The emergence of the pandemic 2009 H1N1 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages raised concern over the safety of pork. Pigs experimentally infected with pandemic 2009 H1N1 influenza A virus developed respiratory disease; however, there was no evidence for systemic disease to suggest that pork from pigs infected with H1N1 influenza would contain infectious virus. These findings support the WHO recommendation that pork harvested from pandemic influenza A H1N1 infected swine is safe to consume when following standard meat hygiene practices.
Citation: Vincent AL, Lager KM, Harland M, Lorusso A, Zanella E, Ciacci-Zanella JR, et al. (2009) Absence of 2009 Pandemic H1N1 Influenza A Virus in Fresh Pork. PLoS ONE 4(12): e8367. doi:10.1371/journal.pone.0008367
Editor: Linqi Zhang, Tsinghua University, China
Received: October 12, 2009; Accepted: November 24, 2009; Published: December 18, 2009
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: Funding was provided by USDA-ARS and DHHS-CDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The emergence of the pandemic H1N1 2009 influenza A virus in humans and subsequent discovery that it was of swine influenza virus lineages  raised many questions about this novel virus. One such concern relates to food safety, if swine were to become infected with the pandemic virus would the meat be contaminated with virus and be a potential source of human infection? To address this question we tested non-respiratory tract tissues for virus following infection of young pigs with the pandemic H1N1 2009 virus.
Materials and Methods
A total of 30 pigs were inoculated with A/CA/04/2009 (H1N1)v (n = 15 pigs) or A/Mexico/4108/2009 (H1N1)v (n = 15 pigs) as part of ongoing studies to determine the susceptibility of swine to the human virus (Vincent, unpublished). The animals were housed according to the National Animal Disease Center Institutional Animal Care and Use Committee guidelines. Five pigs from each virus challenge group were euthanized on 3, 5, and 7 days post infection (dpi). All pigs were treated similarly receiving an intratracheal challenge with 2×105 50% tissue culture infectious dose (TCID50) as previously described . Postmortem samples including serum, lung, tonsil, liver, kidney, spleen, inguinal lymph node, colon contents (feces), and skeletal muscle from the semitendinosus were collected at necropsy using individual sterile instruments between tissues and between pigs. Non-challenged age-matched negative control pigs were necropsied at 7 dpi (n = 5 pigs).
The tissues were tested for virus by a real-time RT-PCR (qRT-PCR) specific for the pandemic H1N1 matrix gene (Lorusso, submitted) and virus isolation on Madin Darby Canine Kidney (MDCK) cells. Briefly, approximately 500 mg of tissue was homogenized in sterile phosphate buffered saline (PBS) with antibiotics using a power homogenizer with sterile generators at 20% w/v. The MagMax Microarray (Ambion) protocol for RNA extraction from tissues was followed using 100 µL of tissue homogenate. The MagMax Viral RNA Isolation (Ambion) kit protocol was used as per manufacturer's instructions for serum by adding 50 µL to the MagMax plate for RNA extraction. Viral RNA samples were tested in duplicate by qRT-PCR.
For virus isolation, 200 µL of the tissue homogenate or serum sample was placed on confluent MDCK cells in 24-well plates to incubate for 1 hr. After 1 hr of incubation the sample was removed and 400 µL MEM w/TPCK trypsin was added. The plate was checked at 24 and 48 hrs for cytopathic effects. After 48 hrs, 200 µL of cell culture supernatant from each well of the 24-well plate after one freeze and thaw cycle was subsequently passed onto a confluent 48 well plate. After 48 hrs, evidence of cytopathic effects was evaluated and presence of virus antigen confirmed by immuno-cytochemical staining. Virus titers in virus isolation positive tissue homogenates were determined on MDCK cells in 96-well plates.
Influenza virus was isolated from the lung tissue of all pigs euthanized on 3 and 5 dpi, and from the tonsil tissue of 1 pig in each virus challenge group (Table 1). The mean virus titers for lung tissue homogenates from pigs infected with CA/09 were 104.0 and 102.8 TCID50 per mL for days 3 and 5 pi, respectively. The mean virus titers for lung tissue homogenates from pigs infected with MX/09 were 103.9 and 103.1 TCID50 per mL for days 3 and 5 pi, respectively. The tonsil samples were below sensitivity limits of titration. The positive lung tissue was consistent with virus isolation from broncho-alveolar lavage fluid (BALF) (data not shown). All pigs were positive in BALF by virus isolation on days 3 and 5 pi, but negative on 7 dpi as demonstrated in the lung tissue. Influenza viral nucleic acid was detected by qRT-PCR in all of the 20 virus-positive lung tissues but from neither of the virus isolation positive tonsil samples (Table 2). In addition, viral nucleic acid was detected in the lung samples of all pigs at 7 dpi as well as one lymph node sample at 3 dpi, but these samples were virus isolation negative. The mean copy numbers by qRT-PCR for lung tissue homogenates from pigs infected with CA/09 were 104.3, 104.7, and 103.3 for days 3, 5, and 7 pi, respectively. The mean copy numbers by qRT-PCR for lungs infected with MX/09 were 104.9, 103.9, and 102.7 for days 3, 5, and 7 pi, respectively. The copy number of the positive lymph node sample was 102.4. No infectious virus or viral nucleic acid was detected in any of the remaining tissue samples from any of the virus challenge pigs or from any of the negative control pigs. qRT-PCR was more sensitive for viral RNA in the lungs at 7 dpi, at which time the pigs were recovering clinically and viral shedding was declining. Importantly, qRT-PCR did not detect viral RNA in any internal organs or muscle tissue samples.
Clinical disease was induced in all infected pigs and will be reported elsewhere in detail (Vincent, unpublished). Infectious virus was detected in lungs from all experimentally infected pigs necropsied on 3 and 5 dpi, confirming infection for both CA/09 and MX/09. These observations are consistent with what has been reported with German and British experiments in which clinical disease was induced and infectious virus and viral nucleic acid could be detected in tissue samples associated with the respiratory tract , . Neither infectious virus nor viral nucleic acid was detected in plasma samples collected on days −1 through 7 dpi . Except for plasma collected from pigs in the British experiment, no other non-respiratory tract tissues were reported to have been tested for virus.
Experimental infections of swine with the pandemic H1N1 virus have described a clinical disease where pigs develop pyrexia, anorexia, and dyspnea within several days following challenge ,  that is similar to what has been reported in endemic swine influenza virus experiments. Likewise, there have been reports of swine becoming infected in the field with the pandemic H1N1 virus in which the pigs displayed mild respiratory disease (http://www.oie.int/eng/en_index.htm). In these cases it is believed that the pigs became infected following contact with infected people. Collectively, this data suggests the pandemic H1N1 virus replicates in swine and produces clinical illness that is indistinguishable from typical swine influenza virus.
In contrast to highly pathogenic avian influenza virus infections in poultry , existing evidence suggests that swine influenza virus does not induce a systemic infection contaminating the meat, although there is limited data to support this assumption. To the authors' knowledge there are only two reports that describe an infrequent viremia in pigs during the acute phase of the infection with swine influenza virus , . One of these papers also describes sporadic isolation of influenza virus from “other tissues such as intestine and muscle and from faeces,” however, the methodology is not well described and it is unclear during the acute infection which tissues were positive at which times . PCR was not utilized in these studies and it is unknown if tissues would have been positive by this method. Infection of swine with highly pathogenic avian influenza virus induced minimal disease with no evidence for a systemic infection based on virus isolation and PCR . Similarly, infection of swine with the 1918 Spanish flu virus resulted in minimal pulmonary disease with no virus being isolated from a variety of non-respiratory tissues .
In this study, tissues outside the respiratory tract were found to be negative by virus isolation on days 3, 5 and 7 pi for both isolates of 2009 pandemic H1N1 evaluated. Only lung and tonsil samples from days 3 and 5 pi were positive by virus isolation. In addition, 7 dpi lung samples and inguinal lymph node from one pig were positive for viral RNA, but were negative by virus isolation. This may be due to the increased sensitivity of the qRT-PCR in detecting viral RNA over the sensitivity of detecting viable virus by tissue culture techniques. By 7 dpi, viable virus is typically cleared from the lung in pigs with uncomplicated infection with influenza A virus, including 2009 pandemic H1N1 (Vincent, unpublished), thus the viral RNA is likely remains following activation of the host innate immune response. Two virus isolation positive tonsil samples were found to be negative by qRT-PCR. This is likely due to the low quantity of viable virus and/or viral RNA being at the threshold of sensitivity for both assays.
In summary, the 2009 pandemic H1N1 virus can induce respiratory disease in swine that is consistent with influenza illness. However, there was no evidence for systemic infection that would contaminate meat with infectious virus. It is important to note that ill swine would not be allowed entry into the U.S. food supply as per USDA Food Safety and Inspection Service criteria. However, the findings reported in this study support the WHO recommendation that pork harvested from 2009 pandemic influenza A H1N1 infected swine would be safe to consume when following standard meat hygiene practices (http://www.who.int/mediacentre/news/statements/2009/h1n1_20090430/en/index.html).
The authors thank Hillary Horst for technical assistance and Dr. Becky Jepsen, Brian Pottebaum and Jason Huegel for assistance with animal studies. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Conceived and designed the experiments: ALV KML MEK. Performed the experiments: ALV KML MH AL EZ JRCZ. Analyzed the data: ALV. Contributed reagents/materials/analysis tools: ALV AIK. Wrote the paper: ALV KML MEK AIK.
- 1. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, et al. (2009) Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans. Science 325: 197–201.
- 2. Vincent AL, Swenson SL, Lager KM, Gauger PC, Loiacono C, et al. (2009) Characterization of an influenza A virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the United States. Vet Microbiol 137: 51–59.
- 3. Lange E, Kalthoff D, Blohm U, Teifke JP, Breithaupt A, et al. (2009) Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J Gen Virol 90: 2119–2123.
- 4. Brookes SM, Irvine RM, Nunez A, Clifford D, Essen S, et al. (2009) Influenza A (H1N1) infection in pigs. Vet Rec 164: 760–761.
- 5. Swayne DE, Beck JR (2005) Experimental study to determine if low-pathogenicity and high-pathogenicity avian influenza viruses can be present in chicken breast and thigh meat following intranasal virus inoculation. Avian Dis 49: 81–85.
- 6. Brown IH, Done SH, Spencer YI, Cooley WA, Harris PA, et al. (1993) Pathogenicity of a swine influenza H1N1 virus antigenically distinguishable from classical and European strains. Vet Rec 132: 598–602.
- 7. Romijn PC, Swallow C, Edwards S (1989) Survival of influenza virus in pig tissues after slaughter. Vet Rec 124: 224.
- 8. Lipatov AS, Kwon YK, Pantin-Jackwood MJ, Swayne DE (2009) Pathogenesis of H5N1 influenza virus infections in mice and ferret models differs according to respiratory tract or digestive system exposure. J Infect Dis 199: 717–725.
- 9. Weingartl HM, Albrecht RA, Lager KM, Babiuk S, Marszal P, et al. (2009) Experimental infection of pigs with the human 1918 pandemic influenza virus. J Virol 83: 4287–4296.