North American triple reassortant and Eurasian H1N1 swine influenza viruses do not readily reassort to generate a 2009 pandemic H1N1-like virus

Abstract

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.

FIG 1

Identification of origin of genes from KS07 and SP04 H1N1 viruses. The enzymes used and size of each gene after enzyme digestion are shown at the bottom.

FIG 2

Reassortant viruses detected in continuous cell lines coinfected with KS07 and SP04 viruses. (A) Viruses found in coinfected MDCK cells. (B) Viruses found in coinfected PK15 cells. The origin of each of the eight gene segments (shown at the top of each panel) was determined; the segments from the SP04 virus are shown in blue, and those from the KS07 virus are in red. The numbers to the right of each panel show the number of viruses with the genotype/number of viruses analyzed.

FIG 3

Viruses detected in BALF and nasal swab (NS) samples from pigs coinfected with KS07 and SP04 viruses and 2 groups of contact animals. Six reassortant viruses with 1 to 3 genes from the SP04 virus as well as the parental KS07 virus were detected in lungs of coinfected pigs. Only the parental KS07 and reassortant KS07 virus containing NA from SP04 virus were found in the BALF and nasal swab samples of the first contact group of pigs and in the lungs of the second group of contact pigs; however, only the parental KS07 virus was detected in nasal swab samples from the second group of pigs. The origin of each of the eight gene segments (shown to the left of each panel) was determined; segments from the SP04 virus are shown in blue, and segments from the KS07 virus are in red. The numbers at the top of each panel show the number of viruses with the genotype/number of viruses analyzed.

FIG 4

Viruses detected in BALF and nasal swab (NS) samples from vaccinated pigs coinfected with KS07 and SP04 viruses and 1 group of contact animals. A total of 109 viruses, including 8 reassortant viruses and parental SP04 virus, were isolated from BALF samples of vaccinated pigs that were coinfected with KS07 and SP04 viruses (top). Although only the parental SP04 virus was found in nasal swabs from the original coinfected pigs, 3 reassortant viruses and the parental SP04 virus were detected in lungs of contact pigs that underwent necropsy 4 days postcontact (D4 Contact group 1). Only the parental SP04 virus and reassortant SP04 virus with the HA gene from the KS07 virus were found in BALF and nasal swab samples from contact animals that underwent necropsy 6 days postcontact (D6 Contact group 1). The origin of each of the eight gene segments (shown to the left of each panel) was determined; segments from the SP04 virus are shown in blue, and segments from the KS07 virus are labeled in red. The numbers at the top of each panel show the number of viruses with the genotype/number of viruses analyzed.