A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes.

African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.

Obstetric anesthesia clinic childbirth course combined with labor epidural analgesia is associated with a decreased risk of postpartum depression : a prospective cohort study.

BACKGROUND: Postpartum depression (PPD) is a serious complication commonly seen in postnatal women. In this paper, an investigation was conducted to see if obstetric anesthesia clinic childbirth course combined with labor epidural analgesia (LEA) was associated with a decreased risk of PPD. METHODS: Six hundred fifty-five nulliparous women were enrolled in this prospective cohort study. The parturients were divided into 4 groups, with Group C being the control group, Group AC received the obstetric anesthesia clinic childbirth course only, Group LEA received LEA only, and Group AC + LEA received both the obstetric anesthesia clinic childbirth course and LEA. Maternal and neonatal variables in the perinatal period were recorded. PPD at 6 weeks was assessed using the Chinese version of the Edinburgh Postpartum Depression Scale (EPDS), where a score ≥ 10 is the threshold for PPD. Multivariate logistic regression analysis was performed to assess the association between obstetric anesthesia clinic childbirth course combined with LEA and postpartum depression. RESULTS: A total of 124 maternities had EPDS ≥10 points, the incidence of PPD was 18.9%。The incidence of PPD and EPDS scores were significantly lower in Group AC + LEA than in Group C (12.1% vs 26.8%, P <  0.05; 6 (5, 7) vs 7 (5, 11), P <  0.05). Received an anesthesia clinic childbirth course combined with LEA was associated with a decreased risk of PPD (OR 0.273, 95% CI, 0.100-0.743, P = 0.013). Multivariate logistic regression analysis identified 5 other independent factors for PPD, including maternal SAS score in the delivery room, W-DEQ score in the delivery room, living in a confinement center, EPDS score at 1st week postpartum and perinatal care satisfaction . CONCLUSIONS: Received an obstetrics anesthesia clinic childbirth course combined with LEA for nulliparous women with a single term cephalic pregnancy was associated with a decreased risk of PPD at 6 weeks. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000039163. Registered on 20/10/2020.

Isolation and molecular characterization of bovine herpesvirus 4 from cattle in mainland China.

Bovine herpesvirus 4 (BoHV-4) is one of the most important of the known viral respiratory and reproductive pathogens of both young and adult cattle. However, BoHV-4 has not been isolated or detected in mainland China prior to this study. In 2019, BoHV-4 strain 512 was isolated from cattle in Heilongjiang Province, China, using MDBK cells, and characterized by PCR, nucleotide sequence analysis, and transmission electron microscopy. Two other unknown herpesvirus strains, BL6010 and J4034, which were isolated from cattle in 2009 in China and stored at -70℃, were also propagated in MDBK cells and identified as BoHV-4 by PCR. Phylogenetic analysis based on partial nucleotide sequences of the thymidine kinase (TK) gene and glycoprotein B (gB) gene for the three isolates indicated that these three Chinese strains belong to BoHV-4 genotype 1. A preliminary virus neutralization test revealed that 64% of the 70 bovine sera (45/70) collected from Inner Mongolia Autonomous Region, China, had anti-BoHV-4 antibodies and that natural BoHV-4 infection occurred in cattle in China. Here, we report for the first time the isolation and molecular characterization of BoHV-4 from cattle in mainland China.

Pathogenesis of a Chinese strain of bovine adenovirus type 3 infection in albino guinea pigs.

Bovine adenovirus type 3 (BAV-3) is considered one of the most important respiratory tract agents of cattle and is widespread among cattle around the world. A BAV-3 strain was isolated from a bovine nasal swab for the first time in China in 2009 and named HLJ0955. Subsequently, BAV-3 has frequently been isolated from calves with respiratory diseases in China. To date, only limited study on the pathogenesis of BAV-3 infection in cotton rats has been conducted, and the pathogenesis of BAV-3 infection in guinea pigs has not been reported. Therefore, sixteen albino guinea pigs were inoculated intranasally with HLJ0955. All of the infected guinea pigs had apparently elevated rectal temperatures (39.2 °C-39.9 °C) at 2-7 days post-inoculation (PI). Consolidation and petechial hemorrhage were also observed in guinea pigs experimentally infected with HLJ0955. Viral replication was detectable by virus isolation and titration and by immunohistochemistry in the lungs of guinea pigs as early as 24 h PI. Viral DNA was detectable in the lungs of infected guinea pigs during 11 days of observation by real-time PCR. Virus-neutralizing antibodies against BAV-3 were detectable from 11 days PI and reached a peak titer at 15 days PI. Histopathological changes mainly occurred in the lungs of infected guinea pigs and were characterized by thickening of alveolar septa, mononuclear cell infiltration, hemorrhage and alveolar epithelial necrosis. These results indicate that HLJ0955 can replicate in the lungs of guinea pigs and cause fever and gross and histological lesions. The guinea pig infection model of BAV-3 would serve as a useful system for monitoring the infection process and pathogenesis of the Chinese BAV-3 strain HLJ0955, as well as immune responses to BAV-3 vaccines.

Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies.

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as 48WDCQDGICKNKITESRFIDS67, and 122GDHQQLSIKQ131, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

A highly efficient blocking ELISA based on p72 monoclonal antibody for the detection of African swine fever virus antibodies and identification of its linear B cell epitope.

Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies.

The attenuated African swine fever vaccine HLJ/18-7GD provides protection against emerging prevalent genotype II variants in China.

Genetic changes have occurred in the genomes of prevalent African swine fever viruses (ASFVs) in the field in China, which may change their antigenic properties and result in immune escape. There is usually poor cross-protection between heterogonous isolates, and, therefore, it is important to test the cross-protection of the live attenuated ASFV vaccines against current prevalent heterogonous isolates. In this study, we evaluated the protective efficacy of the ASFV vaccine candidate HLJ/18-7GD against emerging isolates. HLJ/18-7GD provided protection against a highly virulent variant and a lower lethal isolate, both derived from genotype II Georgia07-like ASFV and isolated in 2020. HLJ/18-7GD vaccination prevented pigs from developing ASF-specific clinical signs and death, decreased viral shedding via the oral and rectal routes, and suppressed viral replication after challenges. However, HLJ/18-7GD vaccination did not provide solid cross-protection against genotype I NH/P68-like ASFV challenge in pigs. HLJ/18-7GD vaccination thus shows great promise as an alternative strategy for preventing and controlling genotype II ASFVs, but vaccines providing cross-protection against different ASFV genotypes may be needed in China.

African swine fever virus pCP312R interacts with host RPS27A to shut off host protein translation and promotes viral replication.

African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Šresolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis.

Identification of two novel B cell epitopes on E184L protein of African swine fever virus using monoclonal antibodies.

African swine fever virus (ASFV) is a large double-stranded DNA virus with a complex structural architecture and encodes more than 150 proteins, where many are with unknown functions. E184L has been reported as one of the immunogenic ASFV proteins that may contribute to ASFV pathogenesis and immune evasion. However, the antigenic epitopes of E184L are not yet characterized. In this study, recombinant E184L protein was expressed in prokaryotic expression system and four monoclonal antibodies (mAbs), designated as 1A10, 2D2, 3H6, and 4C10 were generated. All four mAbs reacted specifically with ASFV infected cells. To identify the epitopes of the mAbs, a series of overlapped peptides of E184L were designed and expressed as maltose binding fusion proteins. Accordingly, the expressed fusion proteins were probed with each E184L mAb separately by using Western blot. Following a fine mapping, the minimal linear epitope recognized by mAb 1A10 was identified as 119IQRQGFL125, and mAbs 2D2, 3H6, and 4C10 recognized a region located between 153DPTEFF158. Alignment of amino acids of E184L revealed that the two linear epitopes are highly conserved among different ASFV isolates. Furthermore, the potential application of the two epitopes in ASFV diagnosis was assessed through epitope-based ELISA using 24 ASFV positive and 18 negative pig serum and the method were able to distinguish positive and negative samples, indicating the two epitopes are dominant antigenic sites. To our knowledge, this is the first study to characterize the B cell epitopes of the antigenic E184L protein of ASFV, offering valuable tools for future research, as well as laying a foundation for serological diagnosis and epitope-based marker vaccine development.

Comprehensive mapping of antigenic linear B-cell epitopes on K205R protein of African swine fever virus with monoclonal antibodies.

African swine fever virus causes an acute, highly contagious swine disease with high mortality, leading to enormous losses in the pig industry. The K205R, a nonstructural protein of African swine fever virus, is abundantly expressed in the cytoplasm of infected cells at the early stage of infection and induces a strong immune response. However, to date, the antigenic epitopes of this immunodeterminant have not been characterized. In the present study, the K205R protein was expressed in a mammalian cell line and purified using Ni-affinity chromatography. Furthermore, three monoclonal antibodies (mAbs; 5D6, 7A8, and 7H10) against K205R were generated. Indirect immunofluorescence assay and western blot results showed that all three mAbs recognized native and denatured K205R in African swine fever virus (ASFV)-infected cells. To identify the epitopes of the mAbs, a series of overlapping short peptides were designed and expressed as fusion proteins with maltose-binding protein. Subsequently, the peptide fusion proteins were probed with monoclonal antibodies using western blot and enzyme-linked immunosorbent assay. The three target epitopes were fine-mapped; the core sequences of recognized by the mAbs 5D6, 7A8, and 7H10 were identified as 157FLTPEIQAILDE168, 154REKFLTP160, and 136PTNAMFFTRSEWA148, respectively. Probing with sera from ASFV-infected pigs in a dot blot assay demonstrated that epitope 7H10 was the immunodominant epitope of K205R. Sequence alignment showed that all epitopes were conserved across ASFV strains and genotypes. To our knowledge, this is the first study to characterize the epitopes of the antigenic K205R protein of ASFV. These findings may serve as a basis for the development of serological diagnostic methods and subunit vaccines.

Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs.

African swine fever virus (ASFV) poses a great threat to the global pig industry and food security. Currently, 24 ASFV genotypes have been reported but it is unclear whether recombination of different genotype viruses occurs in nature. In this study, we detect three recombinants of genotype I and II ASFVs in pigs in China. These recombinants are genetically similar and classified as genotype I according to their B646L gene, yet 10 discrete fragments accounting for over 56% of their genomes are derived from genotype II virus. Animal studies with one of the recombinant viruses indicate high lethality and transmissibility in pigs, and deletion of the virulence-related genes MGF_505/360 and EP402R derived from virulent genotype II virus highly attenuates its virulence. The live attenuated vaccine derived from genotype II ASFV is not protective against challenge of the recombinant virus. These naturally occurring recombinants of genotype I and II ASFVs have the potential to pose a challenge to the global pig industry.

Overexpressed mutant optineurin(E50K) induces retinal ganglion cells apoptosis via the mitochondrial pathway.

Mutations in the coding region of the OPTN gene are associated with certain glaucomas. Although the function of the optineurin protein is yet to be elucidated, the most common mutation, E50K, is associated with a severe phenotype. Plasmids expressing wild-type Optineurin (WT) and mutant Optineurin(E50K) were transfected into RGC-5 and monitored by immunofluorescence staining and western blotting. The mutant Optineurin(E50K) induced the death of retinal ganglion cells by generation of reactive oxygen species accompanied disruption of mitochondrial transmembrane potential, down-regulation of bcl-2, and up-regulation of bax, which led to the release of cytochrome C from the mitochondria into the cytosol, which, in turn, resulted in the activation of caspase-9 and caspase-3, indicating that mutant Optineurin(E50K) acquired the ability to induce cell death through the mitochondrial caspase-dependent cell death pathway.

Transgenic mice with overexpression of mutated human optineurin(E50K) in the retina.

In the present work, Site-directed mutagenesis to insert the Glu50Lys amino acid substitution was achieved by PCR using plasmid pBluescript-OPTN. Mutated human OPTN(E50K) gene-driven mouse c-kit promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and DNA dot blot were used to screen the positive transgenic mice. RT-PCR analyzed the RNA level and location of mutated human OPTN(E50K) mRNA expression in transgenic mice. Western blot and immunohistochemistry were used to detect the level and location of mutated human OPTN(E50K) expression in transgenic mice. A transgenic mouse model with overexpression of mutated human OPTN(E50K) in retina was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Mutated human OPTN(E50K) gene was controlled by c-kit promoter and expressed in the retina in mice. Mutated human OPTN(E50K) in transgenic mice was higher than that of wild type C57BL/6J mice. Our studies had provided a new transgenic model for investigating the molecular properties of mutated human OPTN(E50K).

The Anesthetic Effect and Safety of Dexmedetomidine in Cesarean Section: A Meta-Analysis.

Objective: To evaluate the anesthetic effect and safety of dexmedetomidine in cesarean section. Methods: The Cochrane Library, EMBASE, and PubMed databases (established until September 2020) were searched by computer. Two authors independently screened and extracted literature related to the application of dexmedetomidine in the cesarean section according to inclusion and exclusion criteria. The control group received either subarachnoid block (lumbar anesthesia) or combined lumbar anesthesia and epidural anesthesia (combined lumbar epidural anesthesia) with bupivacaine or combined bupivacaine and fentanyl. The observation group was additionally given dexmedetomidine based on the control group, to analyze the anesthetic effect and safety of dexmedetomidine in cesarean section. Results: A total of 580 cesarean delivery women were included in 8 studies, and the results showed that the peak time of sensory block in the observation group was shorter than that in the control group (standard mean difference = -0.28; 95% confidence interval: -0.48, -0.08; P = 0.006), sensory block lasted longer than that in the control group (standard mean difference = 1.49; 95% confidence interval: 1.21, 1.78; P < 0.00001), the sedation rate was higher than that in the control group, the onset of the first postoperative pain was significantly delayed compared with that in the control group, and the incidence of postoperative pain, nausea and vomiting, postoperative chills, and fever was lower than that in the control group (P < 0.05). Conclusion: Dexmedetomidine combined with lumbar anesthesia or combined lumbar epidural anesthesia for women in cesarean section has more clinical benefits and better safety.

Anesthetic Effect of Dexmedetomidine in Clinical Functional Neurosurgery.

Background: Dexmedetomidine is a highly selective and efficient α2-adrenoceptor agonist with good antianxiety, analgesic, hypnotic, and sedative effects without causing respiratory depression. Aim: To investigate the anesthetic effect of dexmedetomidine in clinical neurosurgery. Methods: A total of 94 patients who received functional neurosurgical treatment in our hospital from March 2019 to October 2020 were selected and divided into the study and control groups. Routine anesthesia was adopted in the control group, while dexmedetomidine was used in the study group. Perioperative hemodynamic indicators such as mean arterial pressure, heart rate, and peripheral capillary oxygen saturation, cognitive function score, pain score VAS, stress response index level, and incidence of adverse reactions were compared between the two groups. Results: Before surgery (T0), no significant differences in MAP, HR, and SpO2 were observed between the two groups. However, at the beginning of the operation (T1), 30 min after the operation (T2), and immediately after the operation (T3), these indicators in the study group were significantly higher than in the control group. The postoperative MMSE of the study group 3 d later was significantly higher than that of the control group. The VAS scores after the operation of the study group were lower than those of the control group. The serum cortisol (COR) and aldosterone (ALD) levels in the study group were not significantly different from those in the control group before surgery. The levels of each index in the two groups were higher than those before and 24 h after surgery. The incidence rate of adverse reactions in the study group was lower. Conclusion: The application of dexmedetomidine in clinical functional neurosurgery is safe and can maintain hemodynamic stability and reduce the degree of stress response, cognitive impairment, and pain caused by invasive surgery.

Development of a One-Step Multiplex Real-Time PCR Assay for the Detection of Viral Pathogens Associated With the Bovine Respiratory Disease Complex.

Bovine respiratory disease complex (BRDC) occurs widely in cattle farms. The main viral pathogens include bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV3), and bovine respiratory syncytial virus (BRSV), and the newly emerged influenza D virus (IDV). In this study, we have developed a one-step multiplex real-time Polymerase Chain Reaction (PCR) capable of simultaneously detecting these five viral pathogens causing BRDC. The established assay could specifically detect targeted viruses without cross-reaction with others. The detection limit was ~10 copies/reaction for single real-time PCR and 100 copies/ reaction for multiplex real-time PCR assay. A total of 213 nasal samples from cattle with signs of respiratory tract disease were then collected for performance evaluation of the established platform, proving that the method has good specificity and sensitivity. The surveillance data suggested that BVDV and BoHV-1 infections are the dominant cause of BRDC in the herd, whereas the detection rate of IDV, BIPV3, and BRSV is relatively lower. In summary, the established assay provides technical support for rapid clinical detection of BRDC associated viral pathogens to guide the formulation of BRDC prevention and control measures.

Development of an ELISA Method to Differentiate Animals Infected with Wild-Type African Swine Fever Viruses and Attenuated HLJ/18-7GD Vaccine Candidate.

African swine fever (ASF) is a highly contagious hemorrhagic disease of pigs, posing a significant threat to the world pig industry. Several researchers are investigating the possibilities for developing a safe and efficient vaccine against ASF. In this regard, significant progress has been made and some gene-deleted ASFVs are reported as potential live attenuated vaccines. A seven-gene-deleted live attenuated vaccine candidate HLJ/18-7GD (among which CD2v is included) has been developed in our laboratory and reported to be safe and protective, and it is expected to be commercialized in the near future. There is an urgent need for developing a diagnostic method that can clearly discriminate between wild-type-ASFV-infected and vaccinated animals (DIVA). In the present study, a dual indirect ELISA based on p54 and CD2v proteins was successfully established to specifically distinguish serum antibodies from pigs infected with wild-type ASFV or possessing vaccine immunization. To evaluate the performance of the assay, a total of 433 serum samples from four groups of pigs experimentally infected with the wild-type HLJ/18 ASFV, immunized with the HLJ/18-7GD vaccine candidate, infected with the new lower virulent variant, and specific-pathogen-free pigs were used. Our results showed that the positive rate of immunized serum was 96.54% (p54) and 2.83% (CD2v), and the positive rate of the infection by wild-type virus was 100% (p54) and 97.8% (CD2v). Similarly, the positive rate to infection by the new low-virulent ASFV variant in China was 100% (p54) and 0% (CD2v), indicating the technique was also able to distinguish antibodies from wild-type and the new low-virulent ASFV variant in China. Moreover, no cross-reaction was observed in immune sera from other swine pathogens, such as CSFV, PEDV, PRRSV, HP-PRRSV, PCV2, and PrV. Overall, the developed dual indirect ELISA exhibited high diagnostic sensitivity, specificity, and repeatability and will provide a new approach to differentiate serum antibodies between wild virulent and CD2v-unexpressed ASFV infection, which will play a great role in serological diagnosis and epidemiological monitoring of ASF in the future.

Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China.

BACKGROUND: Bovine adenovirus type 3 (BAV-3) belongs to the Mastadenovirus genus of the family Adenoviridae and is involved in respiratory and enteric infections of calves. The isolation of BAV-3 has not been reported prior to this study in China. In 2009, there were many cases in cattle showing similar clinical signs to BAV-3 infection and a virus strain, showing cytopathic effect in Madin-Darby bovine kidney cells, was isolated from a bovine nasal swab collected from feedlot cattle in Heilongjiang Province, China. The isolate was confirmed as a bovine adenovirus type 3 by PCR and immunofluorescence assay, and named as HLJ0955. So far only the complete genome sequence of prototype of BAV-3 WBR-1 strain has been reported. In order to further characterize the Chinese isolate HLJ0955, the complete genome sequence of HLJ0955 was determined. RESULTS: The size of the genome of the Chinese isolate HLJ0955 is 34,132 nucleotides in length with a G+C content of 53.6%. The coding sequences for gene regions of HLJ0955 isolate were similar to the prototype of BAV-3 WBR-1 strain, with 80.0-98.6% nucleotide and 87.5-98.8% amino acid identities. The genome of HLJ0955 strain contains 16 regions and four deletions in inverted terminal repeats, E1B region and E4 region, respectively. The complete genome and DNA binding protein gene based phylogenetic analysis with other adenoviruses were performed and the results showed that HLJ0955 isolate belonged to BAV-3 and clustered within the Mastadenovirus genus of the family Adenoviridae. CONCLUSIONS: This is the first study to report the isolation and molecular characterization of BAV-3 from cattle in China. The phylogenetic analysis performed in this study supported the use of the DNA binding protein gene of adenovirus as an appropriate subgenomic target for the classification of different genuses of the family Adenoviridae on the molecular basis. Meanwhile, a large-scale pathogen and serological epidemiological investigations for BVA-3 infection might be carried out in cattle in China. This report will be a good beginning for further studies on BAV-3 in China.

Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection.

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene.

A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155-165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.