Mitochondrial genome of Theileria uilenbergi endemic in sheep and goats in China.

Mitochondrial genomes provide new insights that help elucidating biological features, genetic evolution, and classification of protozoans. Theileria uilenbergi (T. uilenbergi), transmitted by Haemaphysalis qinghaiensis and H. longicornis, is considered as highly pathogenic to sheep and goats in China. This study reports and outlines features of its mitochondrial genome. The T. uilenbergi mitochondrial genome is a linear monomeric molecule of 6.0 kb length, which encodes three protein-coding genes named cytochrome c oxidase I (cox1), cytochrome b (cob), and cytochrome c oxidase III (cox3), as well as six large subunit (LSU) rRNA gene fragments, and ends in terminal inverted repeats (TIRs). The array structure and organization of the mitochondrial genome of T. uilenbergi is identical to that of T. parva. Phylogenetic analysis based on the amino acid sequences of cox1, cob, and cox3 genes suggests that T. uilenbergi is distantly related to the group of transforming Theileria species such as T. parva. This study contributes to a comprehensive understanding of the phylogeny and evolution of the mitochondrial genome of piroplasms and provides useful information of diagnostic marker for T. uilenbergi.

Comparative evaluation of two informative markers in the phylogeny of Babesia and Theileria parasites.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. In the study, we assessed the relative resolution capabilities of the DNA sequences of the nuclear genes 40S ribosomal protein S5 (RPS5) and mitochondrial DNA Cytochrome c oxidase subunit III (cox3) gene in the phylogeny of Babesia and Theileria species isolates. We demonstrated that by using the cox3 gene can recover a better supported species tree for some Theileria species than when using the nuclear RPS5 gene alone, it tends to intra-specific diversity and considerable inter-specific difference. Additionally, the combined DNA sequences of the nuclear RPS5 and cox3 gene improved the inference of evolutionary relationships among Babesia and Theileria species. The mitochondrial cox3 gene outperforms nuclear RPS5 gene and yields better resolution on the intra-specific diversity of Babesia and Theileria species. However, the combined RPS5 nuclear DNA and cox3 DNA tree had more advantage in the phylogeny of Babesia and Theileria species than that of single gene alone.

Transcriptome analysis of responses to bluetongue virus infection in Aedes albopictus cells.

BACKGROUND: Bluetongue virus (BTV) causes a disease among wild and domesticated ruminants which is not contagious, but which is transmitted by biting midges of the Culicoides species. BTV can induce an intense cytopathic effect (CPE) in mammalian cells after infection, although Culicoides- or mosquito-derived cell cultures cause non-lytic infection with BTV without CPE. However, little is known about the transcriptome changes in Aedes albopictus cells infected with BTV. METHODS: Transcriptome sequencing was used to identify the expression pattern of mRNA transcripts in A. albopictus cells infected with BTV, given the absence of the Culicoides genome sequence. Bioinformatics analyses were performed to examine the biological functions of the differentially expressed genes. Subsequently, quantitative reverse transcription-polymerase chain reaction was utilized to validate the sequencing data. RESULTS: In total, 51,850,205 raw reads were generated from the BTV infection group and 51,852,293 from the control group. A total of 5769 unigenes were common to both groups; only 779 unigenes existed exclusively in the infection group and 607 in the control group. In total, 380 differentially expressed genes were identified, 362 of which were up-regulated and 18 of which were down-regulated. Bioinformatics analyses revealed that the differentially expressed genes mainly participated in endocytosis, FoxO, MAPK, dorso-ventral axis formation, insulin resistance, Hippo, and JAK-STAT signaling pathways. CONCLUSION: This study represents the first attempt to investigate transcriptome-wide dysregulation in A. albopictus cells infected with BTV. The understanding of BTV pathogenesis and virus-vector interaction will be improved by global transcriptome profiling.

Complete genome sequence of a bovine ephemeral fever virus JT02L strain in mainland China.

In this study, we report the complete genome sequence of bovine ephemeral fever virus (BEFV) JT02L, which has been used in our laboratory, in mainland China, for more than a decade. The genome is 14941 nucleotide (nt), comprising a leader sequence of 50 nt, nucleoprotein (N) gene of 1328 nt, phosphoprotein (P) gene of 858 nt, matrix protein (M) gene of 691 nt, glycoprotein (G) gene of 1897 nt, non-structural glycoprotein (GNS) gene of 1785 nt, α1α2 gene of 638 nt, ß gene of 460 nt, γ gene of 400 nt, large multi-functional enzyme (L) gene of 6470 nt and a trailer sequence of 73 nt. Individual genes are separated by intergenic regions (IGRs) of 26, 44, 47, 51, 37, 39, 68 and -21 nt respectively. The overall organization is similar to an Australian BEFV isolate BB7721 but demonstrates some distinctive features including longer α3 and ß open reading frames, intact termination/polyadenylation (TTP) sequence downstream of the ß open reading frame and a longer ß-γ IGR integrated with a 38 nt AT-rich fragment. To our knowledge, this is the first report describing the complete genome of a BEFV strain of East Asian lineage, which may facilitate studies on genomic diversity among geographic strains of BEFV in China and the world.

Proteomic analysis of sheep primary testicular cells infected with bluetongue virus.

Bluetongue virus (BTV) causes a non-contagious, arthropod-transmitted disease in wild and domestic ruminants, such as sheep. In this study, we used iTRAQ labeling coupled with LC-MS/MS for quantitative identification of differentially expressed proteins in BTV-infected sheep testicular (ST) cells. Relative quantitative data were obtained for 4455 proteins in BTV- and mock-infected ST cells, among which 101 and 479 proteins were differentially expressed at 24 and 48 h post-infection, respectively, indicating further proteomic changes during the later stages of infection. Ten corresponding genes of differentially expressed proteins were validated via real-time RT-PCR. Expression levels of three representative proteins, eIF4a1, STAT1 and HSP27, were further confirmed via western blot analysis. Bioinformatics analysis disclosed that the differentially expressed proteins are primarily involved in biological processes related to innate immune response, signal transduction, nucleocytoplasmic transport, transcription and apoptosis. Several upregulated proteins were associated with the RIG-I-like receptor signaling pathway and endocytosis. To our knowledge, this study represents the first attempt to investigate proteome-wide dysregulation in BTV-infected cells with the aid of quantitative proteomics. Our collective results not only enhance understanding of the host response to BTV infection but also highlight multiple potential targets for the development of antiviral agents.

Genome analysis of an atypical bovine pestivirus from fetal bovine serum.

We report the complete genome sequence of a bovine pestivirus LVRI/cont-1 originated from a commercial batch of fetal bovine serum. Its complete genome consists of 12,282 nucleotides (nt), which contain an open reading frame (ORF) of 11,700 bp flanked by 5' and 3' untranslated regions (383 and 199 bp). The size of the 5'UTR and the individual protein coding region of LVRI/cont-1 are identical to those of the reference virus Th/04_KhonKaen, but it has a deletion of the first 56 nt in the 3'UTR. Alignment of the complete nucleotide sequence and phylogenetic analysis indicate that this viral isolate belongs to atypical pestiviruses.

Identification of three novel linear B-cell epitopes on VP7 of African horse sickness virus using monoclonal antibodies.

African horse sickness (AHS) is an acute and subacute infectious disease of equine species caused by the African horse sickness virus (AHSV). The VP7 of AHSV is a group-specific protein conserved in all serotypes and is an excellent candidate for the serological diagnosis and an AHS vaccine component. However, to date, B-cell epitopes on the AHSV VP7 recognized by humoral immune responses remain unclear. This study expressed the recombinant AHSV VP7 soluble in Escherichia coli and purified it for mouse immunization. Four monoclonal antibodies (mAbs) were screened and identified by hybridoma cell fusion, clonal purification, and immunological assays. The B-cell epitopes, recognized by monoclonal antibodies 4B5, 3G10, 3D7, and 4D6, were identified by a series of truncated overlapping peptides expressed as glutathione S-transferase (GST)-fusion proteins. The results revealed that 4B5 recognized the 124VQTGRYAGA132 motif, 3G10 recognized the 140RYYVPQGRT148 motif, while 3D7 and 4D6 recognized the 292QPINPPIFP300 motif. Amino acid sequence alignment indicated that three novel B-cell epitopes were conserved among various AHSV serotypes but unconserved in other orbiviruses, such as the bluetongue and epidemic hemorrhagic disease viruses. This study informs on the antigenic epitopes of AHSV VP7, facilitating future investigations into the serological diagnosis method and epitope-based vaccines against AHSV.

African swine fever virus RNA polymerase subunits C315R and H359L inhibition host translation by activating the PKR-eIF2a pathway and suppression inflammatory responses.

ASFV C315R is homologous to the transcription factor TFIIB of large unclassified DNA viruses, and H359L is identical to the subunit 3 (RPB3) of eukaryotic RNA polymerase II. The C315R and H359L may play an important role in ASFV replication and transcription. Here, we evaluated the biological function of the C315R and H359L genes during virus replication in vitro and during infection in pigs. Results showed that C315R and H359L are highly conserved among ASFV genotype II strains; quantitative PCR (qPCR) and western blotting analyses revealed that C315R and H359L are early transcribed genes prior to viral DNA replication, but their protein expression is delayed. The immunofluorescence and western blotting analysis revealed that both proteins localized in the cell cytoplasm and nucleus at 24 h post infection, however, pH359L was mainly detected in the cell cytoplasm. Furthermore, overexpression of pH359L in MA104 cells significantly increased viral titer, RNA transcription levels, and viral protein expression levels, while overexpression of pC315R slightly enhanced ASFV replication. In contrast, siRNA targeting ASFV-H359L or C315R reduced replication efficiency in porcine macrophage culture compared to the parent ASFV-CN/SC/2019, demonstrating that C315R and H359L genes are necessary for ASFV replication. Finally, the functional role of C315R or H359L on PKR and eIF2α phosphorylation status and SG formation, as well as cytokine production were evaluated. These studies demonstrated that C315R and H359L are involved in virus replication processes in swine and play important roles in ASFV replication.

The GTPase activity and isoprenylation of Swine GBP1 are critical for inhibiting the production of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is a flavivirus that cause severe neurological deficits. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on many RNA and DNA viruses via diverse mechanisms, however, the roles and the action modes of GBP1 in the antiviral effect on the production of JEV RNA and infectious virions remain to be clarified. In this study, we found that the RNA levels of swine GBP1 (sGBP1) in PK15 cells were up-regulated at the late stage of JEV infection. The overexpression of sGBP1 significantly inhibited the production of JEV while the knockdown of sGBP1 promoted the production of JEV. The GTPase activity and isoprenylation of sGBP1 both are critical for anti-JEV activity. The GTPase activity of sGBP1 is responsible for inhibiting the production of JEV genomic RNA. The isoprenylation of sGBP1 inhibited the expression and cleavage of JEV prM to decrease the yields of infectious virions, which may be associated with the interaction between sGBP1 and cellular proprotein convertase furin. Taken together, the study dissected the action modes of sGBP1with potent anti-JEV activity in more details.

Interferon-stimulated gene 15 facilitates BTV replication through interacting with the NS1 protein.

Bluetongue virus (BTV) infection effectively activates the innate immune response, followed by the expression of interferon (IFN) and multiple interferon-stimulated genes (ISGs). ISG15 is one of the most induced ISGs, and often plays a role in inhibiting virus replication. This study aims to explore the role and specific mechanisms of ovine ISG15 (oISG15) in BTV infection. We found that the transcription level of oISG15 was upregulated in a time-dependent and BTV multiplicity of infection-dependent manner. The overexpression of exogenous oISG15 enhances BTV replication, whereas the knockdown of endogenous oISG15 inhibits BTV replication. The viral protein in wild-type oISG15-overexpressed cells and ISGylation defective oISG15-overexpressed cells have no significant differences, which indicated that oISG15 promoted BTV replication in an ISGylation-independent manner. A co-immunoprecipitation assay showed that four viral BTV proteins-VP3, VP4, VP5, and NS1-interacted with oISG15. We also found that the VP4 and NS1 proteins associated with ubiquitin via co-immunoprecipitation, and that oISG15 overexpression improved the stability of both proteins. Further results showed that the degradation of NS1 was involved in lysine 63-linked polyubiquitin. This suggested that oISG15 may interfere with NS1 degradation via the autophagy pathway. This study provides new insights on the interaction between BTV and ISG15, and enriches our understanding of the regulation and biological function of ISG15 with virus replication.

[Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model].

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-Erns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 106 TCID50 virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

Development and validation of a DIVA ELISA for differentiating BEFV infected from vaccinated animals.

Inactivated vaccine is considered safe and used for prevention of bovine ephemeral fever in several endemic countries. To differentiate between BEFV-infected and vaccinated animals, we developed an ELISA capable of detecting infection-related antibodies against BEFV. Recombinant proteins, including N, P, M, L, GNS, α2, ß and γ, were expressed in E. coli and screened by Western blotting and ELISA. The results showed GNS, α2 and ß specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. A DIVA ELISA based on a C-terminal truncated form of GNS was developed, with 100% sensitivity and 98.0% specificity at a sample to positive-control optical density ratio (S/P) threshold of 0.18. Specificity analysis showed that the assay has no cross-reactivity with antisera of other common bovine viruses. Anti-GNS antibody appears at 3-4 days post infection (dpi) and persists up to 240-300 dpi in the experimentally infected cattle. Sero-epidemiological survey using sera collected from vaccinated cattle in an endemic area in Jiangsu Province revealed sero-positive rate of 2.36% (6/254), indicating that the DIVA ELISA could be used as a reliable diagnostic tool for differentiating BEFV infected from vaccinated animals.

[Using mouse model to evaluate the immune effect of DNA prime-protein boost strategies targeting Japanese encephalitis virus].

In order to evaluate the immune effect of the genotype Ⅰ Japanese encephalitis virus prM-E DNA vaccine and the prM-EⅢ fusion protein subunit vaccine on mice using DNA prime-protein boost strategy, the prM-E gene was inserted into the pVAX1 eukaryotic expression vector. The recombinant expression vector prM-E-pVAX1 was constructed as a DNA vaccine for initial immunity, and the recombinant prM-EⅢ fusion protein was obtained using a prokaryotic expression system as a subunit vaccine for enhanced immunity. Thirty two female BALB/c mice aged 4-6 weeks were randomly divided into four groups, and a prM-E-pVAX1 DNA vaccine group, a DNA prime-protein boost immune group, a prM-EⅢ subunit vaccine group, and a pVAX1 vector control group were set up. The specific antibody level in serum was monitored by ELISA, the neutralizing antibody titer was detected by plaque reduction neutralization, and the cellular immune responses induced by different vaccine immune groups were analyzed by cytokine expression abundance and lymphocyte proliferation experiments. The results showed that the neutralizing antibody titers induced by mice immunized with the DNA prime-protein boost strategy were close to that of the group immunized with the single prM-EⅢ subunit vaccine, but significantly higher than that of the group immunized with the single prM-E-pVAX1 DNA vaccine. DNA prime-protein boost strategies induced effective Th1/Th2 immune responses in mouse models, in particular the Th1 cell-mediated immune responses. This study provides a new immune strategy that may facilitate the prevention of Japanese encephalitis.

ISG20 inhibits bluetongue virus replication.

ISG20 is an interferon-inducible exonuclease that inhibits virus replication. Although ISG20 is thought to degrade viral RNA, the antiviral mechanism and specificity of ISG20 remain unclear. In this study, the antiviral role of ovine ISG20 (oISG20) in bluetongue virus â€‹(BTV) infection was investigated. It was found that BTV infection up-regulated the transcription of ovine ISG20 (oISG20) in a time- and BTV multiplicity of infection (MOI)-dependent manner. Overexpression of oISG20 suppressed the production of BTV genome, proteins, and virus titer, whereas the knockdown of oISG20 increased viral replication. oISG20 was found to co-localize with BTV proteins VP4, VP5, VP6, and NS2, but only directly interacted with VP4. Exonuclease defective oISG20 significantly decreased the inhibitory effect on BTV replication. In addition, the interaction of mutant oISG20 and VP4 was weakened, suggesting that binding to VP4 was associated with the inhibition of BTV replication. The present data characterized the anti-BTV effect of oISG20, and provides a novel clue for further exploring the inhibition mechanism of double-stranded RNA virus by ISG20.

An improved luciferase immunosorbent assay for ultrasensitive detection of antibodies against African swine fever virus.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal infectious disease of pigs and causes great socioeconomic losses globally. The reliable diagnostic method is critical for prevention and control of the disease. In this study, an improved Luciferase immunosorbent assay (LISA) for detecting ASF was developed using the cell lysates containing ASFV p35 protein fused with a reporter Nano-luciferase (p35-Luc protein). The improved method avoids the complicate procedures of immobilizing the serum samples with protein G in the normal LISA method, and replaced by directly coating the serum samples with carbonate buffer, therefore reduces the productive cost and simplifies the operation procedures. The p35-Luc LISA exhibited high specificity for anti-ASFV sera while no cross-reactions with the sera against other swine viruses. The detection limit of the p35-Luc LISA was shown to be at least four times higher than that of the p35 based indirect ELISA established in our lab. The receiver operating characteristic (ROC) analysis showed the 96.36% relative specificity and 96.97% relative sensitivity of the p35-Luc LISA with the cutoff values of 3.55 as compared to the commercial Ingezim p72-ELISA kit. Furthermore, a total of 248 serum samples were tested by both the p35-Luc LISA and commercial Ingezim p72-ELISA kit, and there was a high degree of agreement (97.6%, kappa = 0.9753) in the performance of the two assays. Collectively, the improved LISA based on the p35-Luc protein could be used as a rapid, ultrasensitive, cost-effective and reliable diagnostic tool for serological survey of ASF in pig farms.

The internal transcribed spacer 1 (ITS-1), a controversial marker for the genetic diversity of Trypanosoma evansi.

Seven Trypanosoma evansi isolates from China and a Trypanosoma congolense sp. gifted from Kenya were characterized genetically by the internal transcribed spacer 1 (ITS-1) of nuclear ribosomal DNA (rDNA). The ITS-1 rDNA with the length of 338-342 bp was amplified by polymerase chain reaction (PCR) and sequenced from individual isolates of T. evansi. Although sequence variation between T. evansi isolates from China only was 0.3-3.8%, the constructed phylogenetic tree based on the ITS-1 rDNA sequence by the method of neighbor-joining and maximum parsimony revealed the genetic diversity among T. evansi isolates from China. For T. congolense sp., the most phylogenetically related species was T. congolense IL1180. Although the sequence variation ranged 0.8-14.5% between T. congolense isolates, the phylogenetic tree can not reflected the genetic diversity among T. congolense isolates perhaps because of the fewer number of isolates and sequences. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.

Discrimination between Haemaphysalis longicornis and H. qinghaiensis based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2).

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1-0.4%, nucleotide differences between the tested species ranged 2.1-23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.

[Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen].

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.

Analysis of Microorganism Diversity in Haemaphysalis longicornis From Shaanxi, China, Based on Metagenomic Sequencing.

Ticks are dangerous ectoparasites of humans and animals, as they are important disease vectors and serve as hosts for various microorganisms (including a variety of pathogenic microorganisms). Diverse microbial populations coexist within the tick body. Metagenomic next-generation sequencing (mNGS) has been suggested to be useful for rapidly and accurately obtaining microorganism abundance and diversity data. In this study, we performed mNGS to analyze the microbial diversity of Haemaphysalis longicornis from Baoji, Shaanxi, China, with the Illumina HiSeq platform. We identified 189 microbial genera (and 284 species) from ticks in the region; the identified taxa included Anaplasma spp., Rickettsia spp., Ehrlichia spp., and other important tick-borne pathogens at the genus level as well as symbiotic microorganisms such as Wolbachia spp., and Candidatus Entotheonella. The results of this study provide insights into possible tick-borne diseases and reveal new tick-borne pathogens in this region. Additionally, valuable information for the biological control of ticks is provided. In conclusion, this study provides reference data for guiding the development of prevention and control strategies targeting ticks and tick-borne diseases in the region, which can improve the effectiveness of tick and tick-borne disease control.

Transcriptome Profiling Reveals Features of Immune Response and Metabolism of Acutely Infected, Dead and Asymptomatic Infection of African Swine Fever Virus in Pigs.

African swine fever virus (ASFV) infection can result in lethal disease in pigs. ASFV encodes 150-167 proteins, of which only approximately 50 encoded viral structure proteins are functionally known. ASFV also encodes some nonstructural proteins that are involved in the regulation of viral transcription, viral replication and evasion from host defense. However, the understanding of the molecular correlates of the severity of these infections is still limited. The purpose of this study was to compare host and viral gene expression differences and perform functional analysis in acutely infected, dead and cohabiting asymptomatic pigs infected with ASFV by using RNA-Seq technique; healthy pigs were used as controls. A total of 3,760 and 2,874 upregulated genes and 4,176 and 2,899 downregulated genes were found in healthy pigs vs. acutely infected, dead pigs or asymptomatic pigs, respectively. Additionally, 941 upregulated genes and 956 downregulated genes were identified in asymptomatic vs. acutely infected, dead pigs. Different alternative splicing (AS) events were also analyzed, as were gene chromosome locations, and protein-protein interaction (PPI) network prediction analysis was performed for significantly differentially expressed genes (DEGs). In addition, 30 DEGs were validated by RT-qPCR, and the results were consistent with the RNA-Seq results. We further analyzed the interaction between ASFV and its host at the molecular level and predicted the mechanisms responsible for asymptomatic pigs based on the selected DEGs. Interestingly, we found that some viral genes in cohabiting asymptomatic pigs might integrate into host genes (DP96R, I73R and L83L) or remain in the tissues of cohabiting asymptomatic pigs. In conclusion, the data obtained in the present study provide new evidence for further elucidating ASFV-host interactions and the ASFV infection mechanism and will facilitate the implementation of integrated strategies for controlling ASF spread.