2019년 국내에서 분리한 H1N2 돼지 인플루엔자바이러스 유전자 분석 및 이의 마우스에 대한 감염성

Genetic Analysis of the 2019 Swine H1N2 Influenza Virus Isolated in Korean Pigs and Its Infectivity in Mice

Abstract

돼지인플루엔자는 동물에서 사람에게 감염할 수 있는 인수공통전염병이다. 우리는 2019년 한국 돼지농장에서 호흡기 증상을 보이는 돼지에서 3주의 H1N2형 인플루엔자바이러스를 분리하였다. 유전자 분석결과, 이들 바이러스의 8개 유전자 중 PA 및 NP 유전자는 2009 대유행 H1N1 인플루엔자 유래였고, 나머지 유전자는 돼지에 유행하는 H3N2 및 H1N2 인플루엔자 유래 유전자를 가진 재조합 바이러스 이었다. 분리된 H1N2 바이러스를 마우스에 접종한 결과, 마우스는 17% 정도 체중이 감소하였고, 염증 세포들이 침윤한 간질성 폐렴 증상을 보였다.

Influenza A viruses are circulating in a variety of hosts, including humans, pigs, and poultry. Swine influenza virus is a zoonotic pathogen that can be readily transmitted to humans. The influenza viruses of the 2009 H1N1 pandemic were derived from swine influenza viruses, and it has been suggested that the 1957 H2N2 pandemic and the 1968 H3N2 pandemic both originated in pigs. Pigs are regarded as a mixing vessel in the creation of novel influenza viruses since they are readily infected with human and avian influenza viruses. We isolated three novel H1N2 influenza viruses from pigs showing respiratory symptoms on a Korean farm in 2019. These viruses were reassortants, containing PA and NP genes from those of the 2009 H1N1 influenza virus in addition to PB2, PB1, HA, NA, M, and NS genes from those of triple-reassortant swine H3N2 and classical swine H1N2 influenza viruses circulating in Korean pigs. Mice infected with the isolated H1N2 influenza virus lost up to 17% body weight and exhibited interstitial pneumonia involving infiltration of many inflammatory cells. Results suggest that close surveillance to detect emerging influenza viruses in pigs is necessary for the health of both pigs and humans.

Introduction

Swine influenza virus belongs to influenza A virus and contains negative-sense RNA segmented genome, which contains PB2 (polymerase basic two), PB1 (polymerase basic one), PA (polymerase acidic), HA (hemagglutinin), NP (nucleoprotein), NA (neuraminidase), M (matrix), and NS (non-structural protein). Among them, HA and NA protein on the surface of influenza viruses are used for differentiating serological difference among influenza viruses [1].

H1N1 swine influenza viruses have been circulating in pigs in NorthAmerica since 1930s until novel triple ressortant H3N2 influenza virus appears in the late 1990s [2-4]. This novel ressortant H3N2 influenza virus contain NP, M, NS genes from classical swine H1N1 viruses, PB2 and PA genes from North American avian influenza viruses and HA, NA and PB1 genes from human seasonal H3N2 influenza viruses [4]. Triple reassortant internal gene cassette further diversified swine influenza viruses. H1N1 swine influenza virus contained HA and NA from classical H1N1 and the rest of genes from triple reassortant H3N2 influenza virus, and H1N2 swine influenza virus contained HA from the classical H1N1 and NA from human seasonal H3N2 influenza virus with rest of genes from triple reassortant H3N2 influenza virus [5,6]. The introduction of 2009 pandemic H1N1 influenza virus into pigs enhanced the diversity of influenza viruses circulating in pigs. Most of reassortant swine influenza viruses contained M gene from 2009-pandemic H1N1 influenza virus [7].

In Asian countries including China, Japan, Vietnam and South Korea, H1N1, H1N2 and H3N2-subtypes of influenza viruses are circulating in pig population. Novel reassortant H1N2 swine influenza viruses containing genes from classical swine influenza viruses, triple reassortant swine influenza viruses, and recent human influenza viruses were detected in pigs in China [8]. In Japan, H1N1 influenza viruses containing genes from 2009 pandemic H1N1 influenza viruses, H1N2 influenza viruses containing internal genes from 2009 pandemic H1N1 influenza viruses, and H3N2 influenza viruses containing internal genes from 2009 pandemic H1N1 influenza viruses were reported in pigs of Japan [9]. In Vietnam, H1N1 influenza viruses containing genes of 2009 pandemic H1N1 influenza viruses, and H3N2 influenza viruses containing genes of 2009 pandemic H1N1 influenza viruses and triple reassorant H3N2 influenza viruses are found in pigs in Vietnam [10]. In South Korea, H3N2 influenza viruses containing genes from classical swine H3N2 influenza viruses and genes from 2009 pandemic H1N1 influenza viruses were detected in pigs in South Korea [11]. H1N2 influenza viruses containing genes of triple reassortant H3N2 swine influenza viruses were also detected in pigs in South Korea [12].

In this study, we isolated three H1N2 influenza viruses in pigs suffering from respiratory distress on a local farm in South Korea, 2019, and genetically analysed these isolates. We also determined the pathogenicity of an isolate in mice.

Materials and Methods

Isolation of swine influenza viruses

Pigs (n=3) showing the respiratory distress on the farm in Chungnam province in South Korea were swabbed in PBS (pH 7.4) supplemented with Antibiotic Antimycotic Solution (100 units penicillin, 1 mg streptomycin and 2.5 μg amphotericin B) (Sigma), and the swabbed samples were transported to our laboratory. The samples were inoculated into the confluent Madin-Darby Canine Kidney (MDCK) cells in a 6-well cell culture plate with MEM with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK-treated trypsin (1 μg/ ml) (Sigma). The presence of virus was confirmed by hemagglutination assay with turkey red blood cells before they were sequenced. The isolated viruses were designated as A/swine/Korea/s1902/2019 (H1N2), A/swine/Korea/s1903/ 2019 (H1N2) and A/swine/Korea/s1904/2019 (H1N2).

Sequencing of the isolated swine influenza A viruses

A viral RNA mini kit (Qiagen) was used to isolate RNAs of isolated swine influenza viruses before cDNA was synthesized with ImProm-IITM Reverse Transcription System (Promega, Madison, USA) with Influenza A virus Uni 12 primer (AGCAAAAGCAGG). Viral eight genomes, PB2, PB1, PA, HA, NP, NA, M and NS, were amplified using GoTaq® Green Master Mix (Promega, Madison, USA) with as described [13]. The amplified eight segments were cloned into TA vectors (Promega, Madison, USA) and were sequenced at Cosmo Co (Daejeon, Korea).

Phylogenetic analysis of viral genes

The nucleotides of isolates were compiled and edited with the sequence analysis software package DNA Star version 4.0 (Madison, USA). The phylogenic analysis was performed with neighbor joining trees and molecular evolutionary genetics analysis software MEGA4 version 7.0. The nucleotide sequences of 2019 swine H1N2 influenza viruses were deposited into GenBank under accession numbers: MN598604-MN598611, MN598627-MN598626, MN598618-MN 598619.

Pathogenicity in mice

Six-week-old female mice (BALB/c) (n=10 per group) were intranasally (i.n.) infected with representative swine influenza virus, A/swine/Korea/s1902/2019 (H1N2) (50 μl of 10⁶ TCID₅₀ /ml). The infected mice were observed for bodyweight change and mortality for 14 days post infections (p.i.). Three mice were euthanized on day 5 post infections (p.i.) by intravenously injecting T61 (Intervet). The lung tissues were used for the measurement of viral titers, histopathology, and antibody staining in lung tissues.

Viral titers, histopathology, and immunohistochemistry in the lung tissues of mice

Half of lung tissues (about 0.1 g per lung sample) was homogenized in 1 ml of PBS (pH 7.4) for measuring viral titers and the rest of lung tissues was used for histopathology and antibody staining.

Viral titers in lung tissues were measured in MDCK cells with MEM supplemented with TPCK-treated trypsin (1 μg/ ml) by log₁₀ tissue culture infectious doses ₅₀ per 1g (log₁₀ TCID₅₀ /1 g). Mouse lung tissues were put in 10% neutral buffered formalin (10%) and then were embedded with paraffin. Lung tissues were cut into 5μm sections which were stained with haematoxylin (H) solution for 4 minutes. The stained tissues were washed with tap water for 10 minutes and then they were stained with eosin (E) solution. The stained tissue sections were recorded under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).

Mouse lung tissues were stained with rabbit anti- influenza A virus nucleoprotein antibody (Sino Biological, China). Tissue sections which were treated with antigen retrieval solution in a microwave oven were blocked with normal rabbit serum in PBS (pH 7.4), were labeled with a rabbit antibody against influenza A virus nucleoprotein (1:100 dilution), and were treated with biotin-labeled goat anti-rabbit imunoglobulin (Vector, USA), Vectastain ABC-AP (Vector, USA), and Vector Red alkaline phosphatase substrate (Vector, USA). The labeled mouse lung sections were counterstained with hematoxylin QS (Ve Laboratories, Burlingame, CA) and were observed under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).

Ethical approval

Internal Animal Use Committee in Chungnam National University (CNU) approved the protocol (201906A-CNU-105) for the pathogenicity in mice, and collection of clinical samples. All studies were carried out with approval and in accordance with relevant legal guidelines and regulations in CNU, Republic of Korea.

Results

Genetic analysis of swine isolates

We isolated 3 H1N2 influenza viruses from pigs showing the respiratory signs such as sneezing and runny nose on the farm in South Korea in 2019. Genetic information on the isolated swine H1N2 influenza viruses were analysed. Phylogenetic analysis revealed that PB2, PB1, HA, NA, M, and NS genes of 3 isolates were grouped together with those of H1N2 and triple reassortant H3N2 influenza viruses circulating in pigs (Fig. 1). PA and NP genes of 3 isolates were clustered with those of 2009 pandemic H1N1 influenza viruses (Fig. 1).

Fig. 1. Phylogenetic analysis of eight genes of swine H1N2 influenza viruses isolated from Korean pigs in 2019. The tree was compiled using the neighbor-joining method in MEGA 7.0 (www.megasoftware.net) with 1,000 bootstrap replicates. Scale bar shows nucleotide substitutions per site.

We performed a blast search in GenBank using 8 gene segments of a representative, A/swine/Korea/s1902/2019 (H1N2) (Table 1). PB2, PB1, HA, NA, M and NS genes of A/swine/Korea/s1902/2019 (H1N2) were closely related to that of A/swine/Korea/CY03-19/2012 (H3N2) with 98.33% homology, that of A/swine/Korea/CY03-13/2012 (H3N1) with 97.23% homology, that of A/swine/Korea/CY03-11/ 2012 (H1N2) with 96.83% homology, A/swine/Korea/CY03-11/2012 (H1N2) with 96.03% homology, that of A/swine/Korea/CY03-19/2012 (H3N2) with 98.78% homology, and that of A/swine/Ohio/11TOSU559/2011 (H3N2) with 98.33% homology, respectively. PA and NP genes of A/swine/Korea/s1902/2019 (H1N2) were closely related to that of A/Korea/CJ24/2009 (H1N1) with 97.21% homology, and that of A/Chile/3760/2009 (H1N1) with 97.10% homology, respectively.

Table 1. Nucleotide homology of H1N2 (A/swine/Korea/s1902/2019 (H1N2) swine influenza virus to the closest related influenza viruses

PB2: RNA polymerase basic subunit 2; PB1: RNA polymerase basic subunit 1; PA: RNA polymerase acidic subunit; HA: haemagglutinin; NP: nucleoprotein; NA: neuraminidase; M: Matrix gene; NS: non-structural gene.

Pathogenicity of swine isolates in mice

We i.n. inoculated mice (n=10 per group) with a representative isolate, A/swine/Korea/s1902/2019 (H1N2) and monitored the change of body weights and mortality. On day 5 after infections, 3 mice per group were euthanized for measuring viral titers in lung tissues, staining lung tissues with H&E, and detecting antigens in the lung tissues with influenza antibody. No mortality was observed in the infected mice, but the infected mice lost body weights up to 17.6% off the original body weights on day 6 after infections (Fig. 2A). When viral titer was measured in lung tissues of infected mice, it was 4.75 TCID₅₀ /g on day 5 p.i. (Fig. 2B). We stained the lung tissues of mice with H&E to find out the pathological damage. The interstitial pneumonia with infiltrations of inflammatory cells were found in the lung tissue in the infected mouse (Fig. 2C) in contrast to that in PBS-mock infected control mouse (Fig. 2D). Viral antigens were detected in bronchiolar cells and alveolar cells in the lung tissue of infected mouse (Fig. 2F) in contrast to that in PBS-mock infected control mouse (Fig. 2E).

Fig. 2. Pathogenicity of mice infected with 2019 swine H1N2 influenza virus. Six-week-old female mice (BALB/c) (n=10 per group) were i.n. infected with 50 μl of 10⁶TCID₅₀ of a representative virus, A/Swine/Korea/s1902/2019 (H1N2). Body weight change were monitored for 14 days (A). On day 5 after infection, mice (n=3 per group) were euthanized and their lung tissues were collected for viral titers (B), H&E staining (C:PBS-mock infected mouse lung, D: H1N2-infected mouse lung, arrow: interstitial pneumonia), (×400), and NP antibody staining (E: PBS-mock infected mouse lung , F: H1N2-infected mouse lung, arrow: antigen staining) (×400).

Discussion

Three novel H1N2 influenza viruses were isolated from pigs on the farm in South Korea in 2019. Genetic analysis showed that these isolates contained PA and NP genes derived from 2009-pandemic H1N1 influenza viruses with rest of genes from triple reassortant H3N2 or classical swine H1N2 circulating in Korean pigs. In 2012 and 2013, swine H1N2 influenza viruses containing the internal genes, PB2, PB1, PA, NP, M, and NS from triple reassortant swine H3N2 influenza viruses of which M gene were from 2009 pandemic H1N1 influenza virus were isolated Korean pigs [12,14]. It was reported that reassortant H1N2 influenza viruses were isolated in pigs in Asian countries such as China, Japan and Vietnam [15-17]. Two novel triple-reassortant H1N2 influenza viruses were isolated from swine in China in 2012[15]. Their HA gene were derived from Eurasian avian-like swine H1N1 influenza virus and their NA gene were derived from North American swine H1N2 influenza virus. Their internal six genes, PB2, PB1, PA, NP, M and NS were originated from those of 2009-pandemic H1N1 influenza viruses [15]. In Japan, two swine H1N2 influenza viruses isolated in 2013, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013 were reassortants. Their HA and NP genes were from a classical swine lineage circulating in Japanese pigs, their NA genes were from human-like H3N2 swine influenza viruses, and the rest of genes were from 2009-pandmeic H1N1 influenza viruses [16]. In Vietnam, eight H1N2 influenza viruses were isolated from pigs in 2013 and 2014 [17]. These viruses were reassortants containing HA from 2009-pandemic H1N1 influenza virus, and the rest of genes from triple reassortant H3N2 swine influenza viruses

Mouse pathogenicity study showed that mice infected with an H1N2 isolate suffered from the loss of body weight without mortality. Lung tissues showed quite a severe interstitial pneumonia. Previous study showed that mouse infected with 2013 Korean swine H1N2 influenza virus containing Eurasian avian-origin HA suffered from severe pneumonia, and that mouse infected with 2012 Korean swine H1N2 influenza virus containing classical swine-origin HA showed mild pneumonia [14].

The role of PA and NP genes in reassortant swine influenza viruses is needed to be studied in a further study.

In conclusion, our isolates are reassorant swine H1N2 influenza viruses containing 2009-pandemic H1N1 genes, and causes the loss of body weights and severe interstitial pneumonia in the infected mice. The results suggest that the close surveillance to detect emerging influenza viruses in pigs is needed for the health of pigs and humans.