Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains

Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.     

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].     

A new porcine reproductive and respiratory syndrome virus strain with highly conserved molecular characteristics in its parental and attenuated strains

Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial economic losses to many swine-producing regions. In this study, PRRSV strain NT0801-F80 was derived from its parental isolate NT0801 by 80 passages in Marc-145 cells. Experimental infection of piglets clearly demonstrated that strain NT0801-F80 is less virulent than NT0801. However, whole genome sequencing showed that the genomes of the parental and attenuated strains are highly conserved compared with those of four other pairs of virulent parental/attenuated vaccine strains (VR2332 and RespPRRS MLV, JA142 and Ingelvac^® ATP MLV, CH-1a and CH-1R, and JXA1 and JXAR). The attenuated strain NT0801-F80 has only 21 nucleotide changes, producing only 14 amino acid changes in NSP2, GP2, GP3, and GP5, compared with those aa sequences of the virulent parental strain. These mutated aa in the attenuated virus may be involved in virulence. These data provide valuable information on the attenuation mechanism of PRRSV that should be useful in future research.     

Complete genome characterization of a East European Type 1 subtype 3 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. PRRSV strains are divided into European (Type 1) and North-American (Type 2) genotypes. Within the European PRRSV genotype, three subtypes have been delineated. Full genome sequences for North American and European subtype 1 strains have been described. Here, the first complete genomic characterization of a European subtype 3 strain (Lena) is described. Amplification of Orf1a and Orf1b fragments was achieved using a set of degenerate oligonucleotides. Using RT-PCR with Lena-specific primers, the full length sequence (15001 nt) was obtained. Alignment of Lena with European subtype 1 reference strain Lelystad showed variation over the entire length (84% identity/89% similarity at amino acid level) with the most variation in Orf1a (Nsp2/NSP2) with a deletion of 29 amino acids. Phylogenetic relationships using different Orfs supported Lena’s genetic distinction from European subtype 1 strains. The availability of the European subtype 3 PRRSV full genome may be important for the understanding of PRRSV evolution and the more pronounced pathogenic nature of Lena.     

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.     

Recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo

Porcine reproductive and respiratory syndrome virus (PRRSV) is characteristic of genetically extensive variation. In this study, five SPF pigs were co-infected with two strains of PRRSV (JXwn06-81c and HB-1/3.9c), and 352 viruses were cloned by plaque assay from the sera of the infected pigs on days 3, 5, 7, 10, 14, 21 postinfection (pi), and the recombinant events between the two viruses were systematically investigated by sequencing the ORF5, ORF3 and Nsp2 genes of each cloned virus and using SimPlot and Genetic Algorithm for Recombination Detection (GARD) analysis. Totally, 133 recombinant viruses out of the plaque viruses were acquired from four of five infected pigs during days 7-21pi upon co-infection with JXwn06-81c and HB-1/3.9c. The intragenic recombination and intergenic fragment exchange of the ORF5, ORF3 and Nsp2 genes between the two viruses exhibited different patterns, and the recombination for ORF5 gene and Nsp2 occurred as early as on day 7pi. The recombination between the ORF5, ORF3 or Nsp2 gene resulted in the generation of chimeric GP5, GP3 or Nsp2. Of the three genes, Nsp2 gene exhibited more complicated recombination situation. Meanwhile, the putative recombination breakpoints and hotspots for the three genes were analyzed. Our findings not only provide valuable evidences for understanding that recombination is an important genetic mechanism contributing to the variation and evolution of PRRSV, but also suggest that extensive use of attenuated vaccine of PRRSV undoubtedly contributes to the increased diversity of PRRSV in field.     

Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype

Summary.  Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1β, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype. Received August 28, 1999 Accepted December 16, 1999     

Analysis of genome determinants of virulence and setting-up of a library of full-size genome copies of the attenuated virus strain of the porcine reproductive and respiratory syndrome virus North American type

Primary genome structures of 3 variants of the NADC-8 North American virulent strain of porcine reproductive and respiratory syndrome virus (PRRSV) were compared for the purpose of detecting any potential genetic virulence determinants of genus Arterivirus. Apart from the virulent variant, we also investigated the attenuated variant, obtained after 251 passages in cell culture, and the intermediate variant isolated from a pig after a partial reversion of the attenuated virus. The attenuated variant genome acquired a 3-nucleotide deletion and 50 mutations versus its virulent precursor. A comparison of the attenuated and intermediary virus variants denoted 8 nucleotide mutations entailing substitutions of 6 amino acids in 3 open reading frames (ORF1a, ORF1b and ORF6). A 32-clone library was constructed in the pACYC177 plasmid vector, which comprised full-size copies of the genome of the NADC-8 attenuated variant strain (251), virus PPCC, for the purpose of experimentally verifying the functional role of the obtained mutations. Full-size analogues ((+)-chain of RNA) of the viral genome, comprising the CAP-structures and polyadenylated ones were obtained in vitro on the basis of the cloned DNA. Seven of the 8 analyzed clones of the viral genome were infected and their insertion into the MARC-145 cell resulted in obtaining of infectious PRRSVs. Four of the constructed recombinant viruses had delayed growth parameters, and 3 of them were similar to the parental strain. The described technology (inverse genetics) would make it possible to introduce changes into the viral genome in applied and fundamental research of Arteriviruses.     

Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates belonging to the European genotype 1 have recently emerged in South Korea, suggesting potential problems for disease control. In the present study, we attempted to determine the complete nucleotide sequence of the first Korean type 1 PRRSV isolate, designated KNU-07. The full-length genome of KNU-07 was found to be 15,038 nucleotides in length, which was 60 nucleotides shorter than the type 1 prototype strain Lelystad due to a notable 60-bp deletion within the nonstructural protein 2 (NSP2). The KNU-07 genome was shown to consist of a 221-nucleotide (nt) 5′ untranslated region (UTR), a 14,703-nt protein-coding region, and a 114-nt 3′ UTR, followed by a 42-73-bp poly(A) tail. A nucleotide sequence comparison of the KNU-07 genome with 20 complete PRRSV genomes revealed a 10.5–13.3% and 39.5–40.3% divergence from type 1 and type 2 strains, respectively, at the genome level, indicating a high similarity to the virus strains commonly identified as the European genotype. In order to investigate genetic variation and to understand the molecular evolution of the type 1 isolate in Korea, extensive phylogenetic analyses were performed using the ORF5 and ORF7 nucleotide sequences of published type 1 PRRSV isolates. The data further indicated that the newly emerging type 1 isolate KNU-07 belongs to the recently proposed pan-European subtype 1. Taken together, the results of this study describe the genomic characterization of the type 1 PRRSV isolated in South Korea, suggesting a recent introduction of the virus typical for this genotype that has commonly appeared worldwide.     

Gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.     

Evolution of ORF5 of Spanish porcine reproductive and respiratory syndrome virus strains from 1991 to 2005

ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) were analysed to determine genetic diversity, codon usage, positive and negative selection sites and potential changes in the predicted glycoprotein 5 (GP5). A hypothetical GP5 containing all selected sites was constructed to determine its characteristics. These sequences corresponded to isolates obtained 10 years apart (1991-1995, 18 strains) and a second set (n = 46) from 2000 to 2005. Similarity to Lelystad virus (LV) decreased from 95.5% in 1991-1995 to 89.5% in 2000-2005. Three highly variable regions were found in ORF5. Codon usage was different in both sets for leucine, glutamine, serine and proline. Thus, 2000-2005 sequences used codons more similar to those present in highly expressed pig genes compared to the 1991-1995 set. Twenty four sites of positive selection and 20 sites of negative selection were found in GP5, most of them in transmembrane regions. Additional glycosylation in N37 of GP5 was common in 2000-2005 but some sequences lack a glycosylation site in N46. The hypothetical GP5 was only 88.1% similar to LV and was less hydrophobic. Taking together these results suggest that PRRSV is still adapting to pig cells.     

Genomic comparison between attenuated Chinese equine infectious anemia virus vaccine strains and their parental virulent strains

A lentiviral vaccine, live attenuated equine infectious anemia virus (EIAV) vaccine, was developed in the 1970s, and this has made tremendous contributions to the control of equine infectious anemia (EIA) in China. Four key virus strains were generated during the attenuation of the EIAV vaccine: the original Liao-Ning strain (EIAV(LN40)), a donkey-adapted virulent strain (EIAV(DV117)), a donkey-leukocyte-attenuated vaccine strain (EIAV(DLV121)), and a fetal donkey dermal cell (FDD)-adapted vaccine strain (EIAV(FDDV13)). In this study, we analyzed the proviral genomes of these four EIAV strains and found a series of consensus substitutions among these strains. These mutations provide useful information for understanding the genetic basis of EIAV attenuation. Our results suggest that multiple mutations in a variety of genes in our attenuated EIAV vaccines not only provide a basis for virulence attenuation and induction of protective immunity but also greatly reduce the risk of reversion to virulence.     

Induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication

Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway that acts in the maintenance of cellular homeostasis and plays important functions in viral replication and pathogenesis. In this study, we investigated the role of autophagy in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), an agent that has caused devastating losses in the international swine industry since the late 1980s. Using protein quantification and microscopy, we observed that PRRSV infection results in LC3-I/II conversion, an increased accumulation of punctate GFP-LC3-expressing cells, and a higher number of autophagosome-like double-membrane vesicles in the cytoplasm of host cells. Inhibition of autophagy using 3-methyladenine (3-MA) or small interfering RNAs targeting ATG7 and Beclin-1 led to a significant reduction in PRRSV titers and protein expression. Conversely, induction of autophagy by rapamycin resulted in increased viral replication. These results demonstrate that PRRSV infection induces autophagy which, in turn, enhances viral replication efficiency.     

板蓝根多糖对猪繁殖与呼吸综合征疫苗免疫猪T细胞亚群的影响

目的：研究板蓝根多糖对猪繁殖与呼吸综合征灭活疫苗免疫猪T细胞亚群及抗体的影响.方法：以板蓝根多糖作为免疫增强剂联合猪繁殖与呼吸综合征灭活疫苗免疫仔猪,通过流式细胞仪检测猪外周血CD3^＋、CD4^＋、CD8^＋细胞百分数,并用ELISA法检测猪血清中抗猪繁殖障碍与呼吸道综合征病毒抗体水平.结果：板蓝根多糖能显著提高仔猪的CD3^＋、CD8^＋淋巴细胞的百分数和特异性抗体滴度.结论：板蓝根多糖能显著增强猪对常规灭活病毒疫苗的免疫应答能力.     

Complete genome sequence of virulent duck enteritis virus (DEV) strain 2085 and comparison with genome sequences of virulent and attenuated DEV strains

We here report the complete genome sequence of the duck enteritis virus (DEV) wild-type strain 2085, an avian herpesvirus (GenBank ID: JF999965). The nucleotide sequence was derived from the 2085 genome cloned as an infectious bacterial artificial chromosome (BAC) clone. The DEV 2085 genome is 160,649-bp in length and encodes 78 predicted open reading frames (ORFs), a number identical to that identified for the attenuated DEV VAC strain (GenBank ID: EU082088.2). Comparison of the genome sequences DEV 2085 and VAC with partial sequences of the virulent CHv strain and the attenuated strain Clone-03 was carried out to identify nucleotide or amino acid polymorphisms that potentially contribute to DEV virulence. No amino acid changes were identified in 24 of the 78 ORFs, a result indicating high conservation in DEV independently of strain origin or virulence. In addition, 39 ORFs contain non-synonymous nucleotide substitutions, while 15 ORFs had nucleotide insertions or deletions, frame-shift mutations and/or non-synonymous nucleotide substitutions with an effect on ORF initiation or termination. In 7 of the 15 ORFs with high and 27 of the 39 ORFs with low variability, polymorphisms were exclusively found in DEV 2085, a finding that likely is a result of a different origin of 2085 (Europe) or VAC, Clone-03 and CHv (Eastern Asia). Five ORFs (UL2, UL12, US10, UL47 and UL41) with polymorphisms were identical between the virulent DEV 2085 and CHv but different from VAC or Clone-03. They, individually or in combination, may therefore represent DEV virulence factors. Our comparative analysis of four DEV sequences provides a comprehensive overview of DEV genome structure and identifies ORFs that are changed during serial virus passage.