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In this study, canine IFNγ was fused by a flexible linker with canine serum albumin to construct the fusion protein IFNγ-CSA for the purpose to design a long-acting canine IFNγ. The fusion protein was successfully expressed in baculovirus-infected Sf9 insect cells and was purified by salting-out and ion exchange chromatography. The IFNγ-CSA fusion possessed potent anti-viral assay against vesicular stomatitis virus in cultured cells. IFNγ-CSA was also stable at 37 °C up to 72 h compared with 8 h for IFNγ alone. In vivo pharmacokinetics demonstrated a significantly longer half-life for IFNγ-CSA (15.42 h) than for canine reIFNγ (1.51 h) in KM mice. These results indicate that IFNγ-CSA expression in the baculovirus system was successful and provide a promising long-acting cytokine for veterinary clinical applications.

Porcine circovirus type 3 (PCV3), which currently lacks effective preventive measures, has caused tremendous economic losses to the pig husbandry. Obtaining the strain of PCV3 is the key to preparing related vaccines and developing corresponding antiviral drugs. In this study, according to the linear sequence of PCV3 DNA published on GenBank, the sequence was rearranged with SnapGene gene-editing software, and after rearrangement, the HindIII restriction endonuclease site was added to the end of the linear DNA, so that both ends have the same restriction endonuclease site. On this basis, the rearranged linear DNA is obtained by gene synthesis, PCR amplification, DNA purification, etc., and is digested and connected in vitro to obtain cyclized DNA. PCV3 infectious clones were obtained by transfecting 3D4/21 cell lines. The obtained PCV3 was identified by PCR, Western blotting, and indirect immunofluorescence tests. The results showed that this study successfully obtained the strain of PCV3 in vitro. To further evaluate the pathogenicity of the obtained PCV3 infectious clones, this study established an animal model of Kunming mice infected with PCV3. The results of RT-PCR, Western blotting and immunohistochemistry showed that PCV3 can infect myocardium and alveoli of Kunming mice, but no PCV3 was detected in other tissues. The above studies indicate that PCV3 circular DNA can be used to construct PCV3 infectious clones. This research will provide a new method for the construction of circular DNA viruses and lay the foundation for the research and pathogenesis of PCV3 vaccine.

The objective of this study was to assess the genetic diversity of porcine reproductive and respiratory syndrome virus circulating in Fujian province (southeastern China). Based on 53 ORF5 nucleotide sequences collected from nine sites, both highly pathogenic (sublineage 8.7) and lineage 1 strains were circulating in Fujian in 2009-2014 along with lineages 3 and 5.1. Notably, the lineage 1 strains were closely related to the NADC30 strain circulating in North America and were the predominant strains in 2014. In addition, we found that nonstructural protein 2 (NSP2) was the most variable nonstructural protein in Fujian isolates, with a 36-amino-acid (aa) insertion and seven different deletions detected in the 53 sequences examined. Similarly, analysis of GP5 amino acid sequences showed that the isolates were highly variable in primary neutralizing epitopes. Interesting, FJ3.2 and FJ7-2 strains have the mutation N44K, but they exhibited high replication and high titers in MARC-145 and PAM cells. The complete genome sequences determined for 12 type 2 isolates were 82.1-99.3% identical and were 15,016-15,407 nucleotides (nt), in length excluding the poly(A) tail. The strains also shared 88.2-99.4% identity with strain VR2332 (the prototype North American strain), 83.4-99.2% identity with strain JXA1 (the prototype high-pathogenicity Chinese strain), 88.2-97.1% identity with strain CH-1a (the prototype classical Chinese strain), and 82.9-97.1% identity with strain NADC30 (the prototype NADC30-like strain). Strikingly, phylogenetic and molecular evolutionary analyses indicated that strain FJW05 is a spontaneous recombinant between a circulating lineage 1 virus and the vaccine strain JXA1-R, which is derived from the highly pathogenic strain JXA-1. Collectively, the data highlight the epidemiology of porcine reproductive and respiratory syndrome in Fujian and may aid in selecting a suitable vaccine for use on pig farms.

Porcine circovirus type 2 (PCV2) has been causing huge economic losses in Chinese swine herds since it was first identified in China in 1999. Genotypes of PCV2 except for PCV2c coexist in swine herds in China, which may facilitate virus recombination. In the current study, six novel PCV2 strains were detected in China, and these strains shared high nucleotide similarity of the Rep gene with the PCV2c strain DK1987PMWSfree and high homology of the Cap gene with PCV2d. Genome sequence analysis revealed that the complete genomes of these strains were 1767 nucleotides (nt) in length and shared 99.8%-99.9% nucleotide identity with each other and 91.7%-98.7% with representative strains. Phylogenetic analysis, sequencing analysis, base-by-base comparisons and comprehensive recombination analysis demonstrated that these six strains originated from recombination within the Rep gene between PCV2c and PCV2d strains. Surprisingly, further investigation through theoretical recombination analysis of Chinese PCV2 GenBank sequences showed that these novel patterns of recombinant PCV2 strains have been generated since 2010. Collectively, our findings provide additional evidence of inter-genotypic recombination of PCV2.

From 2014 to 2015, four novel highly pathogenic PRRS virus (HP-PRRSV) strains named 14LY01-FJ, 14LY02-FJ 15LY01-FJ, and 15LY02-FJ were isolated from high morbidity (100%) and mortality (40%-80%) in piglets and sows in Fujian Province. To further our knowledge about these novel virus strains, we characterized their complete genomes and determined their pathogenicity in piglets. Full-length genome sequencing analysis showed that these four isolates were closely related to type 2 (North American type, NA-type) isolates, with 88.1%-96.3% nucleotide similarity, but only 60.6%-60.8% homology to the Lelystad virus (LV) (European type, EU-type). The full length of the four isolates was determined to be 15017 or 15018 nucleotides (nt), excluding the poly(A) tail. Furthermore, the four isolates had three discontinuous deletions (aa 322-432, aa 483, and aa 504-522) within hypervariable region II (HV-II) of Nsp2, as compared to the reference strain VR-2332. This deletion pattern in the four isolates is consistent with strain MN184 and strain NADC30 isolated from America. Phylogenetic and molecular evolutionary analyses indicated that these virulent strains originated from a natural recombination event between the JXA1-like HP-PRRSV (JXA-1 is one of the earliest Chinese HP-PRRSV strains; sublineage 8.7) and the NADC30-like (lineage 1) PRRSV. Animal experiments demonstrated that these four strains caused significant weight loss and severe histopathological lung lesions as compared to the negative control group. High mortality rate (40% or 80%) was found in piglets infected with any one of the four strains, similar to that found with other Chinese HP-PRRSV strains. This study showed that the novel variant PRRSV was HP-PRRSV, and it is therefore critical to monitor PRRSV evolution in China and develop a method for controlling PRRS.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most devastating swine diseases worldwide, resulting in immense economic losses. PRRS virus (PRRSV) is divided into two major genotypes, European (type 1) and the North American (type 2). Type 1 PRRSV have recently emerged in Fujian province (South China), and this might have a significant impact on the Chinese pig industry. From 2013 to 2014, two type 1 PRRSV strains, named FJEU13 and FJQEU14, were isolated from piglets and sows with respiratory problems and reproductive disorders in Fujian province. The full genome length of the two isolates was 14,869-15,062 nucleotides (nt), excluding the poly(A) tail. These isolates shared 86.0-89.9% sequence identity with the prototypic strains Lelystad virus (LV) and 82.8-92% with Chinese type 1 PRRSV strains, but only 59.9-60.1% with the North American reference strain VR-2332. However, they were 82.9% identical to each other. Nonstructural protein 2 (Nsp2) and ORF3-ORF5 were the most variable regions when compared to other type 1 PRRSV strains. Nsp2 and ORF3 contained multiple discontinuous deletions and a 204-bp deletion in NSP2 in isolate FJQEU14, which has never been described in other Chinese type 1 PRRSV strains. All of these results might be useful for understanding the epidemic status of type 1 PRRSV in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) was considered as an important pathogen, which caused huge economic losses for the world swine industry annually. Until now, according to antigenic and genetic characteristics, two genotypes were identified, European (EU genotype, or type 1) genotype and North American genotype (NA genotype, or type 2), respectively. In China, both of them co-existed in swine herds, and even some novel viral strains emerged in the lastest years. The aim of the review was to describe genetic diversity of PRRSV based on non-structural protein 2 (Nsp2) in China, which could help us better understand molecular

Members of the family Anelloviridae are emerging circular DNA viruses infecting many species of vertebrates including pigs. To date, members of two distinct genera, Iotatorquevirus, including torque teno sus virus 1a and torque teno sus virus 1b (TTSuV1a and TTSuV1b), and Kappatorquevirus, including torque teno sus virus k2a and torque teno sus virus k2b (TTSuVk2a and TTSuVk2b), have been identified in domestic pigs and wild boars. The goal of this study was to evaluate the prevalence and genetic diversity of these viruses based on 5' non-coding genes in Chinese swine herds experiencing clinical symptoms. One hundred eighty-five clinical samples from 11 different regions, collected during 2008-2009, were analyzed using a PCR method, and the results revealed a high TTSuV-positive rate of 78.9 % (146/185) in pigs. Moreover, we detected co-infection with multiple TTSuV strains in the same pig. Nucleotide sequencing results revealed greater genetic diversity within the genus Kappatorquevirus than within the genus Iotatorquevirus. In addition, TTSuVk2b, a novel virus discovered in New Zealand in 2012, was also identified in this study. In summary, the present work helps us obtain more knowledge about the epidemiology and genetic diversity of TTSuVs.

Porcine circovirus type 2 (PCV2) is considered the major etiological pathogen of porcine circovirus-associated diseases (PCVADs) in pigs. Recently, PCV2 was also found in non-porcine animals such as cattle, rats, and mice. However, there was no record of PCV2 in rats in China. The goal of this study was to investigate whether PCV2 was present in rats (Rattus norvegicus, RN) on three swine farms, using molecular tools. PCR results showed that 30 of 95 (31.6 %) rat samples were positive for PCV2. Moreover, further genotype analysis suggested that 10 of 30 (33.3 %) were positive for PCV2a, 19 of 30 (63.3 %) were positive for PCV2b, and only one sample (1/30, 3.33 %) was co-infected by PCV2a and PCV2b. To determine the possible origin of PCV2, 60 serum samples were also collected from weaned pigs on those swine farms, and 23 out of 60 samples were positive for PCV2. In addition, two distinct RN-origin and two distinct porcine-origin PCV2 full-length nucleotide sequences were obtained from the farms. Sequence and phylogenetic analysis indicated that they had the highest nucleotide similarity and closest genetic relationships to each other. In this study, we report the infection and genome characterization of PCV2 in rats and compare RN-origin and porcine-origin PCV2 sequences obtained from the same pig farm, revealing possible cross-species transmission of PCV2.

Porcine circovirus 2 (PCV2) has been prevalent in swine herds in China since 2002, causing severe economic loss to the pig industry. The number of live pigs in southeast China is > 20 million. Since information on the genetic variation of PCV2 in the Fujian province is limited, the objective of the present work was to investigate the epidemiological and evolutionary characteristics of PCV2 in southeast China from 2013 to 2017. Of the 685 samples collected from 90 different swine herds from 2013 to 2017, 356 samples from 84 different swine herds were positive for PCV2. PCV2a, PCV2b, PCV2d, and PCV2e co-existed in the Fujian province, with PCV2d being the predominant circulating strain in swineherds and PCV2e being reported for the first time in China. Strikingly, PCV2-FJ-water DNA comes from contaminated river water and not infected animals. Sequence comparison among all isolates indicated that 95 isolates shared approximately 78.7%–100% nucleotide identity and 74.5%–100% amino acid identity for open reading frame 2 (ORF2). Amino acid alignment showed that the Cap protein of PCV2e differed markedly from those of PCV2a, PCV2b, PCV2c, and PCV2d. These results indicated that various PCV2 genotypes exist in China, and that PCV2 is continuously evolving, leading to rapid emergence of new variant stains.

Porcine circovirus type 2 (PCV2) is considered to be the main pathogen in PC-associated diseases, and significantly affects the global pig-producing industry. PCV2 continuously evolves by point mutations and genome recombinations. In the present study, we aimed to further identify recombinant PCV2 strains. We used polymerase chain reaction to detect PCV2 in the carcasses of pigs with suspected infections from different regions of Guangdong Province in China. DNA was extracted from samples with confirmed infection and full- genome amplification, sequencing, phylogenetic tree construction, gene recombination detection, and sequence alignment were performed in gene recombination analysis. Our results show that recombination occurred between the strains SHC (DQ104421) and ZhuJi2003 (AY579893). The recombination resulted in three recombinants: GD003 (KM503044), GD005 (KM487708), and GD008 (KM487709). Further analyses revealed that these novel recombinants appeared to result from recombination between the PCV2a and PCV2b strains, with crossover regions located in ORF2. This study was a comprehensive analysis that used several different methods, which demonstrated that a cluster of PCV2 strains resulted from the same type of inter-genotypic recombination pattern, with a breakpoint in the structural protein coding region. The results of our study provide both information on the recombination mechanism and disease pathogenesis and useful data for the prevention of PCV2 in the swine industry.

Complete genome characterization of porcine circovirus type 2 (PCV2) for bovid origins was still unclear in China. Therefore, in this study, PCV2 full-length genome of buffalo-origin was amplified and analyzed using PCR, DNAStar and MEGA 5.1. Genome size of three distinct PCV2 strains (buffalo1, buffalo2 and buffalo3) was 1767 bp (48.56% G+C), 1767 bp (48.67% G+C) and 1768 bp (48.08% G+C), respectively. At the nucleotide level, their identity varied from 95% to 96% for complete genome, from 97% to 97.8% for ORF1, and from 90.6% to 94.4% for ORF2. At the amino acid level, their identity varied from 98.7% to 99% for ORF1, and from 88% to 94.9% for ORF2. Online Blast analysis showed that buffalo1, buffalo2 and buffalo3 had highest nucleotide identity (varied from 99.77% to 99.83%) with porcine-origin PCV2 strains. Moreover, in the phylogenetic tree, they were divided into three different clusters and belonged to the worldwide accepted genotypes of PCV2b, PCV2c and PCV2a, respectively. To summarize, this study first recorded complete genome information of PCV2 for non-porcine origins in China.

Currently, porcine circovirus type 2 (PCV2) is considered the major pathogen of porcine circovirus associated-diseases (PCVAD) that causes large economic losses for the swine industry in the world annually, including China. Since the first report of PCV2 in 1998, it has been drawing tremendous attention for the government, farming enterprises, farmers, and veterinary practitioners. Chinese researchers have conducted a number of molecular epidemiological work on PCV2 by molecular approaches in the past several years, which has resulted in the identification of novel PCV2 genotypes and PCV2-like agents as well as the description of new prevalence patterns. Since late 2009, commercial PCV2 vaccines, including the subunit vaccines and inactivated vaccines, have already been used in Chinese swine farms. The aim of this review is to update the insights into the prevalence and control of PCV2 in China, which would contribute to understanding the epidemiology, control measures and design of novel vaccines for PCV2.

For the worldwide pig industries, porcine circovirus type 2 (PCV2) is an economically important pathogen. At present, the prevalence of PCV2 is common in Chinese swine herds. However, there is little information on PCV2 prevalence in non-porcine animals in China, such as bovids. Therefore, the goal of this study is to obtain the firsthand prevalence data of PCV2 in bovids in China. Two hundred and eighty serum and muscle samples from dairy cows (n = 180), buffalo (n = 50), and yellow cattle (n = 50) were analyzed by PCR. The detection results show that PCV2 infections (16 %, 8/50) only exist in buffaloes. In addition, there are different PCV2 viral DNAs identified by differential PCR in the same buffalo sample. Nucleotide sequencing and phylogenetic analysis results based on partial ORF1 and ORF2 sequences suggest that PCV2 strains have genetic diversity in buffaloes and they are divided into three different genotypes (PCV2b, PCV2d, and PCV2e, respectively). Moreover, to our knowledge, the PCV2d and PCV2e genotypes have not been previously reported in bovids. Through this study, the firsthand data of PCV2 prevalence in bovids in China was documented.