Genetic Analysis of Influenza A/H1N1 of Swine Origin Virus (SOIV) Circulating in Central and South America

Abstract

Since the first detection of swine origin virus (SOIV) on March 28, 2009, the virus has spread worldwide and oseltamivir-resistant strains have already been identified in the past months. Here, we show the phylogenetic analysis of 63 SOIV isolates from eight countries in Central and South America, and their sensitivity to oseltamivir.

Introduction

The swine origin influenza A/H1N1 virus (SOIV) emerged both in Mexico and the United States in March 2009, and has rapidly spread worldwide.^1,2 This virus is not related to previous or current human seasonal influenza viruses and its genome is a product of a combination of four influenza virus gene segments from North American swine and avian influenza, human influenza, and Eurasian swine influenza.^3,4

Since its emergence, some genotypic mutations have been reported, for example, resistance to Oseltamivir (PROMED) was detected in Denmark, Japan, and Israel.^5–7 In view of the situation, the World Health Organization (WHO) suggests the monitoring of changes in the nucleotide sequence of the virus as part of individual case reports.⁸ This measure is important for case management and vaccine development, and for detecting critical changes in this virus, which may give origin to a more pathogenic strain or to a resistant strain that may be highly transmittable.

Here, we show the phylogenetic analysis of the hemagglutinin gene of 63 SOIV samples and their sensitivity to oseltamivir.

Material and Methods

Specimen collection, isolation, and identification of influenza viruses.

Patient recruitment, sampling, and viral isolation were performed as previously described⁹ by the NMRCD Collaborative Surveillance Network. Five thousand four hundred thirteen nasopharyngeal and throat swab specimens were collected from April to July 2009 from Argentina (N = 215), Ecuador (N = 318), El Salvador (N = 142), Honduras (N = 141), Nicaragua (N = 464), Paraguay (N = 332), Peru (N = 3,702), and Venezuela (N = 99).

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR).

Nucleic acid was extracted with the use of a viral RNA kit (QIAamp, Qiagen, Valencia, CA), and real-time RT-PCR performed for SOIV detection according to WHO recommendations.¹⁰

Sequencing and phylogenetic analysis.

A total of 1,581 samples resulted positive to real-time RT-PCR, an approximate 10% of these specimens were randomly selected from each contributing country and the samples were then amplified by RT-PCR, sequenced, and analyzed. Amplification was carried out using the SUPERSCRIPT III Platinum One-Step RT-PCR (Invitrogen, San Diego, CA) with the specific primers and reaction conditions for the hemagglutinin gene for phylogenetic analysis,¹¹ and for the neuraminidase gene to monitor specific mutations for detecting resistance to Oseltamivir.¹²

Direct sequencing of RT-PCR products was carried out with the use of the Big Dye terminator cycle sequencing kit (version 3.1, Applied Biosystems) on a Genetic Analyzer System (version 3130xL, Applied Biosystems).

Gene sequences were assembled, aligned, and edited using Sequencher (version 4.8, Gene Codes Corp.) and Clustal X softwares, and phylogenetic analyses were performed using the neighbor-joining and maximum likelihood algorithms software (molecular evolutionary genetics analysis [MEGA] version 4). For the neighbor-joining analyses, distance was used and bootstrap values were calculated on the basis of 1,000 replicates to place confidence values on grouping within trees. Phylogenetic analyses and the evolutionary distances were performed using the Kimura two-parameter model as a model of nucleotide substitution and using the neighbor-joining method to reconstruct phylogenetic trees (MEGA version 4). Nucleotide Sequence Database accession nos. on GenBank: CY045101 to CY045142.

Results

The sequences of 63 SOIV A/H1N1 isolates were compared with those originally reported from Mexico and the United States in April, at the beginning of the pandemic (Table 1). Our results show that all isolates were genetically similar to the original strains. The phylogenetic analysis reveals great similarity at the hemagglutinin (Figure 1) and neuraminidase. The strains were homogeneous and showed sequence similarity higher than 99%. The number of samples with identical sequences shown in parenthesis in Figure 1 on the right of one representative strain. No significant differences at the amino acid level were found.

Table 1.

Number of isolates sequenced by country

Country	Total samples	A H1N1 positive	Sequenced
Argentina	215	63	5
Ecuador	318	54	5
El Salvador	142	28	2
Honduras	141	9	2
Nicaragua	464	75	7
Paraguay	332	137	9
Peru	3,702	1,211	32
Venezuela	99	4	1
Total	5,413	1,581	63

Figure 1.

Phylogenetic tree constructed using the neighbor-joining method and bootstrap analysis to determine the best-fitting tree for the gene. This figure is showing the phylogenetic relationship of the Influenza A H1N1 viruses found during an outbreak (based in the hemagglutinin region). The number strains with identical sequences are shown in parenthesis on the right of one representative strain. All the strains were part of only one cluster; hence, they are closely related to the worldwide circulating strains such as A/Texas, A/Mexico. For the comparison we have included strains reported from Mexico, Canada, and California in 2009.

The genetic analysis of the neuraminidase gene shows that all SOIVs analyzed lack the H274Y mutation, which is well known to confer resistance to Oseltamivir. No other substitutions described in the literature as conferring resistance to Oseltamivir were found in these isolates.¹³

Conclusions

Pandemic influenza A viruses were first in Mexico³ during this pandemic and thereafter it spread throughout the country and the entire world in < 4 months. The genetic analysis of SOIVs from Central and South America were similar to the original SOIV strains. Nevertheless, replacement of isoleucine by valine in position 477 was detected, and although this may not generate a critical change in the biological property of this virus, it may be a signal of how rapid this strain can mutate.

Considering the emergence of resistant strains to Oseltamivir in several countries worldwide^5,6 and, although those resistant strains have not been detected in our surveillance network, it is highly important to continue the molecular surveillance of SOIV strains to detect critical genetic changes, which may generate resistant strains to first line therapy.

Disclaimer: The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. None of the authors have a financial or personal conflict of interest related to this study. The corresponding author had full access to all data in the study and final responsibility for the decision to submit this publication.