Introduction

Influenza A viruses are negative-sense single-stranded RNA viruses causing respiratory disorders in humans and animals. Since the genome of influenza A viruses is segmented RNA, the genome can be reassorted through the life cycle of various types of influenza A viruses and cause antigenic shifts (Javanian et al. 2021; Moghadami 2017). The pandemic influenza A H1N1 2009 virus is caused by an antigenic shift mediated by quadruple reassortments of viral genomes, including swine, avian, and human influenza A viruses (Allen et al. 2017; Everitt et al. 2012; Girard et al. 2010; Kim et al. 2020; Schnitzler and Schnitzler 2009). In response to the invasion of influenza A viruses, macrophages are activated to clear pathogens. However, macrophages are also the major reservoir of reactive oxygen species (ROS). The generation of excessive ROS results in the destruction of epithelial cell layers, which are considered the first line of antiviral defense for the host (Lin et al. 2016; Reshi et al. 2014; Kim et al. 2021a, b).

The prion protein (PrP), encoded by the prion protein gene (PRNP), is a glycosylphosphatidylinositol (GPI)-anchored multifunctional protein composed of a nonstructural octapeptide repeat domain and a globular C-terminal domain (Kim et al. 2021c; Prusiner 1998a, b). Previous studies have reported that PrP plays a pivotal role in the protection of oxidative stress in several organs, including the brain, heart and lungs (Castle and Gill 2017). In addition, deficiency of the PRNP gene confers susceptibility and lethality in influenza A virus-infected mice (Chida et al. 2018). Thus, we postulated that genetic variations in the PRNP gene, which are related to the function and expression level of PrP, may be associated with susceptibility to influenza A viruses.

To investigate the association between PRNP polymorphisms and vulnerability to pandemic 2009 swine flu, we investigated the genotype and allele frequencies of the PRNP polymorphisms located on the open reading frame (ORF) and transcriptional regulatory region in 97 healthy control subjects and 30 pandemic 2009 swine flu-infected patients using direct sequencing. We evaluated an association between PRNP polymorphisms and susceptibility to pandemic 2009 swine flu infection by comparing the genotype and allele frequencies of the PRNP polymorphisms between these two groups.

Materials and methods

Ethics statements

All samples were collected with informed consent under institutional review board-approved protocols. All experimental procedures were approved following the guidelines of the institutional review board (IRB) of Jeonbuk National University and the 1964 Helsinki Declaration and its later amendments (approval number: JBNU 2017-08-009). All the samples and information were anonymized prior to study.

Subject

Detailed information on all subjects was explained in a previous study (Kim et al. 2020). In brief, healthy controls and pandemic 2009 swine flu-infected patients have no underlying disease and co-morbidity (Table 1). 

Table 1 Detailed information on the study population
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Genomic DNA extraction

Genomic DNA was isolated from 200 μl of peripheral blood using the Blood Genomic DNA Isolation Kit (Qiagen, Valencia, California, USA) following the manufacturer’s protocol.

Genetic analysis

The PRNP gene was amplified by polymerase chain reaction (PCR) using gene-targeted primers. Detailed information on the primers and experimental conditions are described in Table 2. PCR was carried out using GoTaq® DNA Polymerase (Promega, Fitchburg, Wisconsin, USA) and an S-1000 Thermal Cycler (Bio–Rad, Hercules, California, USA) following the manufacturer’s protocol. The PCR products were directly analyzed with an ABI 3730 automatic sequencer (ABI, Foster City, California, USA) and the results were annotated by Finch TV software (Geospiza Inc, Seattle, USA).

Table 2 Detailed information on the primers used in this study
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Statistical analysis

Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., USA).

Results

Investigation of polymorphisms of the PRNP gene

To investigate the genotype and allele frequencies of PRNP polymorphisms in the Korean population, we performed direct sequencing in 97 healthy controls and 30 pandemic 2009 swine flu-infected patients. The sequenced products were homologous to the PRNP gene of Homo sapiens registered in GenBank (Gene ID: 5621). We performed genoty** for 2 SNPs, M129V and E219K, located on the ORF of the PRNP gene (Table 3), and 3 SNPs, c.1368 T > C, c.1380 T > C and c.1424G > A, located on the transcriptional regulatory region of the PRNP gene (Table 4).

Table 3 Comparison of genotype and allele frequencies of the prion protein gene (PRNP) polymorphisms located on the open reading frame (ORF) between healthy control subjects and pandemic 2009 swine flu -affected patients
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Table 4 Comparison of genotype and allele frequencies of the prion protein gene (PRNP) polymorphisms located in the transcriptional regulatory region between healthy control subjects and pandemic 2009 swine flu-affected patients
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Evaluation of an association between PRNP polymorphisms and vulnerability to pandemic 2009 swine flu

To examine an association between the genetic distribution of the PRNP gene and susceptibility to pandemic 2009 swine flu infection, we compared the genotype and allele frequencies of the PRNP gene polymorphisms between the healthy control subjects and pandemic 2009 swine flu-affected patients.

Interestingly, there is no association of the genotype and allele distributions of 2 SNPs, M129V and E219K, of the PRNP gene located on the ORF with vulnerability to pandemic 2009 swine flu (Table 3). In addition, there is no association of the genotype and allele distributions of 3 SNPs, c.1368 T > C, c.1380 T > C and c.1424G > A, located in transcriptional regulatory region of the PRNP gene with susceptibility to pandemic 2009 swine flu (Table 4).

Discussion

Previous studies have reported that nonsynonymous genetic polymorphisms of the PRNP gene modulate structural alterations of PrP and are related to susceptibility to several types of prion diseases, including Creutzfeldt–Jakob disease (CJD) in humans and chronic wasting disease (CWD) in elks and deer. In humans, genetic variations of the human PRNP gene at codons 129 and 219 play a major role in susceptibility to CJD (Alperovitch et al. 1999; Jeong et al. 2005; Lee et al. 2001; Lloyd et al. 2011; Vollmert et al. 2006). In addition, nonsynonymous SNPs of the cervid PRNP gene at codons 95, 96, and 132 are also associated with vulnerability to CWD (Arifin et al. 2021; Johnson et al. 2006; Robinson et al. 2012). However, we did not find an association between functional genetic variations, including M129V and E219K, and vulnerability to pandemic 2009 swine flu (Table 3). In addition, previous studies have reported that genetic polymorphisms found in the promoter of the PRNP gene are involved in susceptibility to CJD and bovine spongiform encephalopathy (BSE). In humans, c.1368 T > C (rs1029273), located in the promoter region plays a pivotal role in susceptibility to sporadic CJD in British and German populations (Bratosiewicz-Wasik et al. 2012; Mastrianni 2010; Mead et al. 2001). In addition, 23- and 12-bp insertion/deletion polymorphisms located in the transcription regulatory region of the bovine PRNP gene are related to the expression level of the bovine PRNP gene and affect vulnerability to BSE (Haase et al. 2007; Murdoch and Murdoch 2015). Notably, we found no association between promoter polymorphisms of the PRNP gene and vulnerability to the pandemic 2009 swine flu in the present study (Table 4). It indicates that PRNP polymorphisms do not affect the infection mechanism of the pandemic 2009 swine flu. However, since this evaluation has been performed in relatively small cohorts, further confirmation in large cohorts would be highly advantageous in the future. In addition, Koreans have highly heterogeneous genetic background of the PRNP gene compared to other ethnic groups, and further investigation of the association analysis in other ethnic groups is needed in the future. In the present study, only one patient was admitted to the ICU (Table 1). Thus, we could not perform the association analysis between the disease severity and the PRNP polymorphisms. Since the PrP is related to lethality in influenza A virus-infected mice, PRNP polymorphisms may be related to the disease severity. Thus, further association analysis stratified by severity, including the entrance of the intensive care unit and/or pneumonia, is needed to validate the association between entire PRNP polymorphisms and the clinical outcome of the pandemic 2009 swine flu-infected patients (Chida et al. 2018).

In a recent study, the MX1 protein, which showed potent antiviral activity, played a pivotal role in blocking H7N9 influenza viruses (Chen et al. 2021). Furthermore, genetic variations of the MX1 gene are associated with susceptibility to influenza viruses. Since H7N9 influenza viruses showed a similar pathomechanism to H1N1 influenza viruses, further investigation between MX1 polymorphisms and susceptibility to pandemic 2009 swine flu is highly desirable in the future.

Conclusions

In this study, we investigated genetic variations of the PRNP gene in healthy control subjects and the pandemic 2009 swine flu-infected patients using direct sequencing. We evaluated an association between PRNP polymorphisms and susceptibility to the pandemic 2009 swine flu infection. However, we did not find an association between these polymorphisms and susceptibility to pandemic 2009 swine flu.