF-κB signaling-related proteins were regulated by the bacterial UGPase. Besides, the expression level of IκBα was decreased, but the level of p-P65 was significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M- and mock-infected cells, demonstrating that B. melitensis UGPase can significantly inhibit the degradation of IκBα and the phosphorylation of p65, and thus suppressing the NF-κB pathway. CONCLUSIONS: The results of this study showed that Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO, which will provide a new scientific basis for the study of immune mechanisms induced by Brucella.

The Role of Exosome and the ESCRT Pathway on Enveloped Virus Infection.

The endosomal sorting complex required for transport (ESCRT) system consists of peripheral membrane protein complexes ESCRT-0, -I, -II, -III VPS4-VTA1, and ALIX homodimer. This system plays an important role in the degradation of non-essential or dangerous plasma membrane proteins, the biogenesis of lysosomes and yeast vacuoles, the budding of most enveloped viruses, and promoting membrane shedding of cytokinesis. Recent results show that exosomes and the ESCRT pathway play important roles in virus infection. This review mainly focuses on the roles of exosomes and the ESCRT pathway in virus assembly, budding, and infection of enveloped viruses. The elaboration of the mechanism of exosomes and the ESCRT pathway in some enveloped viruses provides important implications for the further study of the infection mechanism of other enveloped viruses.

Immunogenicity evaluation of inactivated virus and purified proteins of porcine circovirus type 2 in mice.

BACKGROUND: Vaccination is considered as an effective and economical way to against PCV2 infection. However, some of commercial available vaccines are based on inactivated viruses, while the others are based on purified protein of PCV2. In the present study, we aimed to compare the immunogenicity of inactivated virus and purified proteins of porcine circovirus type 2 in mice. RESULTS: The results showed that positive antiserum titers were significantly increased after second, third and fourth immunization using inactivated PCV2 or purified proteins as coating antigen. Moreover, the inactivated PCV2 induced significantly higher levels of PCV2-specific antibodies than that of PCV2 subunit proteins. After PCV2 wild strain challenged, the average daily gain was comparable with that of mice in the mock group, and the sera from both inactivated PCV2-immunized animals and subunit protein Cap+ORF3 + Rep immunized animals had significantly higher neutralizing antibody titers than that of the PBS group. As expected, the neutralizing antibody in the inactivated PCV2 group was significantly higher than that of the subunit protein group. These results indicated that positive antiserum induced by the inactivated PCV2 had a better reactivity and specificity than that of the positive antiserum induced by the purified proteins. CONCLUSIONS: The results in the present study demonstrated inactivated PCV2 is more effective than PCV2 subunit proteins in stimulating immune response to against PCV2 infection.

Detection of Kobe-type and Otsu-type Babesia microti in wild rodents in China's Yunnan province.

Babesiosis is an emerging tick-transmitted zoonosis prevalent in large parts of the world. This study was designed to determine the rates of Babesia microti infection among small rodents in Yunnan province, where human cases of babesiosis have been reported. Currently, distribution of Babesia in its endemic regions is largely unknown. In this study, we cataloged 1672 small wild rodents, comprising 4 orders, from nine areas in western Yunnan province between 2009 and 2011. Babesia microti DNA was detected by polymerase chain reaction in 4·3% (72/1672) of the rodents analyzed. The most frequently infected rodent species included Apodemus chevrieri and Niviventer fulvescens. Rodents from forests and shrublands had significantly higher Babesia infection rates. Genetic comparisons revealed that Babesia was most similar to the Kobe- and Otsu-type strains identified in Japan. A variety of rodent species might be involved in the enzootic maintenance and transmission of B. microti, supporting the need for further serological investigations in humans.

Interactions of porcine circovirus 2 with its hosts.

Porcine circovirus 2 (PCV2) can cause porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are widely presented in swine-producing countries. Since the discovery of this virus, considerable efforts have been devoted to understanding this pathogen and its interactions with its host. Here, we review the current state of knowledge on interactions between host cell factors and PCV2 with respect to viral proliferation, virus-induced cell apoptosis and autophagy, and host antiviral defenses during PCV2 infection. We also review mouse model systems for PCV2 infection.

Effect of atovastatin treatment on porcine circovirus 2 infection in BALB/c mice.

The HMG-CoA reductase (HMGCR) pathway is an important metabolic route, which is not only present in almost every organism, but also involves virus infection. It has recently been shown that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells. The aim of this study was to determine whether inhibition of HMGCR by atovastatin would significantly affect Porcine circovirus type 2 (PCV2) infection and immunological reaction in BALB/c mice. The results showed atovastatin significantly stimulated PCV2 replication in vivo. Immunological reaction in atovastatin-treated mice was also significantly enhanced during PCV2 infection. Atovastatin also enhanced PCV2-induced illness in mice. The results of this study will provide new insight into the role of atovastatin in PCV2 infection.

HMG-CoA reductase is negatively associated with PCV2 infection and PCV2-induced apoptotic cell death.

We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication.

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase increases the expression of interferon-responsive genes.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) pathway is an important metabolic route that is present in almost every organism. However, whether HMGCR affects the expression of interferon (IFN)-responsive genes is unclear. In the present study, expression levels of IFN-responsive genes were monitored by real time polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells, indicating that inhibition of HMGCR activates the expression of IFN-responsive genes. The result in this study will provide new insight into the role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in antiviral research.

Complete genome sequence of porcine circovirus 2b strain CC1.

A porcine circovirus 2 (PCV2) strain, designated CC1, was isolated and purified from tissue samples from pigs with wasting syndromes in China. We report the complete genome sequence of PCV2b strain CC1 with a deletion of C at position 1053 resulting in elongation of open reading frame 2 (ORF2) and formation of ORF5. There were 11 ORFs in the genome.