Applying improved ddPCR to reliable quantification of MPXV in clinical settings.

Monkeypox virus (MPXV) poses a global health threat. Droplet digital PCR (ddPCR) holds potential as an accurate diagnostic tool for clinical microbiology. However, there is limited literature on the applicability of ddPCR in clinical settings. In this study, the clinical features of patients with MPXV during the initial outbreak in China in June 2023 were reviewed, and an optimized ddPCR method with dilution and/or inhibitor removal was developed to enhance MPXV detection efficiency. Eighty-two MPXV samples were tested from nine different clinical specimen types, including feces, urine, pharyngeal swabs, anal swabs, saliva, herpes fluid, crust, and semen, and the viral load of each specimen was quantified. A comparative analysis was performed with qPCR to assess sensitivity and specificity and to investigate the characteristics of MPXV infection by analyzing viral loads in different clinical specimens. Consequently, common pharyngeal and gastrointestinal symptoms were observed in patients with MPXV. The optimized ddPCR method demonstrated relatively high sensitivity for MPXV quantification in the clinical materials, with a limit of detection of 0.1 copies/µL. This was particularly evident in low-concentration samples like whole blood, semen, and urine. The optimized ddPCR demonstrated greater detection accuracy compared with normal ddPCR and qPCR, with an area under the curve (AUC) of 0.939. Except for crust samples, viral loads in the specimens gradually decreased as the disease progressed. Virus levels in feces and anal swabs kept a high detection rate at each stage of post-symptom onset, and feces and anal swabs samples may be suitable for clinical diagnosis and continuous monitoring of MPXV. IMPORTANCE: The ddPCR technique proved to be a sensitive and valuable tool for accurately quantifying MPXV viral loads in various clinical specimen types. The findings provided valuable insights into the necessary pre-treatment protocols for MPXV diagnosis in ddPCR detection and the potentially suitable sample types for collection. Therefore, such results can aid in comprehending the potential characteristics of MPXV infection and the usage of ddPCR in clinical settings.

RNA binding motif 4 inhibits the replication of ebolavirus by directly targeting 3'-leader region of genomic RNA.

Ebola virus (EBOV) belongs to Filoviridae family possessing single-stranded negative-sense RNA genome, which is a serious threat to human health. Nowadays, no therapeutics have been proven to be successful in efficiently decreasing the mortality rate. RNA binding proteins (RBPs) are reported to participate in maintaining cell integrity and regulation of viral replication. However, little is known about whether and how RBPs participate in regulating the life cycle of EBOV. In our study, we found that RNA binding motif protein 4 (RBM4) inhibited the replication of EBOV in HEK293T and Huh-7 cells by suppressing viral mRNA production. Such inhibition resulted from the direct interaction between the RRM1 domain of RBM4 and the "CU" enrichment elements located in the PE1 and TSS of the 3'-leader region within the viral genome. Simultaneously, RBM4 could upregulate the expression of some cytokines involved in the host innate immune responses to synergistically exert its antiviral function. The findings therefore suggest that RBM4 might serve as a novel target of anti-EBOV strategy.

Case Report: First case of HIV, hepatitis B, hepatitis C, and Vibrio vulnificus coinfection.

Our patient, a 48-year-old man from Guangdong's coastal region, worked selling and processing oysters and other seafood. He started experiencing swelling and pain in his left knee on October 4, 2022, and they got worse over time. The findings of mNGS test showed Vibrio vulnificus infection. The patient had AIDS, hepatitis A and hepatitis B concurrently. He was admitted to the hospital's intensive care unit (ICU) for treatment as his symptoms worsened. We refrained from performing an amputation because the family members expressed a desire to keep the limb. The limb was managed with regular dressing changes, thorough debridement, wound closure, ongoing VSD drainage, and local antibiotic irrigation. The patient's organ function eventually returned to normal, and the systemic infection got better. On November 1, the wound's new granulation tissue had grown well and had gradually crept to cover 80% of the wound. The tissue's blood flow had also improved, indicating a trend of growth and healing.

Residues 318 and 323 in capsid protein are involved in immune circumvention of the atypical epizootic infection of infectious bursal disease virus.

Recently, atypical infectious bursal disease (IBD) caused by a novel variant infectious bursal disease virus (varIBDV) suddenly appeared in immunized chicken flocks in East Asia and led to serious economic losses. The epizootic varIBDV can partly circumvent the immune protection of the existing vaccines against the persistently circulating very virulent IBDV (vvIBDV), but its mechanism is still unknown. This study proved that the neutralizing titer of vvIBDV antiserum to the epizootic varIBDV reduced by 7.0 log2, and the neutralizing titer of the epizootic varIBDV antiserum to vvIBDV reduced by 3.2 log2. In addition, one monoclonal antibody (MAb) 2-5C-6F had good neutralizing activity against vvIBDV but could not well recognize the epizootic varIBDV. The epitope of the MAb 2-5C-6F was identified, and two mutations of G318D and D323Q of capsid protein VP2 occurred in the epizootic varIBDV compared to vvIBDV. Subsequently, the indirect immunofluorescence assay based on serial mutants of VP2 protein verified that residue mutations 318 and 323 influenced the recognition of the epizootic varIBDV and vvIBDV by the MAb 2-5C-6F, which was further confirmed by the serial rescued mutated virus. The following cross-neutralizing assay directed by MAb showed residue mutations 318 and 323 also affected the neutralization of the virus. Further data also showed that the mutations of residues 318 and 323 of VP2 significantly affected the neutralization of the IBDV by antiserum, which might be deeply involved in the immune circumvention of the epizootic varIBDV in the vaccinated flock. This study is significant for the comprehensive prevention and control of the emerging varIBDV.

Identification and Pathogenicity Evaluation of a Novel Reassortant Infectious Bursal Disease Virus (Genotype A2dB3).

Infectious bursal disease virus (IBDV) is a non-enveloped, bi-segmented double-stranded RNA virus and the causative agent of a poultry immunosuppressive disease known as infectious bursal disease (IBD). The novel variant IBDV (nVarIBDV) recently posed a great threat to the development of the poultry industry. In this study, we identified a novel segment-reassortant IBDV strain, IBDV-JS19-14701 (Genotype A2dB3). Phylogenic analysis showed that Segments A and B of IBDV-JS19-14701 were derived from emerging nVarIBDV (Genotype A2dB1) and long-prevalent HLJ0504-like strains (Genotype A3B3) in China, respectively. The pathogenicity of IBDV-JS19-14701 was further evaluated via animal experiments. IBDV-JS19-14701 exhibited a similar virulence to chickens with the nVarIBDV. The identification of this reassortment event is beneficial for understanding the epidemiology of nVarIBDV and will contribute to the efficient prevention and control of IBD.

TRIM25 inhibits infectious bursal disease virus replication by targeting VP3 for ubiquitination and degradation.

Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3-6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3.

Development of a Viral-Like Particle Candidate Vaccine Against Novel Variant Infectious Bursal Disease Virus.

Infectious bursal disease (IBD), an immunosuppressive disease of young chickens, is caused by infectious bursal disease virus (IBDV). Novel variant IBDV (nVarIBDV), a virus that can evade immune protection against very virulent IBDV (vvIBDV), is becoming a threat to the poultry industry. Therefore, nVarIBDV-specific vaccine is much needed for nVarIBDV control. In this study, the VP2 protein of SHG19 (a representative strain of nVarIBDV) was successfully expressed using an Escherichia coli expression system and further purified via ammonium sulfate precipitation and size-exclusion chromatography. The purified protein SHG19-VP2-466 could self-assemble into 25-nm virus-like particle (VLP). Subsequently, the immunogenicity and protective effect of the SHG19-VLP vaccine were evaluated using animal experiments, which indicated that the SHG19-VLP vaccine elicited neutralization antibodies and provided 100% protection against the nVarIBDV. Furthermore, the protective efficacy of the SHG19-VLP vaccine against the vvIBDV was evaluated. Although the SHG19-VLP vaccine induced a comparatively lower vvIBDV-specific neutralization antibody titer, it provided good protection against the lethal vvIBDV. In summary, the SHG19-VLP candidate vaccine could provide complete immune protection against the homologous nVarIBDV as well as the heterologous vvIBDV. This study is of significance to the comprehensive prevention and control of the recent atypical IBD epidemic.

A reassortment vaccine candidate of the novel variant infectious bursal disease virus.

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is the most important immunosuppressive disease threatening the poultry industry worldwide. Recently, the novel variant IBDV has been emerging in large-scale in Asia including China and is becoming a new threat to the healthy development of the poultry industry, but no ideal vaccine is available. Therefore, it is necessary and urgent to develop a new vaccine against the novel variant IBDV. In this study, based on the skeleton of an attenuated vaccine strain Gt, a reassortment virus strain rGtVarVP2 was constructed for the first time, which could express the main protective antigen VP2 of the novel variant IBDV and replicate well in cell culture. Subsequently, the safety and effectiveness of rGtVarVP2 were further evaluated using animal experiments. The rGtVarVP2 is nonpathogenic to specific-pathogen-free (SPF) chicken. The immunization of rGtVarVP2 could induce the specific neutralizing antibodies against the novel variant IBDV. The challenge protection tests further confirmed the effectiveness of the IBDV reassortment virus rGtVarVP2. No atrophy and obvious lesions were observed in the immunization group while the bursae of non-immunization control group were severely destroyed after challenge, which showed that rGtVarVP2 could provide complete protection against the novel variant IBDV. These data indicate that the vaccine candidate (rGtVarVP2 strain) is safe and effective, which is of great significance for comprehensive control of IBD and healthy breeding.

Novel variant infectious bursal disease virus suppresses Newcastle disease vaccination in broiler and layer chickens.

Newcastle disease (ND) is one of the most important avian diseases that seriously threaten the poultry industry worldwide. Sometimes vaccination might not effectively reduce ND occurrence in some poultry farms for unclear reasons. Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases, and the novel variant IBD virus (IBDV) is threatening chicken farms in China. This study evaluated the influence of the novel variant IBDV (SHG19 strain) on immunization of ND vaccine (LaSota strain) in broiler and layer chickens. In commercial broilers, the hemagglutination inhibition antibody titers against LaSota strain were decreased by the prior infection of the novel variant IBDV. Pathological examination revealed that the novel variant IBDV severely damaged the key immune organs of the bursa and spleen, and the B lymphocytes in the bursa were severely destroyed, which was the primary reason involved in the immunosuppression on ND vaccination. Moreover, the novel variant IBDV dramatically reduced the BW of infected broilers by about 16% compared to that of control, which indicated huge economic losses. Furthermore, this study confirmed the immunosuppression induced by the novel variant IBDV in specific pathogen-free layer chickens. In this study, it was discovered that the novel variant IBDV could interfere with ND vaccination in both broilers and layers, which was one important factor involved in immune failure in poultry farms. This study therefore suggests the urgency to control the novel variant IBDV infection for healthy breeding.

Naturally occurring homologous recombination between novel variant infectious bursal disease virus and intermediate vaccine strain.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD), an important immunosuppressive disease seriously threatening poultry farming worldwide. Since the identification of the classic strain in 1957, variant IBDV, very virulent IBDV, and novel variant IBDV have successively emerged brought severe challenges. Over the years, attenuated, intermediate, and intermediate-plus live vaccines have been developed to control the disease. The coexistence of various strains in flocks increases the probability of homologous recombination, and in this study, a naturally occurring homologous recombination between a novel variant strain and an intermediate vaccine strain of IBDV was first identified. Sequence analyses demonstrated that the IBD16HeN01 strain was a recombinant IBDV incorporating the skeleton of the novel variant IBDV (SHG19-like strain), where the 3' region of segment A (nt 1539-3260) was replaced by an intermediate vaccine strain (W2512-like strain). Pathogenicity experiments indicated that IBD16HeN01 could cause severe bursal lesions and the recombination increased viral pathogenicity to chick embryos compared with the novel variant IBDV. Homologous recombination in IBDV has increased the complexity of disease prevention and control and reminds us that we should use live vaccines more scientifically and cautiously.

Naturally occurring cell-adapted classic strain of infectious bursal disease virus.

Infectious bursal disease virus (IBDV), the etiological agent of infectious bursal disease (IBD), is a variable RNA virus of Avibirnavirus. Some artificially attenuated vaccine strains of IBDV can adapt to cell culture of chicken embryo fibroblast (CEF) cell or its immortalized cell line DF1 in vitro while wild-type IBDV cannot. In this study, for the first time, a naturally occurring cell-adapted classic strain (genogroup 1) of IBDV named IBD17JL01 was identified in China. Animal experiments showed that IBD17JL01 could severely damage the central immune organ of infected chickens. Sequence analysis of the full-length genome revealed the peculiar molecular characteristics of IBD17JL01 with a few amino acid substitutions that might be involved in cell-tropism, antigenicity, and virulence of IBDV. Identification of this novel strain is beneficial to our understanding of the complexity of the epidemiology of IBDV. And the expansion of viral cell-tropism might increase the potential risk of the reassortment of different IBDVs including the live vaccines.

Novel variants of infectious bursal disease virus can severely damage the bursa of fabricius of immunized chickens.

In recent years, atypical infectious bursal disease (IBD) with severe immunosuppression has brought new threats to the poultry industry and has caused considerable economic losses. Novel variant infectious bursal disease virus (IBDV) has been identified as the etiological pathogen and for unknown reasons is widespread in poultry on many chicken farms in China that have been immunized with vaccines against very virulent IBDV (vvIBDV). Using immunoprotection experiments in specific-pathogen-free chickens, we first verified that novel variant IBDV could severely damage the bursa of Fabricius of the important immune organ of immunized chicken in the presence of antibodies induced by three types of vvIBDV vaccines, which is a primary reason for the current epidemic of atypical IBD. Monoclonal antibody reactivity patterns and cross-neutralization assays further confirmed the obvious antigenic mismatch between novel variant IBDV and vvIBDV. Sequence analysis of the genome of novel variant IBDV (SHG19 strain) was performed and the key amino acid residues that might be involved in antigenicity and virulence differences of novel variant IBDV compared to vvIBDV were further analyzed. This study not only determined the primary reason for the atypical IBD epidemic, but also remind us of the urgency for developing new vaccines against novel variant IBDV.

Novel variant strains of infectious bursal disease virus isolated in China.

Infectious bursal disease (IBD) is one of the most important immunosuppressive diseases that seriously threaten poultry farming and food safety worldwide. The variant strain of infectious bursal disease virus (IBDV) has been greatly neglected for more than 30 years. Recently, the subclinical infection of suspected IBD, causing considerable economic losses, occurred in the main chicken-farming regions of China. Through RT-PCR, sequencing, and phylogenic analyses, novel variant IBDVs were first identified in six provinces of eastern China. Immunological detection further confirmed the antigenic variation of the Chinese variant IBDVs. The Chinese IBDV variants were obviously different from the American IBDV variants, with less than a 97.7% (VP1) or 98.7% (VP2) amino acid sequence identity. Animal experiments further confirmed the serious threat of the variant IBDVs to chickens, demonstrating irreversible damage to the central immune organ, obvious immunosuppression, and growth retardation. This study not only identified the pandemic nature of the novel variant IBDVs for the first time but also discovered the distinct molecular epidemiological characteristics of these viruses, which will contribute more to the control of the disease.