Molecular Basis Of Pathogenicity Of Avian H5N1 Influenza A Viruses In Mice

Highly pathogenic avian influenza A viruses (HPAIV) of H5N1 subtype spread around the world and cause great loss to poultry industry. Occasionally HPAIV H5N1 subtype infected people. The first direct transmission of H5N1 from poultry to human occurred in Hong Kong in 1997. Until March 2015,784 cases of human infection of H5N1 were confirmed, with 429 deaths, a death rate up to 55%. H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Understanding molecular mechanism of pathogencity of H5N1 influenza virus in a mammalian model is of great importance to avian influenza prevention and control.In the present study, we characterized two H5N1 HPAIV isolates A/Hunan/316/2005 (HN05) and A/Hubei/489/2004 (HB04) both in vivo and in vitro.Firstly, in chicken HB04 and HN05 with intravenous pathogenicity index (IVPI) of 2.4 and 1.4, respectively, both were highly pathogenic. While in mice, HB04 was nonpathogenic, with a MLD50 of 105.4 TCID50; HN05 was highly pathogenic, with a MLD50 of 102.5 TCID50. HB04 replicated poor in the lung of mice infected. Virus titers in lungs decreased gradually post-infection. At 5-day post infection (dpi), no virus was detected in lungs of mice infected. HN05 replicated efficiently in lung of mice infected and reached its peak at 3 dpi (1055 TCID50/100mg), and maintained high titer until 5 dpi. HN05 also showed a replication advantage and yielded a higher titer in mammalian cell lines compared with HB04. However, no replication difference was observed between HB04 and HN05 in avian DF-1 cell line.Reverse genetic system of HB04 and HN05 were constructed based on 8 plasmids, respectively. A series of recombinant viruses on the background of HB04 or HN05 with one segment replaced with the corresponding one from the other virus were rescued and the pathogencity of recombinant viruses in mice were investigated. The results demonstrated that PB1 and PA from HN05 played a key role in high pathogenicity of recombinant viruses in mice. The recombinant viruses, rHB/HN-PB1 and rHB/HN-PA showed a 1023 and 101.6 fold increase in MLD50 compared with that of HB04, and caused severe inflammation companying plenty of lymphocytes and microphages infiltration, intra-alveolar edema and congestion in primary pulmonary vein. rHB/HN-PB2 caused modest inflammation, and significantly increased viral load in the lung.The mice nonpathogenic HB04 isolate was serially passaged in lungs of mice, and a mice-adapted variant HB04M was obtained. HB04M exhibited enhanced pathogenicity and virulence in mice, with a MLD50 of 10-0.4 TCID50 and MST of 5.6 days. However, similar pathogenicity in chicken was observed from both HB04 and HB04M. HB04M showed boosted replication in lungs of mice. At 1 dpi virus titers in lungs reached its peak, with a titer of 1056 TCID50/100mg. In mammalian cells, HB04M showed enhanced replication and yielded higher titer. However, in avian cells HB04M maintained the growth characteristic of the parental HB04.Reverse genetic system of HB04M was constructed based on 8 plasmids. Genome sequencing analysis showed 23 amino acids differences between HB04 and HB04M, including the well-characterized PB2 E627K associated with adaptation to and pathogenicity in mammalian hosts. We rescued virus by introducing K627E reverse mutation in PB2 of HB04M and tested its pathogencity in mice. The virus, with a MLD50 of 102.9 TCID50, was modest pathogenicity in mice suggesting that other mutations in HB04M contributed to its pathogencity in mice. A series of recombinant viruses were generated on the background of HB04 with one segment replaced with the corresponding one from HB04M, and their pathogencity in mice were tested. Results showed that polymerase genes from HB04M significantly enhanced pathogencity of the recombinant viruses. The MLD50 of rHB/M-PA, rHB/M-PB1 and rHB/M-PB2 were 101.8,1026 and 102.8 TCID50, respectively. The mice infected with rHB/M-PA showed a significant shortened MST (6.6 days).Mutation analysis showed that PB2 E627K, PB1 1205V and PA V474A were the virulence determinants. The MLD50 of the virus with all three mutations in the background of HB04 was similar to that of HB04M. PB2 E627K increased the replication and virus load in the lung and expanded virus tropism in the organs. Mutations PB1 1205V and PA V474A posed less effect on replication and virus tropism but caused severe lung lesion.Our results contribute to understanding the molecular basis of pathogencity in mammals of H5N1 avian influenza virus and prevention the spread of H5N1 viruses.