Isolation and characteristics of influenza viruses circulating among swine populations in Kazakhstan during 2018–2019

Swine influenza is regarded as a highly contagious and acute infection. Swine can serve as a tool for the emergence of new influenza viruses that are potentially dangerous to humans. This paper shows the results of the isolation of influenza virus strains circulating among swine in Kazakhstan during 2018–2019 and the characterization of their biological properties. Biological samples were collected from pigs of 2 to 5 months of age in peasant livestock farms in East Kazakhstan, North Kazakhstan, Almaty, and Pavlodar regions. During the execution of the polymerase chain reaction for 662 collected samples, the genetic material of the influenza virus was found in 3.17 % of cases, of which the influenza A/H1N1 virus RNA was detected in 1.08 % and that of the A/H3N2 virus in 0.30 %. With performing virological studies of the obtained samples, three hemagglutinating agents were retrieved and indicated as influenza A viruses of the H1N1 and H3N2 subtypes. The isolated strains of swine influenza A virus revealed similarities with each other in a number of characteristics (temperature sensitivity of hemagglutinin, rates of adsorption and elution, sensitivity to nonspecific serum inhibitors), but differed in infectious activity. The results achieved in the detection of virus genetic material in the polymerase chain reaction and the data of virological studies confirmed the circulation of influenza A/H1N1 and a/h3n2 viruses among the swine population in different regions of Kazakhstan during 2018–2019. These findings highlight the importance of continuous monitoring of swine influenza viruses for detection of possibility of interspecies transmission of this infectious agent.

Introduction

The influenza virus (IV) belongs to the Orthomyxoviridae family and is the major causative agent of infectious diseases of respiratory tract of humans and animals. Today, it is common to classify influenza viruses into four types: A, B, C, D.

Influenza A virus (IAV) can infect epithelial cells of almost all species of birds and mammals including humans [1]. Swine IV mainly affects the epithelium of the respiratory tract, causing cough, fever, somnolence, and anorexia. IAV is one of the most widespread exciters of respiratory tract diseases in pigs throughout the world [2]. The rate of IAVs infection in swine is high (up to 100 %); the disease is accompanied by low lethality: only 1 % of infected animals die. However, reproductive disorders along with weight loss and exacerbation of secondary infections characteristic of swine influenza result in some serious problems in animals and huge economic losses for the swine industry [3].

Currently, three influenza A subtypes are the most commonly circulating in swine: H1N1, H3N2, and H1N2 [4]. The swine population plays a significant role in the IAVs’ evolution since they are a unique reservoir for the re-assortment of multi-host infectious agents. The presence of cellular receptors for human, mammalian, and avian IAVs in swine explains the possibility of their transmission from humans and birds to swine and vice versa [5]. There is abundant evidence that all IV subtypes can persist in swine. The simultaneous infection of an animal with viruses of various origins can lead to their re-assortment. The resulting IV reassorting strains may give rise to a new influenza pandemic. Such an example is presented by two influenza pandemics of the XX century (IV A/H2N2 and A/H3N2), as well as the triple re-assorting H1N1pdm09, which has overcome the interspecies barrier, rapidly spread among the population of all countries of the world [6].

In recent years, a/h1n1pdm09 virus which caused the 2009–2010 influenza pandemic and subsequently almost displaced the previously circulating seasonal A/H1N1 strains continues to circulate in Kazakhstan. At the same time, the circulation of viruses with the antigenic formula A/H3N2 and two type B lines is going on in parallel with IV A/H1N1pdm09 [7].

Human and swine IVs exhibit a number of common features which suggests their relationship and common origin [8]. The issue of interspecies transmission of human and swine IAV (H1N1) is an important factor in the studies of the evolution, ecology, and epidemiology of the pathogen.

In the Republic of Kazakhstan, influenza epizootics among swine are a matter of great concern. In 1984, as a result of virological examination of piglets with clinical signs of respiratory diseases in swine farms located in East Kazakhstan, three IV A/H1N1 strains were isolated. The circulation of influenza A/H1N1 and a/h3n2 viruses was revealed in 2014–2018 among pigs of different ages in peasant farms located in different regions of the country [9, 10].

Determination of the biological and antigenic characteristics of circulating viruses makes it possible to identify the key directions in variation, phylogenetic relationships of the strains that were observed earlier and constantly appear in different countries and regions. The presence of mechanisms for antigenic variation in the influenza virus can lead to a radical change in their biological properties [11–15].

To understand the origin and structure of pandemic IVs, predict and prevent epidemics and pandemics, the surveillance of influenza infection among swine and the identification of new reassortant variants will therefore never lose their relevance.

In this regard, the goal of this investigation is to obtain and describe swine IV among animal populations in Kazakhstan during 2018–2019.

Experimental

Nasopharyngeal swabs from pigs were gathered in flasks with 2 mL of medium 199; 0.5 % bovine serum albumin and a complex of antibiotic drugs (50,000 units/mL of penicillin; 50 μg/mL of streptomycin; 3,000 μg/mL of gentamicin; 5,000 units/mL of nystatin). The specimens were retained for a day at 4 °C and preserved in liquid nitrogen (–196 °C).

RT-PCR was implemented on a Rotor-Gene Q 6Plex amplifier (QIAGEN, Germany) using RIBO-prep, AmpliSens® Influenza virus A/B-FL, AmpliSens® Influenza virus A-type FL, and AmpliSens® Influenza virus A/H1-swine-FL kits (Moscow).

9–11-day-old developing chicken embryos (ce) were used as a model for the isolation of hemagglutinating agents (HAA). To indicate the virus in the hemagglutination assay, a 0.75 % suspension of chicken and human 0 (1) blood group erythrocytes was used.

New viruses were typed in the hemagglutination inhibition (HAI) assay and the neuraminidase inhibition (NAI) assay according to the WHO recommendations with the panels of polyclonal serums [16].

The infectious activity of the strains was established in accordance with the generally accepted method [17], their titer was manifested in lgEID50/0.2 mL.

Antigenic surface glycoproteins were examined in the HAI assay according to the WHO recommendations [18] with rabbit immune serums.

The adsorbing properties of viruses were studied for 18 hours on formalized chicken erythrocytes with constant stirring at 4 °C. The rate of elution from erythrocytes of the Kazakhstan swine iv strains was determined in buffered saline at 37 °C with a time interval of 30–240 min.

The sensitivity of hemagglutinin (HA) to the temperature factor was determined by the ability of viruses to agglutinate erythrocytes after heating at 56 °C for 5, 10, 15, 30, and 60 min [19].

The spectrum of hemagglutinating activity of the strains was established in the hemagglutination assay with 0.75 % suspensions of guinea pig, hen, ram, and human blood group 0 (1) red blood cells [20].

The responsiveness of strains to nonspecific inhibitors was recognized in the HI assay with guinea pig, hen, and rabbit blood serums with native (without heating) and after heating (at 62 °C for 30 min and 100 °C for 10 min).

Statistical processing of the results was implemented by obtaining the geometric mean of the data collected in the thrice-repeated experiments and determining their standard deviations.

Results and Discussion

To carry out virological studies, in 2018–2019 biological samples were collected from 2–5-month-old pigs in peasant livestock farms located in the Almaty, North Kazakhstan, East Kazakhstan, and Pavlodar regions. A total of 662 nasopharyngeal swabs were obtained. The results of the primary investigation of nasopharyngeal samples for present of genetic material of IVs in RT-PCR assays are presented in Table 1.

Table 1

Characterization and RT-PCR screening of biological samples collected from swine in 2018–2019

As shown in Table 1, while carrying out studies of samples in RT-PCR, the IV genetic material was revealed in 21 one (3.17 % of the total amount of investigated samples). When subtyping IV-positive samples, influenza A/Hsw1N1 virus RNA was detected in seven samples (1.08 %), while influenza A/H3N2 virus RNA in two samples (0.30 %). The subtype could not be identified in nine biological samples (1.36 %).

Thus, the primary screening of nasopharyngeal swabs showed the circulation of influenza A/Hsw1N1 and A/H3N2 viruses among the swine population in Kazakhstan.

In the case of primary infection of CEs with samples collected from different regions of Kazakhstan in 2018–2019, three haas were identified (a/swine/pavlodar/43/19, a/swine/pavlodar/44/19, and a/swine/al- maty/45/19) with hemagglutination titers which varied in the range of 1:32–1:128.

The antigenic formula of the isolates was determined in the HAI and NAI assays and RT-PCR. Table 2 is presented the results of HA subtype identification in the HAI assay.

Table 2

Identification of hemagglutinin subtypes of swine influenza viruses in HAI assay in Kazakhstan 2019

Table 2 shows that the hemagglutinating activity of the A/swine/Pavlodar/43/19 and A/swine/Pavlo- dar/44/19 isolates from 1/16 to 1/4 of the homologous titer (1:40–1:160) was inhibited by diagnostic serums against reference viruses with antigenic formula A/H1N1. This made it possible to assignHAA from the Pavlodar region (43/19 and 44/19) to IAV with the H1 HAsubtype. Isolate A/swine/Almaty/45/19 reacted with immune serums against reference variants with antigenic formula a/h3n2 in 1/8–1/4 of the homologous titer (1:40–1:160), which identified it as iv with the h3ha subtype.

Identification of the neuraminidase subtype, carried out in the NAI assay, showed that the enzymatic activity of isolates with the A/H1 HA subtype (A/swine/Pavlodar/43/19 and A/swine/Pavlodar/44/19) was inhibited by polyclonal diagnostic serum against A/H1N1 virus, while that with the A/H3 HA subtype (A/swine/Almaty/45/19) by the serum against A/H3N2 virus in the titers of 1:100.

RT-PCR studies confirmed the belonging of two strains isolated from biological samples collected in Pavlodar (43/19 and 44/19) to IV A/H1N1 and that of the 45/19 strain isolated from samples obtained in Almaty to influenza A/H3N2 virus.

According to the results of identification in the HAI and NAI assays and RT-PCR, the Pavlodar isolates: 43/19 and 44/19 were authenticated as influenza A/H1N1 viruses, the Almaty isolate 45/19 was allocated to influenza A virus with antigenic formula H3N2.

Isolates were cloned by the limiting dilution method on developing CE. As the results showed, the hemagglutinating activity of strains (A/swine/Pavlodar/43/19, A/swine/Pavlodar/44/19, and A/swine/Al- maty/45/19) was in the range of 1:512–1:1024.

In Table 3 the results of studies of the major biological properties (infectious activity, HA thermal sensitivity, adsorption-elution) of the regional and reference IV strains are shown.

Table 3

Biological properties of swine influenza virus strain isolated in 2019 in comparison with reference viruses

Note — * the geometric mean logarithms to the base 2 for hemagglutinin inverse titers after heating at 56 °C are presented

Table 3 shows that the infectious activity of swine IV strains on EC ranged from 3.8 to 5.4 lg EID50/0.2 mL. On this basis, the Kazakhstan isolates were slightly inferior to the reference strains, the infectivity of which was 6.0–8.7 lg eid50/0.2 mL^.

While studying the HA sensitivity of the Kazakhstan 2019 swine influenza viruses to heating at 56 °C, it was found that, as the reference strains, they were classified as thermostable variants, since after heating for 60 min they retained the ability to agglutinate chicken erythrocytes in high titers.

All examined viruses had good adsorption and elution capacity in relation to chicken erythrocytes, since the percentage of their adsorption on erythrocytes was 90–100 %, the elution process was completed after 30– 60 min of incubation at 37 °C.

While examining the spectrum of hemagglutinating activity with erythrocytes from humans with 0 (1) blood group and various animal species, it was found that the Kazakhstan strains, as the reference variants, actively agglutinated in high titers all types of erythrocytes taken in the experiment (Tab. 4).

Table 4

Hemagglutinating activity of isolates and reference strains in relation to erythrocytes from humans and various animal species

The sensitivity of isolated strains to serum inhibitors was determined in the HAI assay with the guinea pig, chicken, and rabbit blood serums. The data are illustrated in Table 5.

Table 5

Sensitivity of isolates and reference strains to nonspecific inhibitors of blood serums

Note — the reciprocals of specific anti-hemagglutinin titers are presented/

It is indicated in Table 5 that all strains were inhibitor-resistant. Their hemagglutinating activity was not inhibited by native chicken, guinea pig, and rabbit serums. At the same time, the heating of blood serums promoted an increase in their inhibitory activity. Titers increased in the HAI assay with serums heated at 62ºC and even more during boiling (1:20–1:160).

Thereby, influenza virus strains isolated from pigs nasal swabs of Pavlodar and Almaty regions in 2018– 2019 showed similarities with each other in terms of HA thermal sensitivity, adsorption and elution rates, sensitivity to nonspecific serum inhibitors, but differenced in infectious activity.

Conclusions

Pigs as a mixing vessel play a major role in the interspecies adaptation and spread of IVs. In order to reduce the likelihood and minimize the role of these animals as a source of the next pandemic causative agent, a decisive role is therefore assigned to the continuous surveillance of influenza in the populations of these animals. Obtaining the comprehensive picture of the infectious process of the influenza infection process will make it possible to predict the epidemiological and epizootic situation; thus, to choose the correct strategy and tactics for conducting preventive measures, identifying the unique biological properties of swine IV is an important component.

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