Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

The proteomic analysis uncovers the cellular responses to the African swine fever virus membrane proteins p54, p17, and pB117L.

African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.

Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection.

African swine fever (ASF) caused by the African swine fever virus (ASFV) is a fatal and highly contagious disease of domestic pigs characterized by rapid disease progression and death within 2 weeks. How the immune cells respond to acute ASFV infection and contribute to the immunopathogenesis of ASFV has not been completely understood. In this study, we examined the activation, apoptosis, and functional changes of distinct immune cells in domestic pigs following acute infection with the ASFV CADC_HN09 strain using multicolor flow cytometry. We found that ASFV infection induced broad apoptosis of DCs, monocytes, neutrophils, and lymphocytes in the peripheral blood of pigs over time. The expression of MHC class II molecule (SLA-DR/DQ) on monocytes and conventional DCs as well as CD21 expression on B cells were downregulated after ASFV infection, implying a potential impairment of antigen presentation and humoral response. Further examination of CD69 and ex vivo expression of IFN-γ on immune cells showed that T cells were transiently activated and expressed IFN-γ as early as 5 days post-infection. However, the capability of T cells to produce cytokines was significantly impaired in the infected pigs when stimulated with mitogen. These results suggest that the adaptive cellular immunity to ASFV might be initiated but later overridden by ASFV-induced immunosuppression. Our study clarified the cell types that were affected by ASFV infection and contributed to lymphopenia, improving our understanding of the immunopathogenesis of ASFV.

Investigation on pseudorabies prevalence in Chinese swine breeding farms in 2013-2016.

Pseudorabies (PR) has been prevalent in Chinese swine breeding farms since the outbreak at the end of 2011. For investigating current prevalence of PR, a nationwide surveillance has been performed in this study. The swine serum samples were collected from 93, 100, 92, and 91 swine farms in China during 2013-2016, respectively. Since the extensive use of gE-deleted pseudorabies virus (PRV) vaccine, we could apply the PRV-gE antibody for determining wild-type virus infection and the PRV-gB antibody for evaluating vaccine immunization. The results were concluded as follows: (1) Nationally, the positive rate of PRV-gB was maintained at a high level (> 90%), while the positive rate of PRV-gE continued to decrease (from 22.17 to 13.14%). (2) The positive rates of PRV-gE were greatly varied in different geographical regions and swine farms (0~100%), while the positive rate of PRV-gB was generally high (> 90%). (3) The number of imported PRV attenuated vaccines were about twice that of domestic PRV attenuated vaccines, while the positive rate of PRV-gB was not significantly different (P > 0.05). (4) The performance of PR eradication developing or developed farms was better than the performance of common farms, with higher positive rate of PRV-gB (> 90%) and much lower positive rate of PRV-gE (nearly 0%).

Analysis of Genetic Variation of Two NADC30-like Strains of Porcine Reproductive and Respiratory Syndrome Virus in China.

BACKGROUND: NADC30-like strains of porcine reproductive and respiratory syndrome virus first appeared in Chinese swine herds in 2012. OBJECTIVE AND METHOD: To explore the possible genetic diversity of these strains, we sequenced and analyzed the complete genomes of two NADC30-like strains. These isolates shared 95.4% homology with NADC30. RESULT: The two strains displayed a discontinuous deletion of 131 amino acids in NSP2, mutations of amino acids in GP3 and GP5, and a 3-nucleotide deletion in the 3' untranslated region. Phylogenetic analysis showed that the two isolates formed a new branch and clustered in a subgroup with NADC30 isolates from North America. CONCLUSION: We conclude that the above two NADC30-like strains may have been introduced from North America to China, where they acquired new genetic diversity.

Genome characterization of two NADC30-like porcine reproductive and respiratory syndrome viruses in China.

BACKGROUND: The recent emergence of NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) in vaccinated pigs arose more attentions for the high incidents of mutation and recombination of PRRSVs. FINDINGS: In this study, we determined full-length genome sequences of two NADC30-like PRRSV isolates from recent PRRSV outbreaks in China. Phylogenetic analysis showed that these two isolates were clustered in an independent branch together with NADC30, an American isolate in 2008. Genetically, HNjz15 shared 95.6 % nucleotide similarity to NADC30 without any exotic gene insertion. By contrast, HNyc15 shared 93.8 % similarity to NADC30 with recombination with VR-2332 and CH-1a. Two more previously reported NADC30-like PRRSVs were also analyzed and had exotic gene insertions with different PRRSV strains in their nonstructural protein genes. CONCLUSIONS: The above results showed the increased mutation and recombination rates of NADC30-like PRRSV under current vaccination pressure and a more pressing situation for the PRRSV eradication and control in China.

Complete genomic characterization and genetic diversity of four European genotype porcine reproductive and respiratory syndrome virus isolates from China in 2011.

Porcine reproductive and respiratory syndrome (PRRS) is an emerging disease that has caused serious economic losses to the swine industry worldwide. In 2011, a nation-wide surveillance program investigated the prevalence of PRRS viruses (PRRSV) in Chinese breeding swine farms, and four European genotype PRRSV (PRRSV-Type 1) were successfully isolated. To explore the genetic diversity of PRRSV-Type 1 in China, these 4 viral strains were subjected to genome sequencing and analysis. The four isolates shared 87.4-90.7 % nucleotide homology with the Lelystad strain (PRRSV-Type 1 stereotype strain). NSP2, ORF3, and ORF4 were the most variable regions and contained discontinuous deletions or insertions when compared to other PRRSV-Type 1 strains. All isolates fell into separate branches of the subtype 1 of PRRSV-Type 1 phylogenetic tree. This analysis of emerging PRRSV-Type 1 strains revealed previously unrecorded genetic diversity. Close attention should be paid to the prevention and control of this evolving virus.

Assessment of the safety and efficacy of an attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus.

The safety and efficacy of the JXA1-R vaccine, an attenuated strain of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), were examined using an intramuscular challenge model in piglets. The JXA1-R vaccine was obtained by passing HP-PRRSV JXA1 through Marc-145 cells (82nd passage). Genomic sequence comparisons showed that strain JXA1-R and its parental strain, JXA1, differ by 47 amino acids, and most of these differences are scattered throughout the PRRSV genome. Four-week-old PRRSV-free piglets were inoculated intramuscularly with JXA1-R vaccine (10(3.0), 10(4.0), 10(5.0), 10(6.0), and 10(7.0) 50% tissue culture infective doses [TCID50]/ml for groups 1 to 5, respectively) and then challenged intramuscularly with the 5th passage virus of JXA1 virus (JXA1-F5, 3 ml × 10(4.5) TCID50/ml) 28 days after inoculation. The humoral immune response, swine growth, clinical signs, and differential organ lesions were monitored. The results showed that all vaccinated piglets had a perceptible humoral immune response to vaccination after day 7, which then promptly increased, almost reaching the maximum sample/positive (S/P) ratio value at 28 days postimmunization. Viremia detection indicated that the viral replication levels of the challenge virus in the immunized groups (immunization doses ≥10(4.0)/ml) were significantly lower than that of the virus-challenged unvaccinated control group. Piglets in groups 2 to 5 were effectively protected against lethal HP-PRRSV infection and did not show any obvious changes in body temperature or clinical signs of disease at any point during the experiment. However, two of five piglets in group 1 showed mild pathological lesions and transitory high fever. These results suggest that JXA1-R (TCID50/ml ≥10(4.0)) is sufficiently attenuated and can provide effective protection against the lethal wild-type HP-PRRSV.

Identification and genomic characterization of a novel porcine parvovirus (PPV6) in China.

BACKGROUND: Parvoviruses are classified into two subfamilies based on their host range: the Parvovirinae, which infect vertebrates, and the Densovirinae, which mainly infect insects and other arthropods. In recent years, a number of novel parvoviruses belonging to the subfamily Parvovirinae have been identified from various animal species and humans, including human parvovirus 4 (PARV4), porcine hokovirus, ovine partetravirus, porcine parvovirus 4 (PPV4), and porcine parvovirus 5 (PPV5). METHODS: Using sequence-independent single primer amplification (SISPA), a novel parvovirus within the subfamily Parvovirinae that was distinct from any known parvoviruses was identified and five full-length genome sequences were determined and analyzed. RESULTS: A novel porcine parvovirus, provisionally named PPV6, was initially identified from aborted pig fetuses in China. Retrospective studies revealed the prevalence of PPV6 in aborted pig fetuses and piglets(50% and 75%, respectively) was apparently higher than that in finishing pigs and sows (15.6% and 3.8% respectively). Furthermore, the prevalence of PPV6 in finishing pig was similar in affected and unaffected farms (i.e. 16.7% vs. 13.6%-21.7%). This finding indicates that animal age, perhaps due to increased innate immune resistance, strongly influences the level of PPV6 viremia. Complete genome sequencing and multiple alignments have shown that the nearly full-length genome sequences were approximately 6,100 nucleotides in length and shared 20.5%-42.6% DNA sequence identity with other members of the Parvovirinae subfamily. Phylogenetic analysis showed that PPV6 was significantly distinct from other known parvoviruses and was most closely related to PPV4. CONCLUSION: Our findings and review of published parvovirus sequences suggested that a novel porcine parvovirus is currently circulating in China and might be classified into the novel genus Copiparvovirus within the subfamily Parvovirinae. However, the clinical manifestations of PPV6 are still unknown in that the prevalence of PPV6 was similar between healthy pigs and sick pigs in a retrospective epidemiological study. The identification of PPV6 within the subfamily Parvovirinae provides further insight into the viral and genetic diversity of parvoviruses.

Pathogenic pseudorabies virus, China, 2012.

In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic.

Complete genome sequence of a European genotype porcine reproductive and respiratory syndrome virus in china.

Here, we report a novel European genotype porcine reproductive and respiratory syndrome virus (PRRSV) strain with 15 nucleotide deletions in the nonstructural protein 2 region and 3 nucleotide deletions in the overlapping regions of the open reading frame 3 (ORF3) and ORF4 regions. This study will aid in further exploration of the genetic and antigenic diversity of the European genotype of the PRRSV in China.

[Expression and identification of truncated Nsp7 protein of North American and Europe genotype porcine reproductive and respiratory syndrome virus].

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.

Two natural recombinant highly pathogenic porcine reproductive and respiratory syndrome viruses with different pathogenicities.

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a variant of type 2 PRRSV with high virulence. Genetic and pathogenic characteristics of HP-PRRSV vary rapidly during the evolution. In this study, we determined the complete genome of a HP-PRRSV isolate designated 10FUJ-2, which shared 98.34 % nucleotide identity with HP-PRRSV reference strain JXA1. Genomic analyses by phylogenetic tree and recombination detection program confirmed 10FUJ-2 to be a recombinant with 09JS and JXA1 as potential parental viruses. Furthermore, we identified that 10FUJ-2 has high virulence as similar as the parental viruses by animal challenge study. In addition, we found that SY0909 was also a recombination virus probably from JXA1 and NT0801, which has been reported to be low pathogenic. Recombination analysis also revealed that Glycoproteins GP2 to GP5 of HP-PRRSV might contain major virulence determinants. Identification of two natural recombinants with different virulence supports the notion that recombination is a driving force affecting HP-PRRSV pathogenicity and a common mechanism contributing to HP-PRRSV evolution.

A novel porcine circovirus type 2a strain, 10JS-2, with eleven-nucleotide insertions in the origin of genome replication.

Here, we report a novel porcine circovirus type 2a (PCV2a) strain with 11 nucleotides (nt) inserted in the origin of genome replication (Ori). This is the first report of a PCV2a strain with nucleotide insertion in Ori. Our study will help further epidemiological studies and extend our knowledge of evolutionary characteristics of PCV2.

Complete genome sequence of two novel Chinese virulent porcine reproductive and respiratory syndrome virus variants.

A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by a discontinuous 30-amino-acid deletion in its Nsp2-coding region, has been emerging in China since 2006. Here, we report the complete genomic sequence of two novel Chinese virulent PRRSV variants with additional NSP2-gene deletions, which will help us understand the molecular and evolutionary characteristics of PRRSV in Asia.

New genomic characteristics of highly pathogenic porcine reproductive and respiratory syndrome viruses do not lead to significant changes in pathogenicity.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) initially emerged in China and currently prevails in other Asian countries as well, resulting in immense economic losses. HP-PRRS virus (HP-PRRSV) has undergone rapid evolution since its first recognition in 2006. To analyze the genomic and pathogenic characteristics of 2010 HP-PRRSV, we tested 919 clinical samples collected from China, Laos and Vietnam, sequenced 29 complete genomes of HP-PRRSV isolates, and determined the pathogenicity of seven HP-PRRS viruses isolated from 2006 to 2010. HP-PRRSV was detected from 45.2% (415/919) samples, while only 0.1% (1/919) was classical PRRSV, indicating that HP-PRRSV isolates with a unique discontinuous deletion of 30 amino acids (aa) in non-structural protein 2 (Nsp2) are still the predominant viruses. 2010 HP-PRRSV together with 2009 HP-PRRSV isolates form a new evolutionary branch based on phylogenetic analyses. The numbers of potential N-glycosylation sites are variable in major glycoprotein GP5 but are conserved in minor glycoproteins GP2, GP3 and GP4. Pathogenicity studies showed that HP-PRRS viruses isolated from 2006 to 2010 maintain similar level of high pathogenicity, which caused high fever (>41°C for at least four days), 100% morbidity, and 40-100% mortality in 4-10 weeks old pigs. Real time monitoring information from this study could help to understand the genetic and pathogenic evolution of HP-PRRSV and assist in the control of HP-PRRS in Asia.

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.

Natural recombinants derived from different patterns of recombination between two PCV2b parental strains.

Porcine circovirus diseases (PCVD) are globally emerging diseases that have huge economic impacts on swine industry. Porcine circovirus type 2 (PCV2) is considered to be the essential primary causative agent of PCVD. In the present study, recombination analyses of PCV2 identified two possible recombination events with high confidence using recombination detection program, phylogenetic analysis and base-by-base comparison. These recombination events occurred between strains 09CQ (HQ395024) and ZhuJi2003 (AY579893), giving rise to two recombinants 09GS (HQ395028) and HN0907 (GU938303). Phylogenetic analyses of the parental strains at full length level suggest that natural recombination happened between PCV2b strains. Interestingly, recombination of the two parental strains yielded two recombinants through different recombination patterns with crossover regions mainly located in ORF1 and ORF2, respectively. These results demonstrate that recombination between PCV2b strains can occur both in non-structural protein coding region and structural protein coding region. Our study not only indicates that PCV2b strains can undergo recombination through a variety of patterns, but also suggests that recombination contributes to the genetic diversity of PCV2.

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.