Novel host markers in the 2009 pandemic H1N1 influenza a virus

The winter of 2009 witnessed the concurrent spread of 2009 pandemic H1N1 with 2009 seasonal H1N1. It is clinically important to develop knowledge of the key features of these two different viruses that make them unique. A robust pattern recognition technique, Random Forests, was employed to uncover essential amino acid markers to differentiate the two viruses. Some of these markers were also part of the previously discovered genomic signature that separate avian or swine from human viruses. Much research to date in search of host markers in 2009 pandemic H1N1 has been primarily limited in the context of traditional markers of avian-human or swine-human host shifts. However, many of the molecular markers for adaptation to human hosts or to the emergence of a pandemic virus do not exist in 2009 pandemic H1N1, implying that other previously unrecognized molecular determinants are accountable for its capability to infect humans. The current study aimed to explore novel host markers in the proteins of 2009 pandemic H1N1 that were not present in those classical markers, thus providing fresh and unique insight into the adaptive genetic modifications that could lead to the generation of this new virus. Random Forests were used to find 18 such markers in HA, 15 in NA, 9 in PB2, 11 in PB1, 13 in PA, 10 in NS1, 1 in NS2, 11 in NP, 3 in M1, and 1 in M2. The amino acids at many of these novel sites in 2009 pandemic H1N1 were distinct from those in avian, human, and swine viruses that were identical at these positions, reflecting the uniqueness of these novel sites.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .

Clinical Predictors for Diagnosing Pandemic (H1N1) 2009 and Seasonal Influenza (H3N2) in Fever Clinics in Beijing,China

Objective Symptomatic predictors of influenza could assess risks and improve decisions about isolation and outpatient treatment. To develop such predictors, we undertook a prospective analysis of pandemic （HIN1） 2009 and seasonal influenza （H3N2） in patients attending fever clinics. Methods From 1 May 2009 to 1 January 2010, all adult patients admitted to fever clinics for suspected influenza, confirmed by real time RT-PCR, were enrolled. Predictors of influenza virus infection were selected with logistic regression models. Measures of sensitivity, specificity, positive and negative likelihood ratios （LRs） were calculated to identify the best predictors. Results The clinical features and routine blood test results of influenza （H1N1） 2009 and seasonal influenza were similar. The positive and negative LRs of current US CDC influenza-like illness （ILl） criteria were modest in predicting influenza infection. Our modified clinic predictors improved the ability of the positive and negative LRs to recognize pandemic （HIN1） 2009 and seasonal influenza. The revised criteria are： fever ~ 38 ~C accompanied by at least one of the following--cough, arthralgia or relative iymphopenia. Conclusion Patients with symptoms and signs that meet the new criteria are likely to have influenza and timely antiviral therapy may be appropriate. In addition, physicians should ascertain if influenza is circulating within the community or if there is a contact history of influenza and combine this information with the newly developed criteria to clinically diagnose influenza.

New mutational trends in the HA protein of 2009 H1N1 pandemic influenza virus from May 2010 to February 2011

As we enter the year of 2011, the 2009 H1N1 pandemic influenza virus is in the news again. At least 20 people have died of this virus in China since the beginning of 2011 and it is now the predominant flu strain in the country. Although this novel virus was quite stable during its run in the flu season of 2009-2010, a genetic variant of this virus was found in Singapore in early 2010, and then in Australia and New Zealand during their 2010 winter influenza season. Several critical mutations in the HA protein of this variant were uncovered in the strains collected from January 2010 to April 2010. Moreover, a structural homology model of HA from the A/Brisbane/10/2010(H1N1) strain was made based on the structure of A/California/04/2009 (H1N1). The purpose of this study was to investigate mutations in the HA protein of 2009 H1N1 from sequence data collected worldwide from May 2010 to February 2011. A fundamental problem in bioinformatics and biology is to find the similar gene sequences for a given gene sequence of interest. Here we proposed the inverse problem, i.e., finding the exemplars from a group of related gene sequences. With a clustering algorithm affinity propagation, six exemplars of the HA sequences were identified to represent six clusters. One of the clusters contained strain A/Brisbane/12/2010(H1N1) that only differed from A/Brisbane/10/2010 in the HA sequence at position 449. Based on the sequence identity of the six exemplars, nine mutations in HA were located that could be used to distinguish these six clusters. Finally, we discovered the change of correlation patterns for the HA and NA of 2009 H1N1 as a result of the HA receptor binding specificity switch, revealing the balanced interplay between these two surface proteins of the virus.

Subtle differences in receptor binding specificity and gene sequences of the 2009 pandemic H1N1 influenza virus

A recent phylogenetic inference indicated that the 2009 pandemic H1N1 strains circulating from March 2009 to September 2009 could be divided into two closely related but distinct clusters. Cluster one contained most strains from Mexico, Texas, and California, and cluster two had most strains from New York, both of which were reported to co-circulate in all continents. The same study further revealed nine nucleotide changes in six gene segments of the new virus specific for the two clusters. In the current study, the informational spectrum method (ISM), a bioinformatics technique, was employed to study the receptor binding patterns of the two clusters. It discovered that while both groups shared the same primary human binding affinity, their secondary binding preferences were different. Cluster one favored swine binding as its secondary binding pattern, whereas cluster two mostly exhibited the binding specificity of A/South Carolina/1/18 (H1N1) (one of the 1918 flu pandemic strains) as its secondary binding pattern. Besides all the nine nucleotide changes found in the previous study, Random Forests were applied to uncover several new nucleotide polymorphisms in 10 genes of the strains between the two clusters, and several amino acid changes in the HA protein that might be accountable for the discrepancy of the secondary receptor binding patterns of the two clusters. Finally, entropy analysis was conducted to present a global view of gene sequence variations between the two clusters, which illustrated that cluster one had much higher genetic divergence than cluster two. Furthermore, it suggested a significant overall correspondence between the nucleotide positions of high importance in differentiating the two clusters and nucleotide positions of high entropy in cluster one.

Receptor binding specificity and origin of 2009 H1N1 pandemic influenza virus

Recently, a genetic variant of 2009 H1N1 has become the predominant virus circulating in the southern hemisphere, particularly Australia and New Zealand, and in Singapore during the winter of 2010. It was associated with several vaccine breakthroughs and fatal cases. We analyzed three reported mutations D94N, N125D, and V250A in the HA protein of this genetic variant. It appeared that the reason for D94N and V250A to occur in pairs was to maintain the HA binding to human type receptor, so the virus could replicate in humans efficiently. Guided by this interpretation, we discovered a new mutation V30A that could compensate for N125D as V250A did for D94N. We demonstrated that the presence of amino acids 30A and 125N in HA enhanced the binding to human type receptor, while 30V and 125D favored the receptors of avian type and of A/South Carolina/1/18 (H1N1). Furthermore, a combination of 94D, 125D, and 250V made the primary binding preference similar to that of A/South Carolina/1/18 (H1N1) and a combination of 94N, 125D, and 250A resulted in the primary binding affinity for avian type receptor, which clearly differed from that of A/California/07/2009 (H1N1), a strain used in the vaccine for 2009 H1N1. We also re-examined the origin of 2009 H1N1 to refine our knowledge of this important issue. Although the NP, PA, PB1, and PB2 of 2009 H1N1 were closest to North American swine H3N2 in sequence identity, their interaction patterns were closest to swine H1N1 in North America.

Effect of Aluminum Hydroxide Adjuvant on the Immunogenicity of the 2009 Pandemic Influenza A/H1N1 Vaccine:Multi-level Modeling of Data with Repeated Measures

Objective To evaluate the effect of the aluminum hydroxide （Al-OH） adjuvant on the 2009 pandemic influenza A/H1N1 （pH1N1） vaccine. Methods In a multicenter, double-blind, randomized, placebo-controlled trial, participants received two doses of split-virion formulation containing 15 ug hemagglutinin antigen, with or without aluminum hydroxide （N-OH）. We classified the participants into six age categories （〉61 years, 41-60 years, 19-40 years, 13-18 years, 8-12 years, and 3-7 years） and obtained four blood samples from each participant on days 0, 21, 35, and 42 following the first dose of immunization. We assessed vaccine immunogenicity by measuring the geometric mean titer （GMT） of hemagglutination inhibiting antibody. We used a two-level model to evaluate the fixed effect of aluminum Al-OH and other factors, accounting for repeated measures. Results The predictions of repeated measurement on GMTs of formulations with or without Al-OH, were 80.35 and 112.72, respectively. Al-OH significantly reduced immunogenicity after controlling for time post immunization, age-group and gender. Conclusion The Al-OH adjuvant does not increase but actually reduces the immunogenicity of the split-virion pH1N1 vaccine.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China

Objective To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China Methods Recombinant viruses were rescued using reverse genetics. Neuraminidase （NA） and hemagglutinin （HA） segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpoll vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embnjonate survival and antigenicity were compared with those of the respective wild-type viruses. Results The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. Conclusion The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

Intergenic subset organization within a set of geographically-defined viral sequences from the 2009 H1N1 influenza A pandemic

We report a bioinformatic analysis of the datasets of sequences of all ten genes from the 2009 H1N1 influenza A pandemic in the state of Wisconsin. The gene with the greatest summed information entropy was found to be the hemagglutinin (HA) gene. Based upon the viral ID identifier of the HA gene sequence, the sequences of all of the genes were sorted into two subsets, depending upon whether the nucleotide occupying the position of maximum entropy, position 658 of the HA sequence, was either A or U. It was found that the information entropy (H) distributions of subsets differed significantly from each other, from H distributions of randomly generated subsets and from the H distributions of the complete datasets of each gene. Mutual information (MI) values facilitated identification of nine nucleotide positions, distributed over seven of the influenza genes, at which the nucleotide subsets were disjoint, or almost disjoint. Nucleotide frequencies at these nine positions were used to compute mutual information values that subsequently served as weighting factors for edges in a graph net-work. Seven of the nucleotide positions in the graph network are sites of synonymous mutations. Three of these sites of synonymous mutation are within a single gene, the M1 gene, which occupied the position of greatest graph centrality. It is proposed that these bioinformatic and network graph results may reflect alterations in M1-mediated viral packaging and exteriorization, known to be susceptible to synonymous mutations.

Novel H1N1 influenza A virus infection in a patient with acute rejection after liver transplantation

BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.

Severe Novel Influenza A(H1N1) in Shanghai:Clinical Features, Therapeutic Management and Risk Factors for Mortality

Objective To analyze the clinical features,therapeutic management and risk factors for mortality of patients with severe novel A(H1N1)influenza in Shanghai,China.Methods All patients were diagnosed by influenza A(H1N1)virus mRNA detection.Chest CT scan,routine blood,hepatic function,humoral and cellular immunity,sputum smears,and sputum cultures were performed.Logistic analysis was applied to identify risk factors for mortality.Results Total of 68 patients were enrolled in this study,the primary clinical symptoms including cough(66,97.1%),expectoration(41,60.3%),and polypnea(41,60.3%).Altogether,37(54.4%)and 11(16.2%)patients were infected with bacterial and fungal,respectively.CT scan demonstrated that 67(98.6%)patients had pneumonia.Oxygen therapy,oseltamivir,antibiotic and antifungal drugs were performed in 68(100%),66(97.1%),39(57.4%),and 11(16.2%)patients,respectively.Finally,4 of 68 patients died.Logistic analysis demonstrated that there was a significant correlation between the percentage of neutrophils and mortality before therapy and direct bilirubin content and mortality after therapy,respectively.Conclusions Patients with severe H1N1 influenza were susceptible to bacterial and/or fungal infection.The risk factors for mortality may be associated with pre-therapeutic neutrophil percentage and post-therapeutic direct bilirubin content.

Naturally occurring PA^(E206K)point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century.In this study,we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients.Sequence comparison of the three main protein subunits(PB2,PB1,and PA)of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution(E206K)was responsible for the observed impaired replication phenotype.Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo.Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature(39.5℃).Hence,this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.

Correlated mutations in the four influenza proteins essential for viral RNA synthesis, host adaptation, and virulence: NP, PA, PB1, and PB2

The NP, PA, PB1, and PB2 proteins of influenza viruses together are responsible for the transcription and replication of viral RNA, and the latter three proteins comprise the viral polymerase. Two recent reports indicated that the mutation at site 627 of PB2 plays a key role in host range and increased virulence of influenza viruses, and could be compensated by multiple mutations at other sites of PB2, suggesting the association of this mutation with those at other sites. The objective of this study was to analyze the co-mutated sites within and between these important proteins of influenza. With mutual information, a set of statistically significant co- mutated position pairs (P value = 0) in NP, PA, PB1, and PB2 of avian, human, pandemic 2009 H1N1, and swine influenza were identified, based on which several highly connected networks of correlated sites in NP, PA, PB1, and PB2 were discovered. These correlation networks further illustrated the inner functional dependence of the four proteins that are critical for host adaptation and pathogenicity. Mutual information was also applied to quantify the correlation of sites within each individual protein and between proteins. In general, the inter protein correlation of the four proteins was stronger than the intra protein correlation. Finally, the correlation patterns of the four proteins of pandemic 2009 H1N1 were found to be closer to those of avian and human than to swine influenza, thus rendering a novel insight into the interaction of the four proteins of the pandemic 2009 H1N1 virus when compared to avian, human, and swine influenza and how the origin of these four proteins might affect the correlation patterns uncovered in this analysis.

The interaction between the 2009 H1N1 influenza A hemagglutinin and neuraminidase: mutations, co-mutations, and the NA stalk motifs

As the world is closely watching the current 2009 H1N1 pandemic unfold, there is a great interest and need in understanding its origin, genetic structures, virulence, and pathogenicity. The two surface proteins, hemagglutinin (HA) and neuraminidase (NA), of the influenza virus have been the focus of most flu research due to their crucial biological functions. In our previous study on 2009 H1N1, three aspects of NA were investigated: the mutations and co-mutations, the stalk motifs, and the phylogenetic analysis. In this study, we turned our attention to HA and the interaction between HA and NA. The 118 mutations of 2009 H1N1 HA were found and mapped to the 3D homology model of H1, and the mutations on the five epitope regions on H1 were identified. This information is essential for developing new drugs and vaccine. The distinct response patterns of HA to the changes of NA stalk motifs were discovered, illustrating the functional dependence between HA and NA. With help from our previous results, two co-mutation networks were uncovered, one in HA and one in NA, where each mutation in one network co-mutates with the mutations in the other network across the two proteins HA and NA. These two networks residing in HA and NA separately may provide a functional linkage between the mutations that can impact the drug binding sites in NA and those that can affect the host immune response or vaccine efficacy in HA. Our findings demonstrated the value of conducting timely analysis on the 2009 H1N1 virus and of the integrated approach to studying both surface proteins HA and NA together to reveal their interdependence, which could not be accomplished by studying them individually.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Host markers and correlated mutations in the overlapping genes of influenza viruses: M1, M2;NS1, NS2;and PB1, PB1-F2

The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

First isolation and identification of H1N1 swine influenza viruses in Colombian pig farms

The pig industry in Colombia has grown 30% in the last decade achieving high levels of technology and efficiency;in spite of that, respiratory diseases remain a constraint. Since 1970, serological evidence and histological findings suggested the role of swine influenza virus (SIV) as part of the porcine respiratory disease complex;nevertheless, elusive and molecular typing isolates are missing. This study was aimed at isolating SIV from intensive pig farms and to achieve molecular characterization to determine strains circulating in the field. In order to accomplish this goal, 242 samples were taken from nasal swabs, 25 from bronchial washes and 8 from lung tissue. Samples were collected during a period of three years, between 2008 and 2010 and were originated from 78 farms of the three main pig production regions of the country. The samples were transported in BHI broth with 2% antibiotic and antimycotic solution and stored at –70?C until processed. The swabs were inoculated in 9 - 11 days old embryo chicken eggs and in MDCK (Madin Darby Canine Kidney) cell cultures with the addition of trypsin. The isolates were identified by the HA (hemoagglutination) test and by RT-PCR targeting the HA (hemagglutinin), NA (Neuraminidase) and M (Matrix) genes. Full length sequence of the HA and NA glycoproteins from four selected virus isolates was conducted (Macrogen?. USA). As a result, fifteen SIV isolates from nine farms distributed in the three regions were obtained. Twelve of the isolates are related to the swine origin H1N1 virus that caused the 2009 influenza pandemic. The remaining three viruses were related to classical swine influenza viruses.