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Tissue expression of porcine transient receptor potential mucolipin protein channels and their differential responses to porcine reproductive and respiratory syndrome virus infection in vitro

Zhiqiang Xia

Zhiqiang Xia

  • School of Biological and Food Processing Engineering, Huanghuai UniversityZhumadian, China
  • Zhumadian Huazhong Chia Tai Co., Ltd.Zhumadian, China
  • Henan Topfond Pharmaceutical Company LimitedZhumadian, China
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Xia, Zhiqiang
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Denggao Long

Denggao Long

Sixteenth Middle School of Yiyang City, YiyangHunan Province, China
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Long, Denggao
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Xinyi Hong

Xinyi Hong

School of Biological and Food Processing Engineering, Huanghuai UniversityZhumadian, China
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Hong, Xinyi
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Ying Lan

Ying Lan

School of Biological and Food Processing Engineering, Huanghuai UniversityZhumadian, China
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Lan, Ying
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Lixia Xie

Lixia Xie

School of Biological and Food Processing Engineering, Huanghuai UniversityZhumadian, China
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Xie, Lixia
  
23 mar 2024
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Pubblicato online: 23 mar 2024
Pagine: 45 - 53
Ricevuto: 15 set 2023
Accettato: 06 mar 2024
DOI: https://doi.org/10.2478/jvetres-2024-0014
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© 2024 Zhiqiang Xia et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

Identification of primer specificity for transient receptor potential mucolipin (TRPML) genes in quantitative reverse-transcription PCR (qRT-PCR) and their distribution in peripheral porcine tissues. A – Melting curves of qRT-PCR for TRPML1 using the designed primers, and relative threshold cycle (Ct) values of TRPML1 obtained with different templates; 1/100(0) dilution – dilution of the constructed TRPML1 plasmid; vehicle – negative control containing no template; B – Melting curves of qRT-PCR for TRPML2 using the designed primers, and relative Ct values of TRPML2 obtained with different templates; C – Melting curves of qRT-PCR for TRPML3 using the designed primers, and relative Ct values of TRPML3 obtained with different templates; D–F – levels of porcine TRPML1, TRPML2 and TRPML3 messenger RNA (mRNA) in different porcine tissues determined by qRT-PCR. Data are presented as the means ± standard deviation
Identification of primer specificity for transient receptor potential mucolipin (TRPML) genes in quantitative reverse-transcription PCR (qRT-PCR) and their distribution in peripheral porcine tissues. A – Melting curves of qRT-PCR for TRPML1 using the designed primers, and relative threshold cycle (Ct) values of TRPML1 obtained with different templates; 1/100(0) dilution – dilution of the constructed TRPML1 plasmid; vehicle – negative control containing no template; B – Melting curves of qRT-PCR for TRPML2 using the designed primers, and relative Ct values of TRPML2 obtained with different templates; C – Melting curves of qRT-PCR for TRPML3 using the designed primers, and relative Ct values of TRPML3 obtained with different templates; D–F – levels of porcine TRPML1, TRPML2 and TRPML3 messenger RNA (mRNA) in different porcine tissues determined by qRT-PCR. Data are presented as the means ± standard deviation

Fig. 2.

Effects of porcine reproductive and respiratory syndrome virus (PRRSV) infection of Meat Animal Research Center 145 (MARC-145) monkey kidney cells on the expression of three TRPML proteins. A – Cell morphology with increased multiplicity of infection (MOI) of PRRSV infection. Morphology of MARC-145 cells at a magnification of 100× (scale bars 100 μm); B – Messenger RNA level of PRRSV open reading frame 5 assessed by quantitative reverse-transcription PCR analysis; C – Differential intracellular expression of three TRPML proteins regulated by PRRSV infection. For quantitative analyses, all data were obtained from at least three independent experiments and data are presented as the means ± standard deviation; Con – MARC-145 cells without virus infection; * – P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001; ns – no significance
Effects of porcine reproductive and respiratory syndrome virus (PRRSV) infection of Meat Animal Research Center 145 (MARC-145) monkey kidney cells on the expression of three TRPML proteins. A – Cell morphology with increased multiplicity of infection (MOI) of PRRSV infection. Morphology of MARC-145 cells at a magnification of 100× (scale bars 100 μm); B – Messenger RNA level of PRRSV open reading frame 5 assessed by quantitative reverse-transcription PCR analysis; C – Differential intracellular expression of three TRPML proteins regulated by PRRSV infection. For quantitative analyses, all data were obtained from at least three independent experiments and data are presented as the means ± standard deviation; Con – MARC-145 cells without virus infection; * – P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001; ns – no significance

Fig. 3.

Inhibitory activity of mucolipin synthetic agonist 1 (ML-SA1) against porcine reproductive and respiratory syndrome virus (PRRSV) in Meat Animal Research Center 145 (MARC-145) monkey kidney cells. A – Cytotoxicity of ML-SA1 in MARC-145 cells. Viability of cells treated with concentrations of ML-SA1 from 0 μM to 200 μM measured by a cell-counting kit 8 assay; B – Messenger RNA level of PRRSV open reading frame 5 with different concentrations of ML-SA1 treatment in MARC-145 cells incubated with 10 μM or 50 μM of ML-SA1; C – MARC-145 cell morphology in PRRSV infection after addition of different concentrations of ML-SA1 and incubation with 10 μM or 50 μM ML-SA1. For quantitative analyses, all data were obtained from at least three independent experiments and data are presented as the means ± standard deviation; Mock –MARC-145 cells without virus or ML-SA1 treatment; * – P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001; ns – no significance
Inhibitory activity of mucolipin synthetic agonist 1 (ML-SA1) against porcine reproductive and respiratory syndrome virus (PRRSV) in Meat Animal Research Center 145 (MARC-145) monkey kidney cells. A – Cytotoxicity of ML-SA1 in MARC-145 cells. Viability of cells treated with concentrations of ML-SA1 from 0 μM to 200 μM measured by a cell-counting kit 8 assay; B – Messenger RNA level of PRRSV open reading frame 5 with different concentrations of ML-SA1 treatment in MARC-145 cells incubated with 10 μM or 50 μM of ML-SA1; C – MARC-145 cell morphology in PRRSV infection after addition of different concentrations of ML-SA1 and incubation with 10 μM or 50 μM ML-SA1. For quantitative analyses, all data were obtained from at least three independent experiments and data are presented as the means ± standard deviation; Mock –MARC-145 cells without virus or ML-SA1 treatment; * – P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001; ns – no significance

Primers used in this experiment

Primer name Primer type Primer sequence (5′ to 3′) Product size (base pairs)
Pig β-actin Forward CGGGACATCAAGGAGAAGC 132
Reverse CTCGTTGCCGATGGTGATG
Pig TRPML1 Forward CAGTTACAAGAACCTCAC 111
Reverse CAGTCAGGGATTTCATTG
Pig TRPML2 Forward AATCAGTATCATCGTCTA 140
Reverse ATATTCAGTGTCTCATTAG
Pig TRPML3 Forward TTACTTGGTCTGGCTGTT 129
Reverse TCCTCTTGGTAATGCTTAATTG
Monkey GAPDH Forward TCAACGACCACTTTGTCAAGCTCA 97
Reverse GCTGGTGGTCCAGGGGTCTTACT
Monkey TRPML1 Forward CGGATGACACCTTCGCAGCCTAC 108
Reverse ACGCATACCGGCCCAGTGACAC
Monkey TRPML2 Forward CTGTGGCTGGATTGTCTTAGG 121
Reverse CTGGATTTGGGCAAAGGTTG
Monkey TRPML3 Forward CCAGAAATTGAAACTGAGTGTT 205
Reverse ATGTTATAGTCAGAGTAAAGTC
PRRSV ORF5 Forward GAAATGCTTGACCGCGGGCT 261 (16)
Reverse GTGTCAAGGAAATGGCTTGT
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Scienze biologiche, Biologia molecolare, Microbiologia e virologia, Scienze della vita, altro, Medicina, Medicina veterinaria
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