Comparative Analysis of Biological Properties of Influenza А (Н1N1) pdm09 Virus Strains isolated in the Pandemic of 2009 and the Post-Pandemic Period in the Asian Part of Russia

Comparative analysis of influenza A (H1N1) pdm09 virus strains isolated in Russian Asia in the pandemic of 2009 and the postpandemic period of 2011 was carried out. It was shown that pandemic influenza А (H1N1) pdm09 virus strain isolated in the pandemic period is more virulent and able to cause more severe pathological processes in lung tissue of experimentally infected mice compared to the strain А (H1N1) pdm09 isolated in the post-pandemic period. It is probably associated with mutations in genes encoding surface proteins that occurred at the time of influenza A (H1N1) pdm09 virus circulation from 2009 to 2011 and that resulted in emergence of new genetic variants of virus with lower virulence.


Introduction
The first pandemic of the 21st century began in March of 2009 and was caused by new influenza А(H1N1)pdm09 virus strain of swine origin (CDC, 2010;Yatsyshina, 2011).This virus drew increased attention due to possible emergence of disastrous influenza pandemic comparable to Spanish influenza of 1918 having taken the lives of about 0.2-8% infected people from different countries (up to50 million people worldwide) (Murray, 2006).
Influenza a (H1N1) pdm09 virus is thought to have occurred in Mexico in the middle of February, 2009, however, exact place and conditions of its origin are still unclear (Fraser, 2009).New A (H1N1) pdm09 virus probably emerged because of two genotypes (American and Eurasian) reassortment of swine influenza A (H1N1) virus ( Lvov, 2010) and therefore may be rereferred as "quadruple" reassortant.High speed of epidemic development during March and April of 2009 affirmed that new antigenic influenza virus variant entered human population, crossed species barrier and was highly contagious (Baz, 2007;WHO, 2010;Yatsyshina, 2011).
Pandemic influenza viruses isolated in Russia from May 2009 to April 2010 were highly homologous to each other and to isolates from abroad, including references train А/California/04/2009(H1N1) pdm09 (Yatsyshina, 2011;Ilyicheva, 2011).Studies carried out in Russia revealed that during the 2009-2010 season influenza had monoetiological nature that is typically in the pandemic period (Chan, 2010).
Influenza А (H1N1) pdm09 morbidity in Russia can be divided into the following periods: pre-pandemic (April 27-June 11, 2009), initial pandemic (June 12 -August 15, 2009), and pandemic (August 15-December 1, 2009) (McCauley, 2012).During the prepandemic period the first case of human infection was identified in a Russian having returned from New Yorkto Moscow on May 21, 2009 (Lvov, 2010).Monitoring data of pandemic influenza virus in Ural, Siberian, and the Far Eastern Federal Districts of Russia showed that the first cases of А (H1N1) pdm09 in the Far East was detected 2-2.5 months later in comparison with Russian European part, and large increase occurred3 months later (Shchelkanov, 2010).From May 2009 to March 2010 samples from 862 patients suspected of having pandemic influenza were received from different regions of Russia by SRC VB "Vector".Eighty nine strains А (H1N1) pdm09 (Agafonov, 2011) were isolated from those samples, including 56 isolates from residents of Siberian and the Far East regions (Ilyicheva, 2011).The total number of detected cases of infection according to The Center of Influenza Ecology and Epidemiology (Ivanovsky Institute of Virology, Moscow) reached 3053 by the beginning of December, 2009(Chan, 2010).
On August 10, 2010 WHO declared cancellation of the 6th threat level for the world and the beginning of the post-pandemic period (WHO, 2010) when influenza А (H1N1) pdm09 virus circulation continued after changing into seasonal infection.
This study objectives were to compare biological characteristics of influenza А (H1N1) pdm09 virus strains having circulated in the pandemic of 2009 and in the post-pandemic period in Russian Asia.

Materials and Methods
In the season of 2009-2010 in Russian Asia we isolated 56 pandemic influenza А (H1N1) pdm09 virus strains (Ilyicheva, 2011).Antigenicity and biomolecular features were similar for all isolates.In our study we used strain A/Novosibirsk/13/2009 (H1N1) pdm09.During the season of 2010-2011 we isolated 41 influenza А (H1N1) pdm09 virus strains (Sobolev, 2012).Antigenicity and biomolecular features were slightly different for strains within one group.In the study we used strain А/Novosibirsk/KSH/2011 (H1N1) pdm09 isolated at the very beginning of influenza epidemic in Western Siberia in January, 2011.All manipulations with laboratory animals were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the SRC VB "Vector" in accordance with the research protocol approved by the Bioethical Committee of VECTOR.

Preparation of Lung Tissue Suspension.
To detect virus concentration in lung tissue of infected animals 100mg of lung tissue were homogenated in porcelain jar with following addition of 9ml 3 of Hanks' solution with antibiotics of the gentamicin and benzylpenicillin.The suspension was clarified by centrifugation.
Cells.Determination of virus titer was carried out in Madin-Darby canine kidney (MDCK) cell culture.MDCK cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) (BioloT, Saint-Petersburg) with addition of 10% cow fetal serum ("HyClone", Erembodegem, Belgium), 100 units per ml of benzylpenicillin in the atmosphere containing 5% of CO2 at 37ºС.Virus cytopathic effect was registered every day.Hemagglutination reaction was positive in each well where cytopathic effect was registered.
Antibody-Mediated Methods.Hemagglutination inhibition (HI) and microneutralization (MN) assays were carried out in compliance with protocols developed by the Influenza Division at the Centers for Disease Control and Prevention (CDC), Atlanta, USA (WHO, 2011).
MID50 and TCID50 values were calculated by the Reed and Muench method (Reed, 1938).
Reference Sera.We used reference serum to virus А (H1N1) pdm09 kindly furnished by WHO Collaborating Centre for influenza (CDC, Atlanta, USA).
Strain titration was conducted in the HI assay using standard practice recommended by WHO with diagnostic serum to influenza A (H1N1) pdm09 virus and goose red blood cells.Real time polymerase chain reaction (PCR) was also carried out to subtype influenza A viruses and differentiate influenza А (H1N1) pdm09 virus.
Genome Sequencing.RNA isolation was carried out using standard practice with "A set of reagents for the detection of swine influenza A/H1 using PCR with hybridizationfluorescence detection "AmpliSens ® Influenza virus A/H1-swine-FL" (Ilyicheva, 2011).PCR amplification and following sequencing were carried out using specific primerpairs recommended by WHO for work with influenza А (H1N1) pdm09 viruses (WHO, 2009).
The analysis of amino-acid changes in hemagglutinin gene (coding part using numeration typical for H1) and neuraminidase (coding part) was carried out with the program Bi-oEdit ver.7.0.9.0.
Phylogenetic analysis of gene sequences was performed with the method of neighbor joining with the program MEGA 5.1.To evaluate reliability bootstrap test (1000 replications) was used.When building phylogenetic trees sequences from GISAID and GenBank databases were used.
Light Microscopy.Excised tissues of the lung of euthanized mice by dislocation of the cervical vertebrae at 1, 3, 6, and 10 days post infection were preserved in 10% phosphatebuffered formalin.Tissues were then processed for paraffin embedding and cut into 5μm-thick sections.The section from each tissue sample was stained using a standard hematoxylin-and-eosin procedure.Lightoptical study and microphotography were carried out using Axioskop 40 (Zeiss, Germany) microscope.

Results
We investigated virulent and immunogenic features of influenza virus strains N13-2009 and KSH-2011 in a mouse model.When mice were infected with virus strains symptoms of disease in both groups were registered since the first day.Disease was accompanied by conjunctivitis, decreased activity, decrease of body weight, breathlessness.The virustiters in lung were registered up to 6 days (Table 1).In sera from mice infected with strain А/Novosibirsk/KSH/2011 (H1N1) pdm09 antibodies were detected only to "their own" strain and only using microneutralization assay.We detected nucleotide sequence of hemagglutin in (HA) and neuraminidase (NA) genes and protein amino-acid sequence of influenza virus strains under study.Also was carried out comparative analysis between aminoacid sequences in HA and NA proteins of studied strains and proteins of one of the first influenza virus strains A/Mexico/InDRE4487/2009 (H1N1) pdm09 and the vaccine strain А/California/07/2009 pdm09 (Table 4).Strain N13-2009 proteins slightly differed from HA and NA of compared strains.Thus, amino-acid changes were detected in: antigenic site of hemagglutinin (Ca) I166T, S203T, R205K and in neuraminidase -V106I, N248D.Strain KSh-2011 had significantly more amino-acid changes: in subunit HA1-S143G, A197T, K211N, A215E, including changes in antigenic sites-(Ca) S203T, (Cb) L70P, (Sb) S185T; in subunit HA2-Е374К, S451N; and in neuraminidase -V106I, N248D.As a result, in the case of influenza А (Н1N1) pdm09 virus isolated in the post-pandemic period, 2011, concentration of amino-acid changes was registered in HA gene compared to influenza virus circulated during the pandemic season of 2009, that was caused by virus antigenic drift under the influence of herd immunity.On the first day post infection there were no significant changes.However, in lung tissue of mice infected with strain of 2009 we registered small concentration of erythrocytes in alveoli, spasm of minute vessels and small in duration of surrounding respiratory tissue (Fig. 3 A, B).
On the third day after infection inflammatory process began in bronchial epithelium in both groups.Hygroscopic dystrophy of lung epithelium developed.However, in lung tissue of mice infected with isolates of 2009 pathologic process was visible not only in large and medium bronchi, but also in bronchial tubes.This points, in turn, at generalization of infection and viral particles invasion into lower respiratory tract.
On the sixth day post infection respiratory tissue airway obstruction was observed.Hypersecretion of mucin-producing cells of bronchioles was registered.Cell debris, multinucleated cells and mucus were visible in bronchi lumen (Fig. 3 C, E, F).We registered the focuses of leucocytic infiltration comprising monocytes, lymphocytes and neutrophiles located in interalveolar septums, focuses of atelectasis and perivascularly.In interstitial tissue surrounding airways and alveolar sacs edema and multilobular necrosis were found.Plasmorrhagia were observed not only on blood vessel walls, but also in interstitium.
On the tenth day post infection we observed marked inflammatory changes in lung tissue of animals experimentally infected with strain N13-2009 (Fig. 3 G).Parenchyma airway obstruction dramatically decreased by type of atelectases and emphysematously dilatated alveoli.In the group of mice infected with strain KSh-2011 together with destruction processes in alveolar tissue we could discern the beginning of reparative processes that was confirmed by the presence of fibroblasts and collagen fibers (Fig. 3 H).Submucous layer edema was reduced.

Discussion
During this study we carried out comparative analysis of influenza А (H1N1) pdm09 virus strains isolated in Russian Asia in the pandemic of 2009 and in the postpandemic period of 2011.Strain N13-2009 was isolated in Russia at the beginning of the pandemic period from 28-years-old woman having returned from Canada.She recovered.Another strain KSh-2011 was isolated in Russia in post-pandemic period (January, 2011) from 27-years-old man who had not been abroad.He recovered, too.
Comparison of virological characteristics of these strains revealed that both strains actively replicated in mice lung without previous adaptation.and predominantly peribronchial interstitial pneumonia, in a whole inflammatory process was more severe when mice were infected with the strain of 2009.We suppose that mutation accumulation in genotype of pandemic influenza virus led to decrease of its pathogenicity by 2011 that had an impact on the course of inflammatory process in tissue of lung as the target organ.
Our research supported by date of the Federal State Budgetary Institution «Research Influenza Institute» Ministry of Health and Social Development of Russia (Karpova, 2011).The epidemic 2011, in contrast to the pandemic period 2009, was lower intensity of involvement in the age groups population of the cities; for the duration of the epidemic; the incidence in the period of the epidemic of the whole population.Decreased hospitalization rate from the number of patients with influenza and acute respiratory viral infections and the percentage of deaths.However, the data presented Rodrígueza and others (2011) indicate significant differences in the postpandemic period in 2011 between Russia and Spain.According to them, in patients admitted to the intensive care unit in the post-pandemic period of 2011 had higher frequency of severe comorbidities, severity of illness and increased mortality when compared with those observed in the 2009 pandemic outbreak.
In summary, we showed that pandemic influenza virus strain N13-2009 is more virulent and is able to cause more severe pathological processes in lung tissues compared to strain KSH-2011 isolated in the post-pandemic period.It seems that during influenza A(H1N1)pdm09 virus circulation from 2009 to 2011 in the structure of surface proteins mutations occurred, that caused change of genetic clade on phylogenetic tree and decrease of virulent features and presumably immunogenicity in virus biology.
_______________ E. A. Prokopeva, S. G. Sayfutdinova, A. V. Glushchenko, O. G. Kurskaya, A. V. Zaykovskaya, A. G. Durymanov, T. N. Ilyicheva, L. V. Shestopalova and A. M. Shestopalov.(2013), Journal of Virology & Microbiology, Bold type and underlined type represent amino-acids that are thought to be typical changes for described strains in their circulation period.Italic type indicates unique amino-acid changes.Nucleotide sequences comparison of HA and NA of studied strains with relevant sequences of other strains from GISAID and GenBank databases helped us carry out phylogenetic analysis and build phylogenetic trees (Fig. 1, 2).Sequences were chosen taking into account the division into genotypes offered in the report of WHO Collabo-rating Centre for Reference and Research on Influenza in London (McCauley, 2012).According to this document 8 different genetic clades of HA and NA are distinguished on the base of amino-acid changes emerged during virus circulation in human population in 2009-2011._______________ E. A. Prokopeva, S. G. Sayfutdinova, A. V. Glushchenko, O. G. Kurskaya, A. V. Zaykovskaya, A. G. Durymanov, T. N. Ilyicheva, L. V. Shestopalova and A. M. Shestopalov.(2013), Journal of Virology & Microbiology, DOI: 10.5171/2013.890660

Fig. 2 :
Fig. 2: NA Phylogenetic Tree Phylogenetic features of HA and NA genes of the studied strain N13-2009 (bold type in the figure), isolated in 2009, are very similar to those of virus genes isolated in