Microfluidic-LAMP chip for the point-of-care detection of gene-deleted and wild-type African swine fever viruses and other four swine pathogens.

Introduction: Different pathogens causing mixed infection are now threatening the pig industry in the context of the African Swine Fever (ASF) circulating especially in China, and it is crucial to achieving the early diagnosis of these pathogens for disease control and prevention. Methods: Here we report the development of a rapid, portable, sensitive, high-throughput, and accurate microfluidic-LAMP chip detection system for simultaneous detection and differentiation of gene-deleted type and wild-type African swine fever virus (ASFV), pseudorabie virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome (PRRSV). Results and discussion: The newly developed system was shown to be sensitive with detection limits of 101 copies/µl for ASFV-MGF505-2R/P72, PPV, and PCV2, 102 copies/µl for ASFV-CD2v, PRV, and PRRSV. The system was highly specific (100%) and stable (C.V.s < 5%) in its ability to detect different pathogens. A total 213 clinical samples and 15 ASFV nucleic acid samples were collected to assess the performance of the detection system, showing highly effective diagnosis. Altogether, the developed microfluidic-LAMP chip system provides a rapid, sensitive, high-throughput and portable diagnostic tool for the accurate detection of multiple swine pathogens.

Protection efficacy of the H1 and H3 bivalent virus-like particle vaccine against swine influenza virus infection.

Swine influenza (SI) is widely prevalent in pig herds worldwide, causing huge economic losses to the pig industry and public health risks. The traditional inactivated swine influenza virus (SIV) vaccines are produced in chicken embryos, and egg-adaptive substitutions that occur during production process can impact vaccine effectiveness. Thus, developing an SI vaccine that can decrease the dependence on chicken embryos with a high immunogenicity is urgently needed. In this study, the utility of insect cell-derived SIV H1 and H3 bivalent virus-like particle (VLP) vaccines containing HA and M1 proteins of Eurasian avian-like (EA) H1N1 SIV and recent human-like H3N2 SIV were assessed in piglets. Antibody levels were monitored, and the protection efficacy of the vaccine after viral challenge was evaluated and compared with the inactivated vaccine. Results show that piglets produced high hemagglutination inhibition (HI) titers of antibodies against H1 and H3 SIV after immunization with SIV VLP vaccine. The neutralizing antibody level was significantly higher in SIV VLP vaccine than in the inactivated vaccine at 6 weeks post vaccination (p < 0.05). Furthermore, piglets immunized with the SIV VLP vaccine were protected against the challenge of H1 and H3 SIV, displaying inhibition of viral replication in piglets, and reduced lung damage. These results show that SIV VLP vaccine has good application prospects, thus laying the foundation for further research and commercialization of SIV VLP vaccine.

Genetic characterization of four strains porcine circovirus-like viruses in pigs with diarrhea in Hunan Province of China.

In this study, we detected a circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus [named Po-Circo-like (PCL) virus] in intestinal tissue and fecal samples of pigs. PCL virus contains a single-stranded DNA genome, and ORF1 encodes the Rep and not the typical capsid protein encoded in PCV. The Rep protein may be responsible for viral genome replication. In addition, PCL virus may be one of the pathogens causing diarrhea symptoms in pigs. We identified four strains of PCL virus in two different pig farms with severe diarrhea outbreaks in Hunan Province, China. The strains in this study share 85.7-99.7% nucleic acid identity and 84.7-100% amino acid identity with Rep of the reference strains. A multiple sequence alignment of these PCL viruses and Bo-Circo-like CH showed a identity of 93.2% for the Rep protein, and the nucleotide identity was 86.7-89.3%. Moreover, Bo-Circo-like CH and HN75, HN39-01, HN39-02 had similar stem-loop sequences. In conclusion, the present study is the first detailed report of the PCL virus in Hunan provinces, which is a potential new virus in pigs that might be involved in cross-species transmission. Further investigation is needed to determine the pathogenesis of this virus and its epidemiologic impact.

Panax Notoginseng Saponins Suppress Type 2 Porcine Reproductive and Respiratory Syndrome Virus Replication in vitro and Enhance the Immune Effect of the Live Vaccine JXA1-R in Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate immune response in the host, reducing and delaying neutralizing antibody production against PRRSV infection and promoting viral infection. Here, we aimed to assess the potential of Panax notoginseng saponins (PNS) for improving the immune response exerted upon PRRSV-2-modified live virus (MLV) vaccine administration. Thirty piglets were randomly divided into six groups. Group 1 piglets were injected with medium 0 days post vaccination (dpv). Group 2 piglets were fed PNS 0-28 dpv. Group 3 and group 4 piglets were administered the JXA1-R vaccine 0 dpv. Group 4 piglets were also fed PNS 0-28 dpv. Group 1-4 piglets were challenged intranasally with the PRRSV JXA1 strain 28 dpv. Group 5 piglets were fed with PNS without challenge. Group 6 piglets served as controls. During the experiment, the samples were collected regularly for 49 days. Compared with group 1 piglets, group 3 piglets showed significantly reduced viremia and clinical scores, and significantly increased average daily gain (ADWG). Compared with group 3 piglets, group 4 piglets showed significantly improved neutralizing antibody titers, IFN-α and IFN-ß mRNA expression, and significantly decreased viremia and viral load in the lungs and lymph nodes, but did not demonstrate any further improvement in PRRSV-specific antibody titer, rectal temperature, ADWG, or clinical scores. PNS upregulates neutralizing antibodies against PRRSV-2 and enhances the expression of IFN-α and IFN-ß, which may reduce PRRSV viremia upon PRRSV-2 MLV vaccine administration. PNS may serve as an effective immunomodulator for boosting the immune defense against PRRSV.

The Prevalence of Porcine Circovirus-like Viruses in China Presents New Challenges to the Diagnosis of Diarrhea-Associated Viruses. Comment on Yang et al. Epidemiology and Evolution of Emerging Porcine Circovirus-like Viruses in Pigs with Hemorrhagic Dysentery and Diarrhea Symptoms in Central China from 2018 to 2021. Viruses 2021, 13, 2282.

Recently, a report in Viruses has highlighted the problem of porcine circovirus-like (PCL) virus [...].

Analysis of Interaction Network Between Host Protein and M Protein of Swine Acute Diarrhea Syndrome Coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enterovirus that can cause acute diarrhea and death in piglets and cause serious economic losses to the pig industry. SADS-CoV membrane (M) protein mainly plays a key role in biological processes, such as virus assembly, budding, and host innate immune regulation. Understanding the interaction between M protein and host proteins is very important to define the molecular mechanism of cells at the protein level and to understand specific cellular physiological pathways. In this study, 289 host proteins interacting with M protein were identified by glutathione-S-transferase (GST) pull-down combined with liquid chromatography-mass spectrometry (LC-MS/MS), and the protein-protein interaction (PPI) network was established by Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways analysis. Results showed that SADS-CoV M protein was mainly associated with the host metabolism, signal transduction, and innate immunity. The Co-Immunoprecipitation (CO-IP) validation results of six randomly selected proteins, namely, Rab11b, voltage-dependent anion-selective channel 1 (VDAC1), Ribosomal Protein L18 (RPL18), RALY, Ras Homolog Family Member A (RHOA), and Annexin A2 (ANXA2), were consistent with LC-MS results. In addition, overexpression of RPL18 and PHOA significantly promoted SADS-CoV replication, while overexpression of RALY antagonized viral replication. This work will help to clarify the function of SADS-CoV M protein in the life cycle of SADS-CoV.

Protective effect of bivalent H1N1 and H3N2 VLP vaccines against Eurasian avian-like H1N1 and recent human-like H3N2 influenza viruses in a mouse model.

Eurasian avian-like (EA) H1N1 swine influenza viruses (SIVs) are currently the most prevalent SIVs in Chinese swine populations, but recent human-like H3N2 SIV subtypes have also been frequently isolated. Hence, there is an urgent need to develop an effective vaccine against both EA H1N1 and recent human-like H3N2 infections. In this study, we utilized the baculovirus expression system to produce virus-like particles (VLPs) containing hemagglutinin protein (HA) and matrix protein (M1) based on A/Swine/Guangdong/YJ4/2014 (H1N1) and A/swine/Guangdong/L22/2010 (H3N2). An immunological experiment showed that in a mouse model, bivalent VLP vaccines against H1N1 and H3N2 can induce stronger humoral and cellular immune responses than whole influenza virus vaccines. Compared with monovalent inactivated vaccines that cannot offer protection against different SIV subtypes, monovalent H1N1 or H3N2 VLP vaccines can provide partial protection against lethal challenge by viruses of different subtypes. Meanwhile, bivalent VLP vaccines against H1N1 and H3N2 can provide full protection against lethal doses of homologous and heterologous viruses belonging to the EA H1N1 or recent human-like H3N2 lineage. These results suggest a promising approach to the development of vaccines against SIVs.

A Method for the Analysis of African Swine Fever by Viral Metagenomic Sequencing.

In 2018, there was an outbreak of African swine fever (ASF) in China, which spread to other provinces in the following 3 years and severely damaged China's pig industry. ASF is caused by the African swine fever virus (ASFV). Given that the genome of the African swine fever virus is very complex and whole genome information is currently inadequate, it is important to efficiently obtain virus genome sequences for genomic and epidemiological studies. The prevalent ASFV strains have low genetic variability; therefore, whole genome sequencing analysis provides a basis for the study of ASFV. We provide a method for the efficient sequencing of whole genomes, which requires only a small number of tissues. The database construction method was selected according to the genomic types of ASFV, and the whole ASFV genome was obtained through data filtering, host sequence removal, virus classification, data assembly, virus sequence identification, statistical analysis, gene prediction, and functional analysis. Our proposed method will facilitate ASFV genome sequencing and novel virus discovery.

The Addition of Nature Identical Flavorings Accelerated the Virucidal Effect of Pure Benzoic Acid against African Swine Fever Viral Contamination of Complete Feed.

African swine fever virus is one of the most highly contagious and lethal viruses for the global swine industry. Strengthening biosecurity is the only effective measure for preventing the spread of this viral disease. The virus can be transmitted through contaminated feedstuffs and, therefore, research has been conducted to explore corresponding mitigating measures. The purpose of the current study was to test a combination of pure benzoic acid and a blend of nature identical flavorings for their ability to reduce African swine fever viral survival in feed. This virus was inoculated to feed with or without the supplementation of the test compounds, and the viral presence and load were measured by a hemadsorption test and quantitative real time polymerase chain reaction. The main finding was that the combination of pure benzoic acid and nature identical flavorings could expedite the reduction in both viral load and survival in a swine feed. Therefore, this solution could be adopted as a preventive measure for mitigating the risk of contaminated feed by African swine fever virus.

Ser-Leu substitution at P2 position of the hemagglutinin cleavage site attenuates replication and pathogenicity of Eurasian avian-like H1N2 swine influenza viruses.

Swine influenza viruses not only constitute a potential economic problem for livestock, but also pose a substantial threat to human health. Mutation in the proteolytic cleavage site of hemagglutinin (HA) is recognized as an essential factor of tissue tropism and viral pathogenicity. However, the molecular properties of the cleavage site of Eurasian avian-like swine (EA) H1N2 virus remain largely unknown. In this study, we found a serine-leucine (Ser-Leu) substitution at the P2 position of the HA cleavage site (S328 L) in naturally occurring EA H1N2 virus. To study the effect of this substitution, we used reverse genetics to generate recombinant wild-type and mutant viruses containing a single amino acid mutation at the P2 position in A/swine/Guangdong/YJ28/2014 (YJ28) or A/swine/Guangdong/DG2/2015 (DG2) background. In vitro experiments showed that the Ser-Leu substitution at the P2 position attenuated the viral replication and HA cleavage efficiency. In vivo analyses revealed that, while all mice inoculated with r/DG2-S328 L or r/YJ28 viruses survived, the survival rates of r/DG2- and r/YJ28-L328S-inoculated animals were 20 % and 40 %, respectively. Furthermore, the Ser-Leu substitution at the P2 position attenuated the replication in nasal turbinate and lungs. In summary, this amino acid change may be useful to understand the molecular properties of the cleavage site and be valuable for vaccine development.

Effects of physical and chemical factors on pseudorabies virus activity in vitro.

BACKGROUND: Pseudorabies (PR) is latent and can persist in infected sows for a long time, and thus, convalescent sows can carry the virus throughout life, causing severe economic losses to farmers and posing a tremendous challenge to PR prevention and control. Here, to investigate the biological characteristics of pseudorabies virus (PRV), a variety of physical and chemical factors were analyzed under controlled conditions. RESULTS: The results showed that a high ambient temperature and dry environment led to faster virus inactivation. PRV had a certain resistance to weakly acidic or alkaline environments and was rapidly inactivated in strongly acidic or alkaline environments. The effect of ultraviolet (UV) radiation on PRV activity primarily depended on the frequency, intensity, and irradiation time of the UV exposure. Exposure to sunlight inactivated PRV via multiple factors, including temperature, sunlight intensity, UV intensity, and environmental humidity, and any shielding from sunlight strongly lowered the killing effect. Conventional disinfectants had a good disinfection effect on PRV. CONCLUSIONS: The biological characteristics of different PRV strains are variable. Generally, the activity of PRV is affected by multiple factors, which can show both synergy and antagonism. Real-world conditions should be taken into consideration to guide pork production.

Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies.

Pseudorabies, also known as Aujezsky's disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

Phylogeography, phylodynamics and the recent outbreak of lineage 3 porcine reproductive and respiratory syndrome viruses in China.

Novel highly pathogenic porcine reproductive and respiratory syndrome viruses (PRRSVs) have attracted increasing attention owing to their continual high emergence and recent re-emergence. Recently, lineage 3 PRRSVs, belonging to the type 2 viruses, with novel characteristics and increased virulence have been continuously re-emerging in China, thereby posing a great threat to pig farming. However, available information about lineage 3 is limited. Here, we carried out molecular epidemiological investigations for PRRSV surveillance in most regions of China from 2007 to 2017. More than 3,000 samples were obtained, amounting to 73 sequences of lineage 3 viruses. The origin, demographic history and spread pattern of lineage 3 PRRSVs were investigated combining with the database globally. Phylogeography and phylodynamic analyses within a Bayesian statistical framework revealed that lineage 3 viruses originated in Taiwan. Followed by subsequent propagation to different areas and geography, it dichotomized into two endemic clusters. South China has become an epicentre for these viruses, which diffused into China's interiors in recent years. Furthermore, viral dispersal route analysis revealed the risk of viral diffusion. Overall, the origin, epidemic history and geographical evolution of lineage 3 PRRSVs were comprehensively analysed in this study. In particular, the epicentre of southern China and the diffusion routines of the viruses are highlighted in this study, and the possible continuous transmission of the novel lineages poses the biggest threat to pig farmers.

The phosphorylation of the N protein could affect PRRSV virulence in vivo.

The porcine respiratory and reproductive syndrome virus (PRRSV) nucleocapsid (N) protein is a multiphosphorylated protein.It has been proved that the phosphorylation of N protein could regulate the growth ability of PRRSV in Marc-145 cells. However, further investigation is needed to determine whether phosphorylation of the N protein could affect PRRSV virulence in piglets. In this study, we confirmed that the mutations could impair PRRSV replication ability in porcine primary macrophages (PAMs) as they did in Marc-145 cells. The animal experiments suggested that the pathogenicity of the mutated virus (A105-120) was significantly reduced compared with parent strain (XH-GD). Our results suggested that the phosphorylation of the N protein contributes to virus replication and virulence. This study is the first to identify a specific modification involved in PRRSV pathogenicity. Mutation of PTMs sites is also a novel way to attenuate PRRSV virulence. The mutations could be a marker in a vaccine. In conclusion, our study will improve our understanding of the molecular mechanisms of PRRSV pathogenicity.

Emergence of novel recombination lineage 3 of porcine reproductive and respiratory syndrome viruses in Southern China.

Lineage 3 of porcine reproductive and respiratory syndrome viruses, which belong to North America type 2, has a long epidemic history in China. The novel lineage 3 viruses constantly emerging in recent years are characterized by a high detection rate and significant pathogenicity. In this study, we investigated the prevalence of lineage 3 in southern China and selected two isolated strains for genome and virulence analyses. A cross-sectional epidemiology investigation indicated that the prevalence of lineage 3 antigens was 35.68% (95% CI: 27.6-44.3%) among 227 samples collected from over 100 infected farms from January 2016 to July 2017 in southern China. Two novel isolates of lineage 3 were selected. After 20 passages, Marc-145 cells were not susceptible to those viruses. Full-length genome analysis indicated that the two strains share 95.2% homology with each other and 95.7%-96.2% with highly pathogenic porcine reproductive and respiratory syndrome viruses (HP-PRRSVs; JXA1-like strain, lineage 8.7). Phylogenetic and molecular evolutionary results showed that for the two isolates, HP-PRRSV provides most of the ORF1 gene. Animal experiment revealed discrepancies in virulence between the strains. Although challenge resulted in 100% morbidity, the isolate carrying most of the HP-PRRSV ORF1 caused severe clinical symptoms and 40% mortality, whereas the other isolate containing part of the ORF1 gene caused no mortality. Overall, these findings suggest that lineage 3 viruses might be commonly circulating in most of southern China. Frequent recombination events within HP-PRRSVs of this lineage with changing virulence could represent potential threats to the pig industry.

Phages bearing specific peptides with affinity for porcine reproductive and respiratory syndrome virus GP4 protein prevent cell penetration of the virus.

Porcine reproductive and respiratory syndrome (PRRS) has caused significant economic losses to the pig industry worldwide over the last 30 years. GP4 is a minor highly glycosylated structural protein composed of 187 and 183 amino acids in types I and II porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The GP4 protein co-localizes with cluster of differentiation 163 (CD163), the major receptor on the target cell membrane, to mediate PRRSV internalization and disassembly. However, it remains to be established whether blocking interactions between GP4 and host cells can inhibit viral proliferation. In the present study, recombinant GP4 protein prepared and purified using the Escherichia coli system effectively recognized PRRSV-positive serum. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish PRRSV from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. The current study expands our understanding of PRRSV GP4 and its receptor CD163 and provides a fresh perspective for the development of novel peptide-based viral inhibition reagents.

Molecular evolution of H1N1 swine influenza in Guangdong, China, 2016-2017.

Swine are the main host of the H1N1 swine influenza virus (SIV), however, H1N1 can also infect humans and occasionally cause serious respiratory disease. To trace the evolution of the SIV in Guangdong, China, we performed an epidemic investigation during the period of 2016-2017. Nine H1N1 influenza viruses were isolated from swine nasal swabs. Antigenic analysis revealed that these viruses belonged to two distinct antigenic groups, represented by A/Swine/Guangdong/101/2016 and A/Swine/Guangdong/52/2017. Additionally, three genotypes, known as GD52/17-like, GD493/17-like and GD101/16-like, were identified by phylogenetic analysis. Importantly, the genotypes including a minimum of 4 pdm/09-origin internal genes have become prevalent in China in recent years. A total of 2966 swine serum samples were used to perform hemagglutination inhibition (HI) tests, and the results showed that the seroprevalence values of SW/GD/101/16 (32.2% in 2016, 32.1% in 2017) were significantly higher than the seroprevalence values of SW/GD/52/17 (18.0% in 2016, 16.7% in 2017). Our study showed that the three reassortant genotypes of H1N1 SIV currently circulating in China are stable, but H1N1pdm09 poses challenges to human health by the introduction of internal genes into these reassortant genotypes. Strengthening SIV surveillance is therefore critical for SIV control and minimizing its potential threat to public health.

Identification and characterization of two linear epitope motifs in hepatitis E virus ORF2 protein.

Hepatitis E virus (HEV) is responsible for hepatitis E, which represents a global public health problem. HEV genotypes 3 and 4 are reported to be zoonotic, and animals are monitored for HEV infection in the interests of public hygiene and food safety. The development of novel diagnostic methods and vaccines for HEV in humans is thus important topics of research. Opening reading frame (ORF) 2 of HEV includes both linear and conformational epitopes and is regarded as the primary candidate for vaccines and diagnostic tests. We investigated the precise location of the HEV epitopes in the ORF2 protein. We prepared four monoclonal antibodies (mAbs) against genotype 4 ORF2 protein and identified two linear epitopes, G438IVIPHD444 and Y457DNQH461, corresponding to two of these mAbs using phage display biopanning technology. Both these epitopes were speculated to be universal to genotypes 1, 2, 3, 4, and avian HEVs. We also used two 12-mer fragments of ORF2 protein including these two epitopes to develop a peptide-based enzyme-linked immunosorbent assay (ELISA) to detect HEV in serum. This assay demonstrated good specificity but low sensitivity compared with the commercial method, indicating that these two epitopes could serve as potential candidate targets for diagnosis. Overall, these results further our understanding of the epitope distribution of HEV ORF2, and provide important information for the development of peptide-based immunodiagnostic tests to detect HEV in serum.