ProtocolsTwo immune-based methods using immortalized porcine kidney macrophages for quantifying neutralizing activity against porcine reproductive and respiratory syndrome virus-2
Introduction
Porcine reproductive and respiratory syndrome (PRRS) is one of the major swine diseases that causes respiratory disorders in all stages of pig farming and reproductive failure in pregnant sows worldwide, thereby leading to devastating economic loss. The etiological agent of PRRS, the PRRS virus (PRRSV), is an enveloped, single-stranded, positive-sense RNA virus of the order Nidovirales, family Arteriviridae, and genus Betaarterivirus. PRRSV can be classified into two distinct species (Betaarterivirus suid 1 or Betaarterivirus suid 2, which were previously known as PRRSV-1 or PRRSV-2, respectively) sharing ∼60 % nucleotide similarity in their 15-kb genome (Allende et al., 1999; Nelsen et al., 1999). The PRRSV genome encodes at least 10 open-reading frames (ORF1a, 1b, 2a, 2b, 3, 4, 5, 5a, 6, and 7) (Lunney et al., 2016; Montaner-Tarbes et al., 2019). Viral glycoproteins encoded by the ORFs reportedly contain neutralizing epitopes in their ectodomains (Li and Murtaugh, 2012; Oleksiewicz et al., 2002; Ostrowski et al., 2002; Vanhee et al., 2009). Since the first viral isolation in 1993 in Japan, isolated field strains have been mostly classified into PRRSV-2 to date (Murakami et al., 1994; Shimizu et al., 1994). Additionally, the high genetic diversity in genome of the field strains in Japan has been demonstrated (Iseki et al., 2011, 2016; Morozumi et al., 2014, 2021). The genetic diversity of the PRRSV genome could affect neutralizing cross-activity (Darwich et al., 2010; Faaberg et al., 2006; Popescu et al., 2017). Therefore, the investigation of how genetic diversity influences cross-neutralization would be required. The evaluation of PRRSV-2 neutralizing titer could contribute to the research on neutralizing antibody against field strains in Japan.
Immune-based methods using specific antibody against viral antigen, such as image cytometry (ICM) and cell-based enzyme-linked immune sorbent assay (ELISA), can evaluate the neutralizing activity of many samples. In several recent reports, ICM and cell-based ELISA have been used to evaluate the neutralizing activity against various viruses, such as feline coronaviruses (Pearson et al., 2020), severe acute respiratory syndrome coronavirus 2 (Conzelmann et al., 2020), and Middle East respiratory syndrome coronavirus (Rosen et al., 2019). Regarding PRRSV, the neutralizing activity of porcine serum has been evaluated using cell-based ELISA (Robinson et al., 2015, 2018); however, ICM has not been used in PRRSV research.
Immortalized porcine kidney macrophages (IPKMs) exhibit higher infectious efficiency and progeny production of PRRSV-2 field strains than MARC-145, which was characterized as a permissive cell line for the virus (Morozumi et al., 2021; Takenouchi et al., 2017). Hence, we used IPKMs for evaluating neutralizing activity against PRRSV-2 field strains, which has been a challenge with other cell strains. A PRRSV-2 field strain spread in Japan was used for evaluating two immune-based methods (ICM and the cell-based ELISA) using IPKMs. Here we report the establishment of two immune-based methods using IPKMs, for evaluating the neutralizing antibody titer against PRRSV-2, including field strains.
Section snippets
Cell culture
IPKMs (Morozumi et al., 2021; Takenouchi et al., 2017, 2021) were cultured at 37 °C with 5 % CO2. The culture medium consisted of Dulbecco’s modified Eagle medium (NACALAI TESQUE, INC., Kyoto, Japan) supplemented with 10 % fetal bovine serum (FBS), 25 μM monothioglycerol (Merck, St. Louis, MO, USA), 10 μg/mL insulin (Merck), and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin; Merck).
Virus and biological samples
During monitoring of virus infection on farrow-to-finish pig farms, a field strain of PRRSV-2 was
Detection range of virus infection of the two immune-based methods in IPKMs
To determine whether ICM and cell-based ELISA could quantify PRRSV-2 infection, the serially diluted K200202 strain was used to infect IPKMs and both immune-based methods were used for detection. In the ICM method, N protein-positive cells and nuclei were detected using immunofluorescence microscopy (Fig. 1a). The number of N protein-positive cells decreased depending on the infection dose and were mostly undetectable at MOI = 0.0001–0.00003 (Fig. 1b; gray bar). However, the number of nuclei
Discussion
To perform the neutralization assay, cells capable of efficiently isolating and proliferating the target virus strains are required. Porcine alveolar macrophages (PAMs) are naturally permissive cells for PRRSV, although MARC-145 cells are preferentially used in PRRSV studies due to reproducibility and ease of maintenance. Inefficient culture of field isolates on MARC-145 cells causes complications in interpreting the results of the neutralization assay (Robinson et al., 2015). We recently
Data availability
Data will be made available on request.
Author contributions
Takumi Terada planned and performed experiments as well as wrote this article. Takeya Morozumi led this study, wrote this article, and discussed the results as corresponding author. Emi Wada performed preliminary experiments and maintained the experimental materials. Shin Sukegawa wrote this article and discussed the experiment plans and results.
Author statement
Takumi Terada: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing–Original Draft, Writing–Review & Editing. Takeya Morozumi: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing–Original Draft, Writing–Review & Editing, Supervision. Emi Wada: Methodology, Resources. Shin Sukegawa: Conceptualization, Methodology, Validation, Writing–Original Draft, Writing–Review & Editing, Project administration.
Funding
This study was conducted with support from a research fund of the Research & Development Center, NH Foods Ltd.
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
This study was conducted with support from a research fund of the Research & Development Center, NH Foods Ltd.
Takumi Terada, Takeya Morozumi, Emi Wada, and Shin Sukegawa are employees of NH Foods Ltd.
Acknowledgments
The authors are most grateful to Dr. Takato Takenouchi (Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization) for the kind provision of IPKMs. The authors would like to thank Enago (www.enago.jp) for the English language review.
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