A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains.

BACKGROUND: Originating in Africa, African swine fever (ASF) was introduced to China in 2018. This acute and highly virulent infectious disease affects domestic pigs. The World Organization for Animal Health has listed it as a statutory reportable disease, and China has listed it as a category A infectious disease. METHODS: Primers and probes were designed for four ASFV genes (B646L, EP402R, MGF505-3R, and A137R). The primers/probes were highly conserved compared with the gene sequences of 21 ASFV strains. RESULTS: After optimization, the calibration curve showed good linearity (R2 > 0.99), the minimum concentration of positive plasmids that could be detected was 50 copies/µL, and the minimum viral load detection limit was 102 HAD50/mL. Furthermore, quadruple quantitative polymerase chain reaction (qPCR) with nucleic acids from three porcine-derived DNA viruses and cDNAs from eight RNA viruses did not show amplification curves, indicating that the method was specific. In addition, 1 × 106, 1 × 105, and 1 × 104 copies/µL of mixed plasmids were used for the quadruple qPCR; the coefficient of variation for triplicate determination between groups was < 2%, indicating the method was reproducible. CONCLUSIONS: The results obtained by testing clinical samples containing detectable EP402R, MGF505-3R, and A137R strains with different combinations of gene deletions were as expected. Therefore, the established quadruple qPCR method was validated for the molecular diagnosis of ASF using gene-deleted ASFV strains.

Development of a quadruple PCR-based gene microarray for detection of vaccine and wild-type classical swine fever virus, African swine fever virus and atypical porcine pestivirus.

BACKGROUND: Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. METHODS: A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. RESULTS: This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875-2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. CONCLUSION: This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

Epitope-specific CD4+, but not CD8+, T-cell responses induced by recombinant influenza A viruses protect against Mycobacterium tuberculosis infection.

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4(+) and CD8(+) T-cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4(+) T-cell epitope from the Ag85B protein (PR8.p25) or CD8(+) T-cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T-cell responses to the M. tuberculosis-specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly-functional CD4(+) T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN-γ-producing M. tuberculosis-specific CD8(+) T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope-specific CD4(+), but not CD8(+) T cells, is essential for protection against acute M. tuberculosis infection in the lung.

Influenza A virus infection impairs mycobacteria-specific T cell responses and mycobacterial clearance in the lung during pulmonary coinfection.

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

miRNA modulation of SOCS1 using an influenza A virus delivery system.

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-ß cytokines.

Infection of domestic pigs with a genotype II potent strain of ASFV causes cytokine storm and lymphocyte mass reduction.

The whole-genome sequence of an African swine fever virus (ASFV) strain (HuB/HH/2019) isolated from Hubei, China, was highly similar to that of the Georgia 2007/1 strain ASFV. After infection with strong strains, domestic pigs show typical symptoms of infection, including fever, depression, reddening of the skin, hemorrhagic swelling of various tissues, and dysfunction. The earliest detoxification occurred in pharyngeal swabs at 4 days post-infection. The viral load in the blood was extremely high, and ASFV was detected in multiple tissues, with the highest viral loads in the spleen and lungs. An imbalance between pro- and anti-inflammatory factors in the serum leads to an excessive inflammatory response in the body. Immune factor expression is suppressed without effectively eliciting an immune defense. Antibodies against p30 were not detected in acutely dead domestic pigs. Sequencing of the peripheral blood mononuclear cell transcriptome revealed elevated transcription of genes associated with immunity, defense, and stress. The massive reduction in lymphocyte counts in the blood collapses the body's immune system. An excessive inflammatory response with a massive reduction in the lymphocyte count may be an important cause of mortality in domestic pigs. These two reasons have inspired researchers to reduce excessive inflammatory responses and stimulate effective immune responses for future vaccine development.

A non-toxic recombinant Clostridium septicum α toxin induces protective immunity in mice and rabbits.

Clostridium septicum alpha toxin (CSA) plays significant roles in ruminant's braxy. Genetically engineered CSA has been shown to function as a potential vaccine candidate in the prevention of the disease caused by Clostridium septicum. In the present study, we synthesized a non-toxic recombinant, rCSAm4/TMD by introducing four amino acid substitutions (C86L/N296A/H301A/W342A) and 11-amino-acid deletion (residues 212 to 222). Compared to recombinant CSA, rCSAm4/TMD showed no cytotoxicity to MDCK cells and was not fatal to mice. Moreover, rCSAm4/TMD could protect immunized mice against 5 × mouse LD100 (100% lethal dose) of crude CSA without obvious pathological change. Most importantly, rabbits immunized with rCSAm4/TMD produced high titers of neutralizing antibodies which protected the rabbits against crude CSA challenge. These data suggest that genetically detoxified rCSAm4/TMD is a potential subunit vaccine candidate against braxy.

Comparative efficacy evaluation of different CSF vaccines in pigs with CSF maternally derived antibodies.

Classical swine fever (CSF) is a highly contagious and important swine disease in China. Sporadic outbreaks with mild clinical signs are still being reported despite massive vaccination with the CSF C-strain vaccine. One possible reason for vaccine failure could be interference from maternally derived antibodies (MDAs) during vaccination in the field. The aim of this study was to evaluate the efficacy of different CSF vaccines in the presence of MDAs and to assess the different vaccination schemes in the field. The results demonstrated that vaccination with a single dose of C-strain-PK vaccine protected pigs against severe clinical signs and significantly reduced viremia. The impact of MDAs was negligible. The interference was also mild during a prime and boost vaccination scheme using the C-strain-ST vaccine. In contrast, a significant influence of MDAs on the efficacy of the subunit E2 vaccine in a one-dose vaccination scheme was observed, with pigs showing severe clinical signs, CSF-associated death, typical pathological lesions and a high level of viremia after challenge, despite robust E2 antibody induction. A field vaccination and challenge study further confirmed the superior effectiveness of a single dose of C-strain-PK vaccine in the presence of MDAs in comparison to a routine prime and boost vaccination scheme applied in the field, with pigs having fever, chronic signs, significant viremia and shedding after challenge. Delaying the vaccination time from the age of 28 days to 45 days, when MDA was low, was beneficial for improving the clinical protection and immunity induced by vaccines. Altogether, the results presented here emphasize that a high-quality vaccine and a scientific design of the vaccination scheme based on serological surveillance are essential pillars to control and eliminate CSF in China.

Boosting BCG with recombinant influenza A virus tuberculosis vaccines increases pulmonary T cell responses but not protection against Mycobacterium tuberculosis infection.

The current Mycobacterium bovis BCG vaccine provides inconsistent protection against pulmonary infection with Mycobacterium tuberculosis. Immunity induced by subcutaneous immunization with BCG wanes and does not promote early recruitment of T cell to the lungs after M. tuberculosis infection. Delivery of Tuberculosis (TB) vaccines to the lungs may increase and prolong immunity at the primary site of M. tuberculosis infection. Pulmonary immunization with recombinant influenza A viruses (rIAVs) expressing an immune-dominant M. tuberculosis CD4+ T cell epitope (PR8-p25 and X31-p25) stimulates protective immunity against lung TB infection. Here, we investigated the potential use of rIAVs to improve the efficacy of BCG using simultaneous immunization (SIM) and prime-boost strategies. SIM with parenteral BCG and intranasal PR8-p25 resulted in equivalent protection to BCG alone against early, acute and chronic M. tuberculosis infection. Boosting BCG with rIAVs increased the frequency of IFN-γ-secreting specific T cells (p<0.001) and polyfunctional CD4+ T cells (p<0.05) in the lungs compared to the BCG alone, however, this did not result in a significant increase in protection against M. tuberculosis compared to BCG alone. Therefore, sequential pulmonary immunization with these rIAVs after BCG increased M. tuberculosis-specific memory T cell responses in the lung, but not protection against M. tuberculosis infection.

Molecular characterization, receptor binding property, and replication in chickens and mice of H9N2 avian influenza viruses isolated from chickens, peafowls, and wild birds in eastern China.

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Serologic evidence of prevalent avian H3 subtype influenza virus infection in chickens.

H3-subtype influenza viruses are known to infect avian and mammalian species, including humans. However, little is known about the prevalence of H3 influenza virus infection in chicken populations in China. Therefore, a serologic survey of chickens was conducted in China to investigate the seroprevalence of avian H3-subtype influenza virus. Anti-H3 antibodies were assayed by using hemagglutination inhibition (HI) and confirmatory virus neutralization (VN) testing of 4598 serum samples, collected between July 2006 and June 2007, from 173 chicken flocks located in 18 areas that included 16 provinces and two municipalities. Seroepidemiologic results indicated that avian H3-subtype viruses were circulating in chickens in some regions of China, regions that included 12 of the 18 test areas, with an overall average prevalence rate of 2.83%. Samples from 44 of 173 flocks were HI/VN seropositive, including 15 flocks with levels that ranged from 10.00% to 41.94%. Significantly higher seroprevalence rates were observed in older chicken flocks and in those sampled in the cooler seasons. Standardized comparisons showed that Guangdong and Jiangsu, located in the south and east of China, respectively, had significantly higher levels of H3 seropositivity. For the first time, these results demonstrated serologic evidence for H3 avian influenza virus infection in chicken populations in several locations throughout China. These observations highlight the need for continued epidemiologic surveillance of the H3 subtype and for other low-pathogenic avian influenza viruses in China and other regions.

Complete Genome Characterization of a Novel Infectious Bursal Disease Virus Strain Isolated from a Chicken Farm in China.

Infectious bursal disease (IBD) is a highly infectious disease in chicken, and vaccination is the best way to prevent outbreak of infectious bursal disease virus (IBDV). In this study, we isolated a variant IBDV strain from a chicken farm with vaccinated chickens. The full genome of this IBDV strain was determined and analyzed.

Complete Genome Sequence of an Avian Polyomavirus Strain First Isolated from a Pigeon in China.

Avian polyomavirus can infect multiple bird species and cause inflammatory disease with high mortality in young psittacine birds. In this study, we sequenced and analyzed an avian polyomavirus isolated from a pigeon in China, strain APV-P, which is closely related to a polyomavirus in psittacine birds.

Complete Genome Sequence of Genotype Psittacine Beak and Feather Disease Virus, a Strain Identified from Budgerigars in China.

Psittacine beak and feather disease virus (PBFDV) has been reported in many countries, such as Australia, Poland, the United States, South Africa, etc. In this study, the complete genome of a PBFDV isolate was determined and characterized from budgerigars in China.

Pulmonary immunization with a recombinant influenza A virus vaccine induces lung-resident CD4+ memory T cells that are associated with protection against tuberculosis.

The lung is the primary site of infection with the major human pathogen, Mycobacterium tuberculosis. Effective vaccines against M. tuberculosis must stimulate memory T cells to provide early protection in the lung. Recently, tissue-resident memory T cells (TRM) were found to be phenotypically and transcriptional distinct from circulating memory T cells. Here, we identified M. tuberculosis-specific CD4+ T cells induced by recombinant influenza A viruses (rIAV) vaccines expressing M. tuberculosis peptides that persisted in the lung parenchyma with the phenotypic and transcriptional characteristics of TRMs. To determine if these rIAV-induced CD4+ TRM were protective independent of circulating memory T cells, mice previously immunized with the rIAV vaccine were treated with the sphingosine-1-phosphate receptor modulator, FTY720, prior to and during the first 17 days of M. tuberculosis challenge. This markedly reduced circulating T cells, but had no effect on the frequency of M. tuberculosis-specific CD4+ TRMs in the lung parenchyma or their cytokine response to infection. Importantly, mice immunized with the rIAV vaccine were protected against M. tuberculosis infection even when circulating T cells were profoundly depleted by the treatment. Therefore, pulmonary immunization with the rIAV vaccine stimulates lung-resident CD4+ memory T cells that are associated with early protection against tuberculosis infection.

Enhanced immunogenicity following miR-155 incorporation into the influenza A virus genome.

Influenza A vaccine efficacy in the elderly is generally poor and so identification of novel molecular adjuvants to improve immunogenicity is important to reduce the overall burden of disease. Short non-coding RNAs, known as microRNAs (miRNAs) are known to regulate gene expression and have the potential to influence immune responses. One such miRNA, miR-155, has been shown to modulate T and B cell development and function. We incorporated miR-155 into the influenza A virus (IAV) genome creating a self-adjuvanting 'live vaccine' with the ability to modify immunogenicity. Infection of mice with a recombinant influenza virus encoding miR-155 in the NS gene segment altered epitope-specific expansion of influenza-specific CD8+ T cells and induced significantly higher levels of neutralising antibody.

Identification of an emerging recombinant cluster in porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is continuously evolving through point mutation and genome recombination. In the present study, genetic affiliations of 40 PCV2 strains were determined by amplification, sequencing and phylogenetic analyses during the nationwide molecular epidemiology investigation from 2009 to 2010 in mainland China. The results revealed that PCV2b was the predominant genotype in mainland China from 2009 to 2010 and PCV2b-1C within PCV-2b genotype was an emerging predominant subtype. More interestingly, five strains (09HaiN-1, 09HaiN-2, 10AH, 10GX and 10QH) were classified into a novel cluster along with the two main PCV2 genotypes PCV2a and PCV2b. Further analyses revealed that this novel cluster arose from recombination between PCV2a and PCV2b stains within the ORF2 gene. Moreover, BLAST search on NCBI website revealed that PCV2 strains of the novel cluster also emerged in Thailand, Indonesia and Laos, indicating that the novel cluster of PCV2 has also been circulating in some other Asian countries. This study is the first time to perform comprehensive analyses to demonstrate a cluster of PCV2 strains derived from the same type of inter-genotypic recombination pattern. Our findings provide valuable information on PCV2 evolution.

The epidemic status and genetic diversity of 14 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolates from China in 2009.

A high-mortality swine disease, the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS), reappeared in some regions of China in 2009. To explore the possible mechanisms underlying the emergence of HP-PRRSV and more fully understand the extent of the genetic diversity of this virus in China, the complete genome of 14 isolates from 10 provinces in China from 2009 were analyzed. Full-length genome sequencing analysis showed that the 14 isolates were closely related to HP-PRRSV, with 98.0-98.9% nucleotide similarity, although 2 of the 14 strains exhibited a new, discontinuous 29-amino acid deletion in the Nsp2 gene. Furthermore, amino acid analysis of the GP5 protein indicated that the 14 isolates had a concurrent mutation in a decoy epitope and different mutations in glycosylation sites. Additionally, the antigenic drift in GP3 and a 1-nucleotide deletion in both the 5'-UTR and 3'-UTR, which are found in almost all highly pathogenic Chinese PRRSV isolates, were examined in all 14 isolates. The phylogenetic analysis showed that the 14 strains belonged to the North American genotype and were clustered in a subgroup with other HP-PRRSV isolates that have been found in China since 2006. However, compared with other Chinese HP-PRRSV isolates collected in 2006-2008, the phylogenetic tree showed that the 14 isolates had a closer relationship with each other. These results indicated that HP-PRRSV remained an extensive pandemic, affecting swine farms in China in 2009 and revealed new genetic diversity.

Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in northern China has not been undertaken since 2004. Here, using the sequences of 22 viruses we isolated from poultry and pigs in northern China during 2003-2008, in combination with sequences available in a public database, we analyzed the evolution of H9N2 influenza viruses in China from 1994 to 2008. Our findings demonstrated that the H9N2 viruses in China underwent extensive reassortment, and novel genotypes continued to emerge. Among 330 viruses, 54 genotypes were observed including 19 novel genotypes that have not been recognized before, and major genotypes were further divided into five series (BJ/94-, G1-, BG-, F/98- and Aq-series). Different epidemiological and biological features among these series were recognized. The BJ/94- and F/98-series viruses were circulating in both southern and northern China, while the other three series viruses were mainly detected in southern China. BJ/94-series influenza viruses predominated in China before 2000 and were gradually replaced by F/98-series viruses that became the predominant viruses since 2004. At least five antigenic groups could be identified over the study period, during which a significant antigenic drift likely occurred between 2002 and 2003. Animal experiments demonstrated that F/98-series viruses were able to replicate and transmit more effectively in chickens than BJ/94-series viruses. The continuing evolution of H9N2 influenza viruses in China emphasizes the importance of H9N2 influenza virus surveillance throughout this region to aid pandemic prediction and prevention.

Genetic analysis of H3 subtype influenza viruses isolated from domestic ducks in northern China during 2004-2005.

The broad distribution and prevalence of H3 subtype influenza viruses in avian and mammalian hosts constitutes a global threat to both human and veterinary health. In this present study, six H3N8 influenza viruses isolated from domestic ducks during 2004-2005 in northern China were genetically and phylogenetically characterized. Sequence analysis showed that HA, NA, and M genes of all the six H3N8 isolates had a close relationship with those of Equine/Jilin/1/89 (H3N8) virus, which once caused outbreak in equine populations in northern China. The PB2 and PA genes of the viruses possessed the highest similarities with highly pathogenic avian H5N1 influenza viruses currently circulating in this region. These findings emphasize the importance of avian influenza virus surveillance in this region for understanding the genesis and emergency of novel reassortants with pandemic potential.