Dataset Information

MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

ABSTRACT: The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10.The work presented here represents an effort to expand and test the capabilities of the MChip to differentiate influenza A/H1N1 of various species origin.The MChip ANN was trained to recognize fluorescence image patterns of a variety of known influenza A viruses, including examples of human H1N1, human H3N2, swine H1N1, 2009 pandemic influenza A H1N1, and a wide variety of avian, equine, canine, and swine influenza viruses. Robustness of the MChip ANN was evaluated using 296 blinded isolates.Training of the ANN was expanded by the addition of 71 well-characterized influenza A isolates and yielded relatively high accuracy (little misclassification) in distinguishing unique H1N1 strains: nine human A/H1N1 (88·9% correct), 35 human A/H3N2 (97·1% correct), 31 North American swine A/H1N1 (80·6% correct), 14 2009 pandemic A/H1N1 (87·7% correct), and 23 negative samples (91·3% correct). Genetic diversity among the swine H1N1 isolates may have contributed to the lower success rate for these viruses.The current study demonstrates the MChip has the capability to differentiate the genetic variations among influenza viruses with appropriate ANN training. Further selective enrichment of the ANN will improve its ability to rapidly and reliably characterize influenza viruses of unknown origin.

SUBMITTER: Heil GL

PROVIDER: S-EPMC3825186 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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Project description:The 2009 pandemic H1N1 virus (pH1N1) was derived through reassortment of North American triple reassortant and Eurasian avian-like swine influenza viruses (SIVs). To date, when, how and where the pH1N1 arose is not understood. To investigate viral reassortment, we coinfected cell cultures and a group of pigs with or without preexisting immunity with a Eurasian H1N1 virus, A/Swine/Spain/53207/2004 (SP04), and a North American triple reassortant H1N1 virus, A/Swine/Kansas/77778/2007 (KS07). The infected pigs were cohoused with one or two groups of contact animals to investigate viral transmission. In coinfected MDCK or PK15 continuous cell lines with KS07 and SP04 viruses, more than 20 different reassortant viruses were found. In pigs without or with preexisting immunity (immunized with commercial inactivated swine influenza vaccines) and coinfected with both viruses, six or seven reassortant viruses, as well as the parental viruses, were identified in bronchoalveolar lavage fluid samples from the lungs. Interestingly, only one or two viruses transmitted to and were detected in contact animals. No reassortant containing a gene constellation similar to that of pH1N1 virus was found in either coinfected cells or pigs, indicating that the reassortment event that resulted in the generation of this virus is a rare event that likely involved specific viral strains and/or a favorable, not-yet-understood environment. IMPORTANCE The 2009 pandemic-like H1N1 virus could not be reproduced either in cell cultures or in pigs coinfected with North American triple reassortant H1N1 and Eurasian H1N1 swine influenza viruses. This finding suggests that the generation of the 2009 pandemic H1N1 virus by reassortment was a rare event that likely involved specific viral strains and unknown factors. Different reassortant viruses were detected in coinfected pigs with and without preexisting immunity, indicating that host immunity plays a relevant role in driving viral reassortment of influenza A virus.

Project description:In June 2009, the World Health Organization declared the first influenza pandemic of the 21st century, due to the emergence and rapid spread of new swine origin H1N1 influenza A virus. In contrast to seasonal influenza infections, which typically cause morbidity and mortality in the elderly, this virus caused severe infection in young adults and not the elderly. This phenomenon was attributed to the presence of cross-neutralizing antibodies acquired by older individuals from previous exposure to swine origin influenza. However, this hypothesis could not be empirically tested using clinical data. To address this question, we investigated viral replication and the development of the immune response in naï12 years old) and aged (20 to 24 years old) female rhesus macaques infected with A/California/04/2009 (H1N1), one of the circulating pandemic strains in 2009. We compared viral loads as well as the kinetics and magnitude of the adaptive immune response in peripheral blood and bronchoalveolar lavage samples (BAL) collected longitudinally for 99 days post-infection. Although, adult animals exhibited earlier T cell responses in peripheral blood, aged animals generated a robust T cell response with comparable kinetics and magnitude as those observed in young animals in BAL. Moreover, aged animals generated a higher hemagglutination inhibition titer compared to young animals. We also measured the concentration of several cytokines in BAL supernatant. With the exception of IL-8, which was higher in aged animals, we found no differences in IFNa, IFNb, TNFa, IL-1r, IL-6, IL-15, IL-17, or MCP1 levels. Finally, we compared gene expression infection using microarray analysis of BAL samples taken on days 0, 4, 7, 10, and 14 pi. Our analyses revealed that the largest difference in host response between aged and young animals was detected day 4 post-infection, with significant enrichment for genes associated with inflammation, the innate immune response, and T cell activation in aged animals. The ability of aged animals to generate a robust immune response, especially antibody response, following infection with 2009 H1N1 virus could explain the lack of morbidity normally observed with seasonal influenza viruses in this vulnerable population.

Dataset Information

MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

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