In this study, pigs in both pH1N1 and rH1N1infected cohorts showed typical SIV clinical signs such as sneezing and coughing from 1-4 DPI [17, 20]. As expected, clinical signs of pigs inoculated with the pH1N1 virus and rH1N1 virus were unable to be distinguished from one another. It should be noted that viral isolation of nasal swabs from both infected groups demonstrated influenza A virus positivity until 6 DPI and only the real time RT-PCR showed positive results on 7-12 DPI suggesting that the duration of infectivity extended to 6 DPI. The modified RT-PCR used in this study appeared more sensitive than viral isolation. However, the infectivity from 7-12 DPI was inconclusive. The viral RNA could be detected as early as 1 DPI in both infected groups. But viral isolation results were only tested positive on 3-6 DPI in the rH1N1-infected group and 5-6 DPI in the pH1N1-infected group. Interestingly, the viral RNA was detected at 7-12 DPI in the pH1N1-infected pigs with mild concurrent clinical signs and histopathological lung lesions. Similar to the pH1N1-infected pigs, the rH1N1-infected pigs also showed prolong period of viral detection from the nasal swabs but in this cohort, the animals also had higher macroscopic lung lesions and the presence of virus antigen was noted in all sampled lung tissue.
Previous study on the pathogenesis of a Thai endemic SIV (H1N1) showed viral shedding between 2-4 DPI and a Thai endemic H3N2 had the shedding period only at 2 DPI . Similarly, a study of human isolate pH1N1 in pigs demonstrated viral shedding as early as 1 DPI which persisted until the end of the experiment at 5 DPI . In the present study, pigs in both infected groups showed detectable live viral shedding from 3-6 DPI based on viral isolation but the viral RNA was only sporadically detected through 12 DPI. The long shedding period may allow viral transmission among pigs as well as interspecies transmission particularly to the humans working in close proximity with infected pigs. In contrast to the rH1N1-infected pigs, there was no SIV antigen detected in the lung of the pH1N1-infected pigs. The sporadic viral detection in the lungs of the pH1N1-infected pigs possibly resulted from limited viral replication and fast viral antigen disappearing. Similar to the previous Thai endemic SIV-infected pigs, the studied viral RNA was detected in the respiratory tract of both infected groups and was not found in any other organ system . In contrast to the study of pH1N1 (human origin) in pigs, viral RNA was also detected in tonsil, and serum .
Interestingly, the rH1N1-infected pigs demonstrated greater severity in term of clinical signs, pathological lesions and the overall number of pigs shedding the virus. As such, the reassortant virus theoretically could better infect pigs in comparison to the pH1N1. The only difference between the two studied viruses is the NA gene responsible for releasing the progeny viral particles from the infected cells . Since rH1N1 obtained the NA gene from the local Thai SIV (97.2% amino acid sequence identity), the virus might be more compatible in Thai pigs when compared with the pH1N1 (99.6% amino acid sequence identity to human pH1N1 but 95% identity to other SIV isolates) . However, the role of NA gene in SIV pathogenesis has not been fully elucidated and would require further investigation.
Importantly, the pH1N1 contains the triple reassortant internal gene (TRIG) cassette composed of swine, avian and human origin genes. It has been speculated that the TRIG cassette may be able to accommodate multiple HA and NA genes providing advantages to the viral infectivity, replication and possibly mutation. As a result, the TRIG cassette might be the cause of the reported increasing genetic variation rate of SIV in the US occurring since 1998 [9, 23, 24]. Since the TRIG cassette was recently introduced into the Thai pig population by the pH1N1 virus, the emergence of the Thai reassortant virus (rH1N1) in pigs has been described . In addition, evidence of interspecies transmission among human and pig populations are occasionally reported [1, 4, 25]. Any novel rH1N1 influenza virus may be able to transmit back to the human population without being noticed and possibly causing another pandemic outbreak. As such, surveillance of influenza virus infections in both pigs and humans is critical for early recognition and prevention of a potential epidemic or pandemic outbreak.