Inefficient transmission of H5N1 influenza viruses in a ferret contact model

Abstract

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two naïve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other naïve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.

Figures

FIG. 1.

Phylogenetic tree based on the HA gene (nucleotides 77 to 1200, 1,124 bp) of H5N1 influenza A viruses. The nucleotide sequences were analyzed by Phylip3.65 software using the neighbor-joining method with 100 bootstraps. The tree was rooted to the A/Gs/Guandong/1/96 HA sequence. Viruses in colored bold type were included in this study. Abbreviations: Ck, chicken; Dk, duck; Gs, goose; Qa, quail; JWE, Japanese white eye; WS, whooper swan; BHG, bar-headed goose.

FIG. 2.

Temperature changes and virus titers of human H5N1 influenza viruses in a ferret contact model. Virus titers (log10 TCID50/ml) are shown (<, below detection limit of 101.5 TCID50/ml). Shown are results for one donor ferret (inoculated) and two recipient (contact) ferrets in two independent tests of each virus. (A and B) HK/03 virus; (C and D) VN/04 virus; (E and F) VN/05 virus; (G) TK/06 virus. Overturned ferret figures indicate time of death (d, day), and the yellow stars indicate sites of virus isolation (virus was detected in the nasal turbinate, olfactory bulb, brain, trachea, lungs, and large intestine of an VN/04-inoculated ferret in panel D; virus was detected in the nasal turbinate, olfactory bulb, brain, and large intestine in panel F).

FIG. 3.

Ferret weight changes during H5N1 influenza virus transmission experiments. The changes in ferret weight are shown for one donor ferret (inoculated) and two recipient (contact) ferrets in two independent tests of each virus. (A and B) HK/03 virus; (C and D) VN/04 virus; (E and F) VN/05 virus; (G) TK/06 virus. Daily weight changes (as a percentage) of each ferret were compared to its own initial weight prior to the initiation of the experiments.

FIG. 4.

Virus titers detected in tissues of inoculated ferrets at 5 days postinoculation. Virus in brain, olfactory bulb, nasal turbinate, trachea, lungs, and large intestine was titrated in MDCK cells. Each data point represents the virus titer (log10 TCID50/gram tissue) (mean ± SD) from two ferrets (data for the Wuhan/95 virus were obtained from one ferret).

FIG. 5.

Histopathologic findings in ferrets with H5N1 virus infection. (A) Views of hematoxylin-and-eosin-stained brain, lung, and liver samples of HK/03- and TK/06-inoculated ferrets and a VN/05-infected contact ferret. Uninfected ferret tissues were included as negative controls. Magnification, ×10. (B) Immunohistochemistry detected H5N1 viral antigen in the brain and liver of VN/04-inoculated or VN/05-infected contact ferrets. Magnification, ×20.