KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/µL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Development and application of a TaqMan-based real-time PCR method for the detection of the ASFV MGF505-7R gene.

African swine fever virus (ASFV), the etiological agent of African swine fever (ASF), causes deadly hemorrhagic fever in domestic pigs. ASF's high mortality and morbidity have had disastrous effects on the world's swine industry. In recent years, the number of African swine virus strains has increased and presented new challenges for detecting classical ASFV-p72-based viruses. In this study, we observed that the ASFV MGF505-7R gene, a member of the multigene family that can enhance ASFV virulence and pathogenesis, has the potential to be a candidate for vaccine formulations. We also developed a real-time PCR assay based on the ASFV MGF505-7R gene and validated it in multiple aspects. The results indicated that the approach could detect standard plasmids with a sensitivity and a specificity of up to 1 × 101 copies/µL. Moreover, the assay had no cross-reactions with other porcine viruses. In laboratory and clinical settings, the assay can detect ASFV-infected samples at an early stage (4 hpi) and show a consistency of 92.56% when compared with classical ASFV detection in clinically ASFV-infected materials. This study's results also indicated that the TaqMan-based quantitative real-time PCR assay we developed for detecting the ASFV MGF505-7R gene is both sensitive and specific. This assay can provide a quick and accurate method for detecting ASFV and has the potential to be used as an optional tool for screening and monitoring ASF outbreaks.

Summary of the Current Status of African Swine Fever Vaccine Development in China.

African swine fever (ASF) is a highly lethal and contagious disease of domestic pigs and wild boars. There is still no credible commercially available vaccine. The only existing one, issued in Vietnam, is actually used in limited quantities in limited areas, for large-scale clinical evaluation. ASF virus is a large complex virus, not inducing full neutralizing antibodies, with multiple genotypes and a lack of comprehensive research on virus infection and immunity. Since it was first reported in China in August 2018, ASF has spread rapidly across the country. To prevent, control, further purify and eradicate ASF, joint scientific and technological research on ASF vaccines has been carried out in China. In the past 4 years (2018-2022), several groups in China have been funded for the research and development of various types of ASF vaccines, achieving marked progress and reaching certain milestones. Here, we have provided a comprehensive and systematic summary of all of the relevant data regarding the current status of the development of ASF vaccines in China to provide a reference for further progress worldwide. At present, the further clinical application of the ASF vaccine still needs a lot of tests and research accumulation.

Attenuated African swine fever virus through serial passaging of viruses in cell culture: a brief review on the knowledge gathered during 60 years of research.

African swine fever virus (ASFV) is a highly pathogenic double-stranded DNA virus. It affects various breeds of pigs, causing serious economic losses and health threats because of its rapid spread and high pathogenicity and infectivity. This situation is not helped by the lack of a validated vaccine or effective therapies. Since the 1960s, different strains of ASFV have been subjected to serial passage in a variety of cell lines. The attenuated ASFV strains obtained through serial passage are not only candidates for ASF vaccine research, but also are useful to study the molecular genetic characteristics and pathogenic mechanism of the virus. This review summarizes related studies on the attenuated strains of ASFV acquired through cell passage over the last 60 years, with the aim of providing inspiration for the rational design of vaccines in future.

Systematic identification and characterization of repeat sequences in African swine fever virus genomes.

African swine fever virus (ASFV) is a large DNA virus that infects domestic pigs with high morbidity and mortality rates. Repeat sequences, which are DNA sequence elements that are repeated more than twice in the genome, play an important role in the ASFV genome. The majority of repeat sequences, however, have not been identified and characterized in a systematic manner. In this study, three types of repeat sequences, including microsatellites, minisatellites and short interspersed nuclear elements (SINEs), were identified in the ASFV genome, and their distribution, structure, function, and evolutionary history were investigated. Most repeat sequences were observed in noncoding regions and at the 5' end of the genome. Noncoding repeat sequences tended to form enhancers, whereas coding repeat sequences had a lower ratio of alpha-helix and beta-sheet and a higher ratio of loop structure and surface amino acids than nonrepeat sequences. In addition, the repeat sequences tended to encode penetrating and antimicrobial peptides. Further analysis of the evolution of repeat sequences revealed that the pan-repeat sequences presented an open state, showing the diversity of repeat sequences. Finally, CpG islands were observed to be negatively correlated with repeat sequence occurrences, suggesting that they may affect the generation of repeat sequences. Overall, this study emphasizes the importance of repeat sequences in ASFVs, and these results can aid in understanding the virus's function and evolution.

A systematic review of genotypes and serogroups of African swine fever virus.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes an acute highly hemorrhagic disease in domestic pigs, with high mortality. Although the overall genome mutation rate of ASFV, a large DNA virus, is relatively low, ASFV exhibits genetic and antigenic diversity. ASFV can be classified into 24 genotypes on the basis of the B646L gene. Cross-protected ASFV strains can be divided into eight serogroups on the basis of antibody-mediated hemadsorption inhibition. Here, we review research progress on ASFV genotyping and serogrouping, and explain how this information assists in the rapid identification of virus origin during ASF outbreaks and will aid in the development of ASF vaccines.

An extra insertion of tandem repeat sequence in African swine fever virus, China, 2019.

On 7 March 2019, African swine fever in a domestic pig farm was detected in Guangxi Province of China. The phylogenetic analysis showed that its causative strain contained two tandem repeat sequence insertions in the intergenic region between the I73R and the I329L genes, and was different from previously reported strains in China and other countries.

Molecular characterization of new emerging sub-genotype VIIh Newcastle disease viruses in China.

Newcastle disease (ND) has been enzootic in China for several decades since the first recognition of the disease in 1946 in China. Continuous surveillance revealed that the sub-genotype VIId Newcastle disease virus (NDV) has been predominantly responsible for most of ND outbreak in China in recent years. But in the present study, three virulent NDVs isolated from poultry in southern China were classified as sub-genotype VIIh, which is highly related to the viruses circulating in some Southeast Asia countries. Continuous isolation of genotype VIIh NDV strains in the region suggests its panzootic potential. This is the first report of the sub-genotype VIIh NDVs in domestic poultry in China. The complete genome length of the three isolates was 15,192 nucleotides, and the motif at the cleavage site of F protein was 112RRRRR/F117 or 112RRRKR/F117, which was typical of virulent NDV. Phylogenetic analysis based on the F gene revealed that the three viruses had close relationship with the sub-genotype VIIh virus isolated from wild bird in 2011 in China. These viruses might have formed a stable lineage in poultry during 2012-2016 and have the potential to cause enzootic in China. Our study revealed the genetic and phylogenetic characteristics of the three sub-genotype VIIh isolates, which could help us to better understand the epidemiological context of these viruses.

Genome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strains.

African swine fever was introduced into China in August 2018 and led to high mortality in domestic pigs. We reported the genome characterization of the China/2018/AnhuiXCGQ strain mainly based on next-generation sequencing and comparison with related European p72 Genotype II strains. The genome was 189,393 bp long, encoding 181 open reading frames. Pair-wise genome sequence comparison revealed 54-107 variation sites between China/2018/AnhuiXCGQ and the other genotype II virulent strains contributing to the change of expression or alteration of amino acid residues in 10-38 genes. China/2018/AnhuiXCGQ strain shared the highest similarity with POL/2015/Podlaskie strain. Phylogenetic analysis based on a 125 kb long conserved central region revealed that the China/2018/AnhuiXCGQ strain and four European genotype II strains were grouped into three clusters. This study expanded our knowledge on the genetic diversity and evolution of ASFV and provided valuable information for diagnosis improvement and vaccine development.

Infection of African swine fever in wild boar, China, 2018.

On 16 November 2018, a wild boar infected with African swine fever was reported in China. The phylogenetic analysis showed that its causative strain belonged to the p72 genotype II, CD2v serogroup 8 and contained no additional tandem repeat sequences between the I73R and the I329L protein genes, which was different from previously reported strains in China.

Molecular Characterization of African Swine Fever Virus, China, 2018.

On August 3, 2018, an outbreak of African swine fever in pigs was reported in China. We subjected a virus from an African swine fever-positive pig sample to phylogenetic analysis. This analysis showed that the causative strain belonged to the p72 genotype II and CD2v serogroup 8.

Genomic Characterizations of Six Pigeon Paramyxovirus Type 1 Viruses Isolated from Live Bird Markets in China during 2011 to 2013.

The genomes of six pigeon paramyxovirus type 1 (PPMV-1) isolated from symptomless pigeons in live poultry markets during the national active surveillance from 2011 to 2013 were sequenced and analyzed in this study. The complete genome lengths of all isolates were 15,192 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. All isolates had the same motif of 112RRQKRF117 at the cleavage site of the fusion protein, which was typical of velogenic Newcastle disease virus (NDV). Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on sequences of complete genomes and six genes revealed that all isolates belonged to genotype VI in class II, but at least 2 sub-genotypes were identified. Most isolates were placed into sub-genotype VIb with the exception of pi/GX/1015/13, which was classified in sub-genotype VIa. The obvious antigenic difference between PPMV-1 isolates and La Sota strain was found based on the R-value calculated by cross hemagglutination inhibition (HI) assay. These results provided the evidence that PPMV-1 could be detected from healthy pigeons, and our study may be useful in designing vaccines used in pigeon, and developing molecular diagnostic tools to monitor and prevent future PPMV-1 outbreaks.

[Detection of an NA gene molecular marker in H7N9 subtype avian influenza viruses by pyrosequencing].

This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.

[A review of research on Schmallenberg virus].

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.

[Progress in development of influenza virus-like particles].

Virus-like particles (VLPs) are composed of multiple copies of one or more expressed recombinant viral structural proteins which spontaneously assemble into particles upon expression. VLPs are non infectious because they assemble without incorporating genetic material. VLPs have structural characteristics and antigenicity similar to the parental virus because they mimick the wild-type virus structure. Hence, they are recognized readily by the immune system which induces strong anti-viral immune responses to stop virus infection. VLPs have therefore shown dramatic effectiveness as candidate vaccines and diagnostic reagent for virus. Here, in order to provide reference to the research of influenza VLPs, we reviewed the current research progress of influenza VLPs, and discussed the characteristics associated with producing VLPs for influenza virus.

Virus-like particles: promising platforms with characteristics of DIVA for veterinary vaccine design.

In general, it is difficult to differentiate infected from vaccinated animals through vaccination with conventional vaccines, thereby impeding the serological surveillance of animal diseases. DIVA (differentiating infected from vaccinated animals) vaccine, originally known as marker vaccine, usually based on the absence of at least one immunogenic protein in the vaccine strain, allows DIVA in conjunction with a diagnostic test that detects antibodies against the antigens lacking in the vaccine strain. Virus-like particles (VLPs), composed of one or more structural proteins but no genomes of native viruses, mimic the organization and conformation of authentic virions but have no ability to self-replicate in cells, potentially yielding safer vaccine candidates. Since VLPs containing either monovalent or multivalent antigen can be produced in compliance with the requirements for serological surveillance, the use of VLP-based vaccines plays a promising role in DIVA vaccination strategies against animal diseases. Here, we critically reviewed VLPs and companion diagnostics with properties of DIVA for veterinary vaccine design, and three different VLPs as promising platforms for DIVA vaccination strategies in animals.

Complete genome sequences of new emerging newcastle disease virus strains isolated from china.

Five Newcastle disease virus strains isolated from geese were classified into a new genotype, designated genotype XII. The complete genome sequences of two strains indicated that these viruses were distinct from viruses of genotype VII. More investigations need to be conducted for us to understand the origin of these new strains.

Novel reassortant highly pathogenic H5N2 avian influenza viruses in poultry in China.

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription-polymerase chain reaction in chicken eggs.

BACKGROUND: Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF) chicken eggs with Influenza virus (AIV) and Newcastle disease virus (NDV) demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for AIV and NDV to specifically detect the viral genomes in mixed infections. RESULTS: Data from this survey showed that when different doses of NDV (Lasota or F48E8) and AIV (F98 or H5N1) were simultaneously inoculated into embryonating chicken eggs (ECE), interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus. CONCLUSION: AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Virus-like particles: potential veterinary vaccine immunogens.

Virus-like particle (VLP) composed of outer shell but no genome of virus mimics the natural configuration of authentic virion and has no characteristics of self-replication. A close resemblance to native viruses in molecular scaffolds and an absence of genomes make VLPs effectively elicit both humoral and cell-mediated immune responses even with no requirement of adjuvant for vaccines. As effective immunogens, characterized by high immunogenicity and safety, VLPs have been employed in production of human vaccines, such as the licensed vaccines of hepatitis B virus and human papillomavirus. However, there has been no report of licensed veterinary VLP vaccine worldwide as yet. Despite the wide application in vaccination, both the conventional inactivated and live attenuated vaccines for animals are subject to potential limitations due to incomplete inactivation and reversion to virulence. Therefore, those conventional vaccines may, to some extent, be replaced with the VLP-based vaccines conferring higher protection and safety to vaccinated animals. Here, we review the current status of VLPs as veterinary vaccines, and discuss the characteristics and problems associated with generating VLPs for different animal viruses.