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United States Department of Agriculture

Agricultural Research Service

Research Project: USDA Swine Influenza Virus Surveillance System: in Vivo Pathogenesis and Transmission and Associated in Vitro Studies
2013 Annual Report


1a.Objectives (from AD-416):
In vitro and in vivo study of swine influenza viruses identified in the USDA Swine Influenza Virus (SIV) Surveillance System.


1b.Approach (from AD-416):
The USDA Swine Influenza Virus (SIV) Surveillance System was initiated in 2009 and roughly 600 SIV isolates have entered into the system. Currently, 3 gene segments (HA, NA, and M) are being sequenced routinely by participating National Animal Health Laboratory Network (NAHLN) laboratories and the sequences submitted to the GenBank database. Approximately 150 isolates have the 3 gene sequences deposited (December 2011). There has not been a systematic approach for analyzing and reporting summarized results of the sequencing efforts on a single gene or whole virus genome level. This is a gap in providing a useful output from the surveillance system for determination of significant virus evolution and identification of viruses of interest. Additionally, the NVSL-APHIS will conduct whole virus sequencing on approximately 600 viruses currently in the SIV repository using the Illumina-based approach developed at St. Jude Children’s Research Hospital and adapted at the NADC-ARS. The NADC will be instrumental in transferring this technique to NVSL for use at the Iowa State University Sequencing and Synthesis core facility. Preliminary data generated from the Illumina sequencing run will be initially assessed and assembled at the NADC with subsequent transfer of the bioinformatics methods developed by the NADC to APHIS personnel. The NADC will then perform a computational evolutionary biology analysis in the context of contemporary and historical SIV for determination of phylogenetic relationships, lineages, and reassortment. From the sequence analysis, novel viruses will be selected for associated in vitro and in vivo study of SIV isolates. Additionally, influenza A virus isolates may be identified through public health investigations of zoonotic transmission events or from the animal health sector for unusual phenotypes or evasion of vaccine immunity. The studies funded in this interagency agreement include in vivo and in vitro pathogenesis and transmission, antigenic characterization, or vaccine protection experiments.


3.Progress Report:

To evaluate the antigenic drift in contemporary viruses identified through the USDA Swine Influenza Virus (SIV) Surveillance System and subsequent sequence analysis, H1 and H3 isolates were requested from the National Veterinary Services Laboratories. The isolates were then analyzed by serologic hemagglutination inhibition (HI) assay cross-reactivity against monovalent serum panels as well as serum raised against commercially available SIV vaccines. These studies showed continued antigenic drift of H1 viruses (notable the delta phylogenetic cluster) and H3 viruses. To investigate the role of the neuraminidase (NA) in vaccine and natural immunity, viruses that could be used in a neuraminidase inhibition (NI) assay were generated by reverse genetics with 1998- or 2002-lineage N2 or the H1N1pdm09 N1 NA proteins on a backbone with irrelevant hemagglutinin (HA) (to reduce confounding effects of the anti-HA antibodies). The three antigens in preparation represent the predominant lineages of NA in the US pig population and will be used in upcoming studies. Working in collaboration with NVSL, a number of additional viruses utilized in NADC studies were submitted for full-genome sequencing.

A study investigating the role of passive antibodies in protecting piglets from homologous or heterologous infection and transmission was conducted. Antibodies from H1N1pdm09 whole inactivated virus (WIV) or live-attenuated influenza virus (LAIV) vaccinated sows were passed to piglets in their respective litters, supplying the piglets with circulating antibody titers. Piglets with and without maternally derived antibodies (MDA) were experimentally challenged with heterologous H1N2 virus or homologous H1N1 virus to their dam's vaccine strain. Indirect contacts were placed 2 days post infection (dpi) to study viral transmission. Regardless of vaccine type, MDA was not effective in protecting piglets from heterologous challenge, and WIV-MDA exacerbated disease. Additionally, MDA failed to prevent transmission of heterologous virus to contact pigs. MDA from either vaccine provided some degree of protection against homologous challenge in terms of reduction of disease, replication and transmission.


Last Modified: 4/23/2014
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