A novel function of African Swine Fever Virus pE66L in inhibition of host translation by the PKR/eIF2α pathway.

African swine fever virus (ASFV) is one of the most contagious and lethal viruses infecting pigs. This virus is endemic in many countries and has very recently spread to China, but no licensed vaccines or treatments are currently available. Despite extensive research, the basic question of how ASFV-encoded proteins inhibit host translation remains. Here, we examined how ASFV interfered with host translation and optimized viral gene expression. We found that 14 ASFV proteins inhibited Renilla luciferase (Rluc) activity greater than 5-fold, and the protein with the strongest inhibitory effect was pE66L, which was not previously reported. Combined with bioinformatical analysis and biochemical experiment, we determined that the transmembrane (TM) domain (amino acids 13-34) of pE66L was required for the inhibition of host gene expression. Notably, we constructed a recombinant plasmid with the TM domain linked to enhanced green fluorescent protein (EGFP) and further demonstrated that this domain broadly inhibited protein synthesis. Confocal and biochemical analyses indicated that the TM domain might help proteins locate to the endoplasmic reticulum (ER) to suppress translation though the PKR/eIF2α pathway. Deletion of the E66L gene had little effect on virus replication in macrophages, but significantly recovered host gene expression. Taken together, our findings complement studies on the host translation of ASFV proteins and suggest that ASFV pE66L induces host translation shutoff, which is dependent on activation of the PKR/eIF2α pathway.Importance African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA virus superfamily that predominantly replicates in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from approximately 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames (ORFs), of which half the encoded proteins have not been explored. Our study showed that 14 proteins had an obvious inhibitory effect on Renilla luciferase (Rluc) gene synthesis, with pE66L showing the most significant effect. Furthermore, the transmembrane (TM) domain of pE66L broadly inhibited host protein synthesis in a PKR/eIF2a pathway-dependent manner. Loss of pE66L during ASFV infection had little effect on virus replication, but significantly recovered host protein synthetic. Based on the above results, our findings expand our view of ASFV in determining the fate of host-pathogen interactions.

African Swine Fever Virus Bearing an I226R Gene Deletion Elicits Robust Immunity in Pigs to African Swine Fever.

African swine fever (ASF) is a severe hemorrhagic infectious disease in pigs caused by African swine fever virus (ASFV), leading to devastating economic losses in epidemic regions. Its control currently depends on thorough culling and clearance of the diseased and surrounding suspected pigs. An ASF vaccine has been extensively explored for years worldwide, especially in hog-intensive areas where it is highly desired, but it is still unavailable for numerous reasons. Here, we report another ASF vaccine candidate, named SY18ΔI226R, bearing a deletion of the I226R gene with a replacement of an enhanced green fluorescent protein (eGFP) expression cassette at the right end of the viral genome. This deletion results in the complete loss of virulence of SY18 as the gene-deleted strain does not cause any clinical symptoms in all pigs inoculated with a dosage of either 104.0 or 107.0 50% tissue culture infective doses (TCID50). Apparent viremia with a gradual decline was monitored, while virus shedding was detected only occasionally in oral or anal swabs. ASFV-specific antibody appeared at 9 days postinoculation. After intramuscular challenge with its parental strain ASFV SY18 at 21 days postinoculation, all the challenged pigs survived, without obvious febrile or abnormal clinical signs. No viral DNA could be detected upon the dissection of any tissue when viremia disappeared. These results indicated that SY18ΔI226R is safe in swine and elicits robust immunity to virulent ASFV infection. IMPORTANCE Outbreaks of African swine fever have resulted in devastating losses to the swine industry worldwide, but there is currently no commercial vaccine available. Although several vaccine candidates have been reported, none has been approved for use for several reasons, especially ones concerning biosafety. Here, we identified a new undescribed functional gene, I226R. When deleted from the ASFV genome, the virus completely loses its virulence in swine. Importantly, pigs infected with this gene-deleted virus were resistant to infection by intramuscular challenge with 102.5 or 104.0 TCID50 of its virulent parental virus. Furthermore, the nucleic acid of the gene-deleted virus and its virulent parental virus was rarely detected from oral or anal swabs. Viruses could not be detected in any tissues after necropsy when viremia became negative, indicating that robust immunity was achieved. Therefore, SY18ΔI226R is a novel, ideal, and efficacious vaccine candidate for genotype II ASF.

Comparison of immune responses to attenuated rabies virus and street virus in mouse brain.

Rabies is a lethal neurological disease caused by the neurotropic rabies virus (RABV). To investigate the innate immune response in the brain during rabies infection, key gene transcripts indicative of innate immunity in a mouse model system were measured using real-time RT-PCR. Mice were infected via the intracerebral or intramuscular route with either attenuated rabies virus (SRV9) or pathogenic rabies virus (BD06). Infection with SRV9 resulted in the early detection of viral replication and the rapid induction of innate immune response gene expression in the brain. BD06 infection elicited innate immune response gene expression during only the late stage of infection. We measured Na-fluorescein uptake to assess blood-brain barrier (BBB) permeability, which was enhanced during the early stage of SRV9 infection and significantly enhanced during the late stage of BD06 infection. Furthermore, early SRV9 replication increased the maturation and differentiation of dendritic cells (DCs) and B cells in the inguinal lymph nodes and initiated the generation of virus-neutralizing antibodies (VNAs), which cooperate with the innate immune response to eliminate virus from the CNS. However, BD06 infection did not stimulate VNA production; thus, the virus was able to evade the host immune response and cause encephalitis. The rabies virus phosphoprotein has been reported to counteract IFN activation. In an in vitro study of the relationship between IFN antagonism and RABV pathogenicity, we demonstrated that SRV9 more strongly antagonized IFN activity than did BD06. Therefore, there is no positive relationship between the IFN antagonist activity of the virus and its pathogenicity.

Rabies virus matrix protein induces apoptosis by targeting mitochondria.

Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination.

Pseudorabies in farmed foxes fed pig offal in Shandong province, China.

Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.

A natural reassortant and mutant serotype 3 reovirus from mink in China.

Mammalian orthoreoviruses (MRVs) are widespread and infect virtually all mammals. We report here the first case of a natural mutant and reassortant serotype 3 reovirus from mink in China, known as MRV3 SD-14. Whole-genome sequence analysis showed that the MRV3 SD-14 may have resulted from a reassortment involving MRVs that infected swine, humans and mink. Interestingly, the S1 segment, which encodes the viral attachment protein σ1, which influences viral virulence and cell tropism in the host, had a stop codon mutation at amino acid 246. Surveillance of the virulence and evolution of MRVs in humans and other animals deserves more attention.

Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

Novel calicivirus from a ferret badger (Melogale moschata) in China.

We describe the isolation and complete genome sequence of a new calicivirus, FBCV-JX12, isolated from a ferret badger (Melogale moschata). Comparison of FBCV-JX12 with other vesiviruses revealed that it shared the highest amino acid sequence identities of 71.6, 60.5, and 59.3% in the nonstructural protein, VP1, and VP2, respectively, with MCV-DL2007 (mink calicivirus). Phylogenetic analysis of the whole genomic sequence showed that it clustered most closely with MCV-DL2007 of the genus Vesivirus, but with low nucleotide similarity in the three open reading frames (62.1-68.5%).

Novel circovirus from mink, China.

A long-established epidemic of enteritis, caused by an unidentified pathogen distinct from parvovirus, has now been recognized in mink. In 2013, we identified a novel circovirus by degenerate PCR and fully sequenced its genome. This virus differs substantially from currently known members of the genus Circovirus and represents a new species.

Molecular characterization of three ferret badger (Melogale moschata) rabies virus isolates from Jiangxi province, China.

Ferret badger (FB) rabies viruses JX09-17(fb), JX09-18 and JX10-37 were isolated from three different regions in Jiangxi province, China, in 2009 and 2010. The complete nucleotide sequence identity between these three isolates was 87-93 %. Compared with the other Chinese rabies virus isolates and vaccine strains, 101 substitutions (53 in JX10-37, 23 in JX09-17(fb) and 25 in JX09-18) in the five structural proteins were observed, and 47 of these substitutions (27 in JX10-37, 14 in JX09-17(fb) and 6 in JX09-18) were unique among lyssaviruses. Amino acid substitutions of S231 and Q333 were noted respectively in the G protein antigenic site I of JX10-37 and site III in JX09-17(fb). Phylogenetic analysis showed that JX09-17(fb) is rooted within the China I lineage, JX09-18 is in China II, and JX10-37 is independent. Evolutionary analysis and comparative sequence data indicate that isolate JX10-37 is a variant virus that diverged from canine rabies viruses around 1933 (range 1886-1963).

Molecular characterization of a rabies virus isolate from a rabid dog in Hanzhong District, Shaanxi Province, China.

A canine rabies virus, Shaanxi-HZ-6, was isolated in Shaanxi Province, China, in 2009. Its genome has been completely sequenced and found to be closely related to the China I rabies virus strains widely circulating in China. The genomic length was 11,923 base pairs, and the overall organization of the genome was similar to that of other rabies virus isolates. Compared with isolates CQ92 and J, 84 amino acid substitutions (7 in the N gene, 15 in P, 6 in M, 25 in G, 31 in L) were observed in strain Shaanxi-HZ-6. Amino acid substitutions of R264H and V332I were noted in the G protein antigenic site I and site III, respectively. Residue 333 of the G protein, which is considered to be associated with pathogenicity, was Arg in Shaanxi-HZ-6. These and other substitutions may help provide an explanation why the China I lineage strain maintains its prevalence in China.

Early diagnosis of Irkut virus infection using magnetic bead-based serum peptide profiling by MALDI-TOF MS in a mouse model.

Early diagnosis is important for the prompt post-exposure prophylaxis of lyssavirus infections. To diagnose Irkut virus (IRKV) infection during incubation in mice, a novel method using magnetic bead-based serum peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been established. For this test, serum peptides were concentrated by adsorption to and elution from the magnetic bead-based weak cation ion exchanger. Mass spectrograms obtained by MALDI-TOF MS were analyzed using ClinProTools bioinformatics software. Construction of the diagnostic model was performed using serum samples from mice infected with IRKV and rabies virus (RABV) BD06, Flury-LEP, and SRV9 (as controls). The method accurately diagnosed sera 2, 4 and 8 days after IRKV and RABV infections. The sensitivity, specificity, and total accuracy of diagnosis were 86.7%, 95.2%, and 92.9%, respectively. However, IRKV could not be differentiated from RABV 1 day after infection. The results of the present study indicate that serum peptide profiling by MALDI-TOF MS is a promising technique for the early clinical diagnosis of lyssavirus infections and needs to be further tested in humans and farm animals.

Deletion of the B125R gene in the African swine fever virus SY18 strain leads to an A104R frameshift mutation slightly attenuating virulence in domestic pigs.

African swine fever (ASF), caused by the ASF virus (ASFV), is a hemorrhagic and fatal viral disease that affects Eurasian wild boars and domestic pigs, posing a substantial threat to the global pig breeding industry. ASFV, a double-stranded DNA virus, possesses a large genome containing up to 160 open reading frames, most of which exhibit unknown functions. The B125R gene of ASFV, located at the 105595-105972 bp site in the ASFV-SY18 genome, remains unexplored. In this study, we discovered that B125R deletion did not affect recombinant virus rescue, nor did it hinder viral replication during the intermediate growth phase. Although the virulence of the recombinant strain harboring this deletion was attenuated, intramuscular inoculation of the recombinant virus in pigs at doses of 102 or 104 TCID50 resulted in mortality. Moreover, sequencing analysis of six recombinant strains obtained from three independent experiments consistently revealed an adenine insertion at the 47367-47375 bp site in the A104R gene due to the B125R deletion, leading to premature termination of this gene. Intriguingly, this insertion did not influence the transcription of the A104R gene between the recombinant and parental strains. Consequently, we postulate that the deletion of the B125R gene in ASFV-SY18 or other genotype II strains may marginally attenuate virulence in domestic pigs.

Deleting the C84L Gene from the Virulent African Swine Fever Virus SY18 Does Not Affect Its Replication in Porcine Primary Macrophages but Reduces Its Virulence in Swine.

(1) Background: African swine fever (ASF) is a highly contagious disease that causes high pig mortality. Due to the absence of vaccines, prevention and control are relatively challenging. The pathogenic African swine fever virus (ASFV) has a complex structure and encodes over 160 proteins, many of which still need to be studied and verified for their functions. In this study, we identified one of the unknown functional genes, C84L. (2) Methods: A gene deficient strain was obtained through homologous recombination and several rounds of purification, and its replication characteristics and virulence were studied through in vitro and in vivo experiments, respectively. (3) Results: Deleting this gene from the wild-type virulent strain SY18 did not affect its replication in porcine primary macrophages but reduced its virulence in pigs. In animal experiments, we injected pigs with a 102 TCID50, 105 TCID50 deletion virus, and a 102 TCID50 wild-type strain SY18 intramuscularly. The control group pigs reached the humane endpoint on the ninth day (0/5) and were euthanized. Two pigs in the 102 TCID50(2/5) deletion virus group survived on the twenty-first day, and one in the 105 TCID50(1/5) deletion virus group survived. On the twenty-first day, the surviving pigs were euthanized, which was the end of the experiment. The necropsies of the survival group and control groups' necropsies showed that the surviving pigs' liver, spleen, lungs, kidneys, and submaxillary lymph nodes did not show significant lesions associated with the ASFV. ASFV-specific antibodies were first detected on the seventh day after immunization; (4) Conclusions: This is the first study to complete the replication and virulence functional exploration of the C84L gene of SY18. In this study, C84L gene was preliminarily found not a necessary gene for replication, gene deletion strain SY18ΔC84L has similar growth characteristics to SY18 in porcine primary alveolar macrophages. The C84L gene affects the virulence of the SY18 strain.

Identification of L11L and L7L as virulence-related genes in the African swine fever virus genome.

Introduction: African swine fever (ASF) is an infectious disease that causes considerable economic losses in pig farming. The agent of this disease, African swine fever virus (ASFV), is a double-stranded DNA virus with a capsid membrane and a genome that is 170-194 kb in length encoding over 150 proteins. In recent years, several live attenuated strains of ASFV have been studied as vaccine candidates, including the SY18ΔL7-11. This strain features deletion of L7L, L8L, L9R, L10L and L11L genes and was found to exhibit significantly reduced pathogenicity in pigs, suggesting that these five genes play key roles in virulence. Methods: Here, we constructed and evaluated the virulence of ASFV mutations with SY18ΔL7, SY18ΔL8, SY18ΔL9, SY18ΔL10, and SY18ΔL11L. Results: Our findings did not reveal any significant differences in replication efficiency between the single-gene deletion strains and the parental strains. Pigs inoculated with SY18ΔL8L, SY18ΔL9R and SY18ΔL10L exhibited clinical signs similar to those inoculated with the parental strains. Survival rate of pigs inoculated with 103.0TCID50 of SY18ΔL7L was 25%, while all pigs inoculated with 103.0TCID50 of SY18ΔL11L survived, and 50% inoculated with 106.0TCID50 SY18ΔL11L survived. Discussion: The results indicate that L8L, L9R and L10L do not affect ASFV SY18 virulence, while the L7L and L11L are associated with virulence.

Molecular characterization of an outbreak-involved Bacillus anthracis strain confirms the spillover of anthrax from West Africa.

BACKGROUND: Anthrax, a zoonotic disease caused by the spore-forming bacterium Bacillus anthracis, remains a major global public health concern, especially in countries with limited resources. Sierra Leone, a West African country historically plagued by anthrax, has almost been out of report on this disease in recent decades. In this study, we described a large-scale anthrax outbreak affecting both animals and humans and attempted to characterize the pathogen using molecular techniques. METHODS: The causative agent of the animal outbreak in Port Loko District, Sierra Leone, between March and May 2022 was identified using the nanopore sequencing technique. A nationwide active surveillance was implemented from May 2022 to June 2023 to monitor the occurrence of anthrax-specific symptoms in humans. Suspected cases were subsequently verified using quantitative polymerase chain reaction. Full-genome sequencing was accomplished by combining long-read and short-read sequencing methods. Subsequent phylogenetic analysis was performed based on the full-chromosome single nucleotide polymorphisms. RESULTS: The outbreak in Port Loko District, Sierra Leone, led to the death of 233 animals between March 26th and May 16th, 2022. We ruled out the initial suspicion of Anaplasma species and successfully identified B. anthracis as the causative agent of the outbreak. As a result of the government's prompt response, out of the 49 suspected human cases identified during the one-year active surveillance, only 6 human cases tested positive, all within the first month after the official declaration of the outbreak. The phylogenetic analysis indicated that the BaSL2022 isolate responsible for the outbreak was positioned in the A.Br.153 clade within the TransEuroAsian group of B. anthracis. CONCLUSIONS: We successfully identified a large-scale anthrax outbreak in Sierra Leone. The causative isolate of B. anthracis, BaSL2022, phylogenetically bridged other lineages in A.Br.153 clade and neighboring genetic groups, A.Br.144 and A.Br.148, eventually confirming the spillover of anthrax from West Africa. Given the wide dissemination of B. anthracis spores, it is highly advisable to effectively monitor the potential reoccurrence of anthrax outbreaks and to launch campaigns to improve public awareness regarding anthrax in Sierra Leone.

Novel orthoreovirus from mink, China, 2011.

We identified a novel mink orthoreovirus, MRV1HB-A, which seems to be closely related to human strain MRV2tou05, which was isolated from 2 children with acute necrotizing encephalopathy in 2005. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent.

Evaluation of rabies biologics against Irkut virus isolated in China.

An Irkut virus (IRKV) was recently isolated from a bat in China. The protective ability of rabies biologics available in the Chinese market and experimental biologics against the rabies virus (RABV) and IRKV were assessed in a hamster model via preexposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) experiments. The results demonstrated that a single dose of rabies vaccine did not induce adequate protection against IRKV infection. However, routine PrEP with three doses of vaccine induced complete protection against IRKV infection. Higher doses of RABV immunoglobulins and alpha interferon were required during PEP to protect hamsters against IRKV versus RABV infection. Experimental recombinant vaccines containing IRKV glycoproteins induced more-reliable protection against IRKV than against RABV infection. Those findings may be explained by limited cross-neutralization of these viruses (confirmed via in vitro tests) in conjunction with antigenic distances between RABV and IRKV. These results indicate that the development and evaluation of new biologics for PrEP and PEP are required to ensure sufficient protection against IRKV infection in China and other territories where this virus is present.

Analysis of the complete genome of the first Irkut virus isolate from China: comparison across the Lyssavirus genus.

The genome of Irkut virus, isolate IRKV-THChina12, the first non-rabies lyssavirus from China (of bat origin), has been completely sequenced. In general, coding and non-coding regions of this viral genome are similar to those of other lyssaviruses. However, alignment of the deduced amino acid sequences of the structural proteins of IRKV-THChina12 with those of other lyssavirus representatives revealed significant variability between viral species. The nucleoprotein and matrix protein were found to be the most conserved, followed by the large protein, glycoprotein and phosphoprotein. Differences in the antigenic sites in glycoprotein may result in only partial protection of the available rabies biologics against Irkut virus, which is of particular concern for pre- and post-exposure rabies prophylaxis.

Motor imagery electroencephalogram classification algorithm based on joint features in the spatial and frequency domains and instance transfer.

Introduction: Motor imagery electroencephalography (MI-EEG) has significant application value in the field of rehabilitation, and is a research hotspot in the brain-computer interface (BCI) field. Due to the small training sample size of MI-EEG of a single subject and the large individual differences among different subjects, existing classification models have low accuracy and poor generalization ability in MI classification tasks. Methods: To solve this problem, this paper proposes a electroencephalography (EEG) joint feature classification algorithm based on instance transfer and ensemble learning. Firstly, the source domain and target domain data are preprocessed, and then common space mode (CSP) and power spectral density (PSD) are used to extract spatial and frequency domain features respectively, which are combined into EEG joint features. Finally, an ensemble learning algorithm based on kernel mean matching (KMM) and transfer learning adaptive boosting (TrAdaBoost) is used to classify MI-EEG. Results: To validate the effectiveness of the algorithm, this paper compared and analyzed different algorithms on the BCI Competition IV Dataset 2a, and further verified the stability and effectiveness of the algorithm on the BCI Competition IV Dataset 2b. The experimental results show that the algorithm has an average accuracy of 91.5% and 83.7% on Dataset 2a and Dataset 2b, respectively, which is significantly better than other algorithms. Discussion: The statement explains that the algorithm fully exploits EEG signals and enriches EEG features, improves the recognition of the MI signals, and provides a new approach to solving the above problem.