scholarly journals Molecular Characterization of the Nsp2 and ORF5 (ORF5a) Genes of PRRSV Strains in Nine Provinces of China During 2016–2018

2021 ◽  
Vol 8 ◽  
Author(s):  
Baishuang Yin ◽  
Shanshan Qi ◽  
Wanli Sha ◽  
Hongyu Qin ◽  
Liming Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Recombination Analysis ◽  
Rt Pcr ◽  
Nsp2 Gene ◽  
Virulent Strains ◽  
Positive Rate ◽  
Wild Strains

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease and brings huge economic losses to commercial pork production worldwide. PRRSV causes severe reproductive failure in sows and respiratory distress in piglets. To trace the evolution of PRRSV in pigs with respiratory diseases in some regions of China, 112 samples were collected from nine provinces in China during 2016–2018. All samples were detected by RT-PCR and analyzed by the Nsp2/ORF5 (ORF5a)-genes-phylogeny. Sequence analysis and recombination analysis were conducted on the Nsp2/ORF5 (ORF5a) genes of the identified strain in the study. The RT-PCR result shown that the positive rate of PRRSV was 50.89% (57/112). Phylogenetic analysis showed that the identified PRRSV strains were all NA genotype and belonged to lineage 1, 3, and 8. The Nsp2 gene of identified PRRSV strains exhibited nucleotide homologies of 53.0 ~ 99.8%, and amino acid homologies of 46.8 ~ 99.7%. The ORF5 gene of identified PRRSV strains exhibited nucleotide homologies of 82.4 ~ 100%, and amino acid homologies of 79.6 ~ 100%. Sequence analysis revealed that a discontinuous 30-amino-acid deletion (positions 481 and 533–561) and a 131-amino-acid discontinuity deletion (positions 323–433, 481, and 533–551) in Nsp2 of PPRSV isolates; all identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. Sequence analysis of ORF5 and ORF5a revealed that the mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region, while the mutation sites of ORF5a were mainly concentrated in the transmembrane and the intramembrane region. The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. This study showed that the prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion; different types of PRRSV strains showed different patterns and variation in China. This study suggested that the monitoring of PRRSV prevalence and genetic variation should be further strengthened.

scholarly journals Genetic Diversity of Porcine Reproductive and Respiratory Syndrome Virus and Evaluation of Three One-Step Real-Time RT-PCR Assays in Korea

2021 ◽  
Author(s):  
Go-Eun Shin ◽  
Ji-Young Park ◽  
Kyoung-Ki Lee ◽  
Mi-Kyeong Ko ◽  
Bok-Kyung Ku ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Real Time ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
The Real ◽  
One Step ◽  
Pcr Assays

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. Methods In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Results A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. Conclusion The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

scholarly journals Complete Genome Sequence of a Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus That Has a Further Deletion in the Nsp2 Gene

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Guobiao Ji ◽  
Yingying Li ◽  
Feifei Tan ◽  
Jinshan Zhuang ◽  
Xiangdong Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Respiratory Syndrome Virus ◽  
Coding Region ◽  
Highly Pathogenic ◽  
Nsp2 Gene

Here, we report the complete genome of a Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) characterized by a further 29-amino acid (87 nucleotides) deletion in its Nsp2-coding region compared to the prototype of the HP-PRRSV JXA1 strain.

scholarly journals Multiplex RT-PCR assay to differentiate genotypes of porcine reproductive and respiratory syndrome virus in swine

2019 ◽  
Vol 18 (06) ◽  
pp. 8-13
Author(s):  
Phat X. Dinh
Keyword(s):  
Real Time ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Swine Industry ◽  
Diagnostic Challenge ◽  
Nsp2 Gene ◽  
Prrs Virus ◽  
Pcr Products

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases to swine industry worldwide. Due to the heterogeneity of field isolates, accurate detection of the PRRS virus is a diagnostic challenge. Recently, co-infection with NA-PRRSV, EU-PRRSV and HP-PRRSV isolates continuously increases in many countries, resulting in a significant impact on PRRSV diagnostics and disease control on farms. To facilitate rapid diagnosis and reliable discrimination of NA-PRRSV, EU-PRRSV and HP-PRRSV, a multiplex RT-PCR assay was established with three pairs of primers targeting highly conservative regions of nsp2 gene with predicted multiplex RT-PCR products of 364 bp, 161 bp and 259 bp, respectively. The primer pairs were optimized to be highly specific for PRRSV genotypes and were able to detect the target gene at the limit of 102 copies/μL for each gene. Clinical samples were used to evaluate this multiplex RT-PCR in parallel with a commercial real-time RT-PCR kit. Results showed over 95.2% (20/21 samples) agreement between the mRT-PCR and the real-time RT-PCR kit. Hence, it indicated that this multiplex RT-PCR could be useful for rapid and deferential diagnosis of NA-PRRSV, EU-PRRSV and HP-PRRSV in swine farms.

Murine Asb-17 expression during mouse testis development and spermatogenesis

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 151-156 ◽  
Author(s):  
Kye-Seong Kim ◽  
Myung-Sun Kim ◽  
Soo-Kyung Kim ◽  
Kwang-Hyun Baek
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Northern Blot ◽  
Northern Blot Analysis ◽  
Mouse Testis ◽  
Rt Pcr ◽  
Testis Development ◽  
Socs Box

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

scholarly journals Cloning, Expression, and Sequence Analysis of the ORF4 Gene of the Porcine Reproductive and Respiratory Syndrome Virus MN-1b

1994 ◽  
Vol 6 (3) ◽  
pp. 293-296 ◽  
Author(s):  
Jimmy Kwang ◽  
Hyun Soo Kim ◽  
Han S. Joo
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Respiratory Syndrome Virus ◽  
European Isolate ◽  
Reading Frame ◽  
Orf4 Protein ◽  
Orf4 Gene

Porcine reproductive and respiratory syndrome virus (PRRSV) MN-1b strain open reading frame 4 (ORF4) has been cloned, sequenced, and expressed in Escherichia coli. The homologies of nucleotide and amino acid sequences between MN-1b (US isolate) and LV (European isolate) are 69% and 64%, respectively. The data also showed that ORF4 of MN-1b is 36 bases shorter than that of LV. Western blot analysis of expressed recombinant ORF4 protein reacted with 65% (26/40) of PRRSV-infected pig sera tested. These results demonstrated that ORF4 of PRRSV may not be a well-conserved region.

Variation analysis of the ORF5 gene of Porcine reproductive and respiratory syndrome virus

2004 ◽  
Vol 1 (3) ◽  
pp. 173-179
Author(s):  
Gao Zhi-Qiang ◽  
Guo Xin ◽  
Cha Zhen-Lin ◽  
Chen Yan-Hong ◽  
Yang Han-Chun
Keyword(s):  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Amino Acid Identity ◽  
Respiratory Syndrome Virus ◽  
Variation Analysis ◽  
Rt Pcr ◽  
Acid Identity ◽  
Pig Farms ◽  
Orf5 Gene

AbstractThree Porcine reproductive and respiratory syndrome virus (PRRSV) isolates (HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002) were obtained from pig farms in Hebei and Jiangxi provinces, China. The complete ORF5 gene of the isolates was amplified using RT-PCR and sequenced. It was shown that ORF5 genes of all isolates encoded 200 amino acids. Comparing ORF5 genes of the three isolates and published sequences for five other PRRSV isolates in China, variation analysis showed that all of the isolates were of the American genotype, with 88.2–99.0% amino acid identity. ORF5 genes among BJ-4, S1 and J1 had higher similarity, sharing 98–99% identity of the deduced amino acids. HB-1(sh)/2002, HB-2(sh)/2002 and JX-1/2002 and CH-1a presented 92–96% identity among their ORF5 genes. Phylogenetic analysis revealed that these isolates could be divided into two subgroups based on the genetic distance of their ORF5 gene: the first subgroup comprised BJ-4, S1 and J1 and was closer to VR2332 and vaccine strains; the second included HB-1(sh)/2002, HB-2(sh)/2002, JX-1/2002 and CH-1a.

scholarly journals Attenuation and Immunogenicity of a Live High Pathogenic PRRSV Vaccine Candidate with a 32-Amino Acid Deletion in the nsp2 Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Wenhui Lu ◽  
Baoli Sun ◽  
Jianyue Mo ◽  
Xiduo Zeng ◽  
Guanqun Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vaccine Candidate ◽  
Clinical Signs ◽  
Respiratory Syndrome Virus ◽  
Amino Acid Deletion ◽  
Lethal Challenge ◽  
Nsp2 Gene ◽  
Amino Acid Mutations

A porcine reproductive and respiratory syndrome virus (PRRSV) QY1 was serially passed on Marc-145 cells. Virulence of different intermediate derivatives of QY1 (P5, P60, P80, and P100) were determined. The study found that QY1 had been gradually attenuated during the in vitro process. Pathogenicity study showed that pigs inoculated with QY1 P100 and P80 did not develop any significant PRRS clinic symptoms. However, mild-to-moderate clinical signs and acute HP-PRRSV symptoms of infection were observed in pigs inoculated with QY1 P60 and P5, respectively. Furthermore, we determined the whole genome sequences of these four intermediate viruses. The results showed that after 100 passages, compared to QY1 P5, a total of 32 amino acid mutations were found. Moreover, there were one nucleotide deletion and a unique 34-amino acid deletion found at 5′UTR and in nsp2 gene during the attenuation process, respectively. Such deletions were genetically stable in vivo. Following PRRSV experimental challenge, pigs inoculated with a single dose of QY1 P100 developed no significant clinic symptoms and well tolerated lethal challenge, while QY1 P80 group still developed mild fever in the clinic trial after challenge. Thus, we concluded that QY1 P100 was a promising and highly attenuated PRRSV vaccine candidate.

scholarly journals First report of a resistance-breaking strain of Tomato spotted wilt orthotospovirus infecting Capsicum annuum carrying the Tsw resistance gene in South Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
Ju-Yeon Yoon ◽  
Nam-Han Her ◽  
In Sook Cho ◽  
Bong Nam Chung ◽  
Seung-Kook Choi
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
South Korea ◽  
Complete Genome ◽  
Chili Pepper ◽  
Rt Pcr ◽  
Genome Sequences ◽  
Specific Primers ◽  
Tomato Spotted Wilt ◽  
Pepper Plants

Tomato spotted wilt orthotospovirus (TSWV) was first reported in 2004 from paprika in South Korea (Kim et al., 2004), where it is currently widespread. TSWV infections were reported in chili pepper, tomato, weeds, and ornamental plant species in South Korea (Choi et al., 2014; Choi and Choi, 2015; Yoon et al., 2016; Yoon et al., 2018; Yoon et al., 2019). One of the best strategies for TSWV management is planting resistant cultivars containing the Tsw gene. In 2019 virus-like symptoms were observed in chili pepper (Capsicum annuum) plants bearing the Tsw gene in Anseong-si, South Korea. The infected chili peppers showed mosaic and wilting followed by necrosis on leaves and fruits in the field. To identify the causal virus, symptomatic leaf samples were analyzed using ImmunoStrip kits (Agdia, USA); we detected three pepper-infecting viruses: Pepper mild mottle virus, Cucumber mosaic virus, and TSWV. TSWV was only detected from 40 naturally infected chili pepper plants exhibiting virus-like symptoms. To further confirm the presence of TSWV (named TSWV-P1), we amplified reverse-transcription polymerase chain reaction (RT-PCR) products for L, M, and S RNA segments using tospovirus-specific and TSWV-specific primers (Batuman et al., 2014). Expected fragments of 445, 868, and 777 bp in length were amplified and sequenced. The complete genome sequences of TSWV-P1 from a symptomatic chili pepper plant were also determined using TSWV-specific primers (Choi et al., 2014; Lian et al., 2013). The complete genome sequences of TSWV-P1 were deposited to GenBank (LC549179, LC549180, and LC549181). The sequences of each fragment were identical to a consensus sequence, showing 99.1%, 98.5%, and 98.6% identity with TSWV-L, M, and S RNA (KP008132, AY744492, and KP008134), respectively. These results clearly showed only a single TSWV infection among the naturally infected chili pepper plants, without reassortment between TSWV and another tospovirus. To confirm whether TSWV-P1 is a resistance-breaking (RB) strain, Nicotiana rustica was mechanically inoculated with sap from leaves of the infected pepper samples to propagate TSWV-P1. A non-RB TSWV isolate (TSWV-Kor-lisianthus) from lisianthus was used as a control (Yoon et al., 2017). Two resistant (with Tsw) and two susceptible chili pepper cultivars (20 plants per cultivar) were mechanically inoculated with sap from leaves of the TSWV-infected N. rustica. The incidence rates of disease caused by TSWV-P1 were 90–100% for resistant and 95–100% for susceptible cultivars. In contrast, TSWV-Kor-lisianthus caused symptoms only in the susceptible pepper cultivars (90–100% incidence). TSWV infection in representative plants was confirmed using the TSWV- ImmunoStrip kit and RT-PCR. The NSs gene of TSWV-P1 consists of 1,404 nucleotides (468 amino acids); sequence analysis of the TSWV-P1 NSs gene showed high nucleotide (99.7%) and amino acid identities (99.8%) with the NSs sequences of two TSWV isolates (FR693035, CBX24121). Protein sequence analysis of TSWV-P1 NSs revealed that no amino acid mutation was associated with those of a representative TSWV RB strain, as previously described (Almási et al., 2017), suggesting that TSWV-P1 is a RB strain. Because this TSWV-P1 can overcome resistance conferred by the Tsw gene in commercially grown chili pepper cultivars, it represents a potential threat to pepper production in South Korea.

scholarly journals Bidens mottle virus and Apium virus Y Identified in Ammi majus in Florida

Plant Disease ◽  
2008 ◽  
Vol 92 (6) ◽  
pp. 975-975 ◽  
Author(s):  
C. A. Baker ◽  
E. N. Rosskopf ◽  
M. S. Irey ◽  
L. Jones ◽  
S. Adkins
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Gene Sequence ◽  
Amino Acid Sequences ◽  
Mottle Virus ◽  
Specific Antiserum ◽  
Rt Pcr ◽  
Ammi Majus ◽  
Pcr Products ◽  
Inclusion Morphology

Ammi majus (bishop's weed), a member of the Apiaceae, is grown from seed for cut flowers in South Florida. In March 2005, plants were found to be showing virus-like symptoms including mosaic, vein clearing, and leaf rugosity (3) that rendered their flowers unmarketable. Inclusion morphology in epidermal strips from these infected plants indicated the presence of one or more potyviruses. This was confirmed by ELISA with commercially available antiserum for potyvirus identification (Agdia, Elkhart, IN). Clover yellow vein virus (ClYVV) was identified by sequencing and confirmed with specific antiserum (4). However, ClYVV was not identified in all potyvirus-infected samples from 2005, indicating the presence of one or more additional potyviruses. Bidens mottle virus (BiMoV) was subsequently identified in one of three potyvirus-infected samples by immunodiffusion tests using specific antiserum for BiMoV (Department of Plant Pathology, University of Florida), cylindrical inclusion morphology in epidermal strips, host range data, and sequencing of cloned reverse transcription (RT)-PCR products from degenerate potyvirus primers (2). Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence (GenBank Accession No. EU255631) were 95 and 98% identical, respectively, to a Florida isolate of BiMoV recently reported from tropical soda apple (1). Similar virus-like symptoms were again observed in A. majus in January 2007 and persisted through March. ELISA testing again indicated the presence of a potyvirus. However, neither ClYVV nor BiMoV were identified in the initial 2007 samples. Instead, sequence analysis of the cloned RT-PCR products amplified with degenerate potyvirus primers (2) from seven potyvirus-infected samples collected on two dates in January and one each in February and March revealed the presence of Apium virus Y (ApVY). The 3′ terminal portion of the genome (GenBank Accession No. EU255632) was found to be 90 to 91% identical to ApVY sequences in GenBank at the nucleotide level. Deduced amino acid sequences of the NIb and CP regions of these RT-PCR products were 96 and 95% identical, respectively, to ApVY sequences in GenBank. One of these seven ApVY-infected samples (collected in March 2007) was determined to be coinfected with BiMoV by sequence analysis of the cloned RT-PCR products. Six clones were sequenced. Three were determined to be ApVY as indicated above. Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence from the other three clones were 95 and 97% identical, respectively, to the 2005 A. majus BiMoV isolate. Although ClYVV and BiMoV have previously been reported in other hosts in Florida, to the best of our knowledge, this is the first report of BiMoV and ApVY in A. majus anywhere and the first report of ApVY in North America. References: (1.) C. A. Baker et al. Plant Dis. 91:905, 2007. (2.) A. Gibbs and A. J. Mackenzie. J. Virol. Methods 63:9, 1997. (3.) M. S. Irey et al. (Abstr.) Phytopathology (suppl.)95:S46, 2005. (4.) M. S. Irey et al. Plant Dis. 90:380, 2006.

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