Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

Abstract

Background: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047-2056.

Results: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression.

Conclusion: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

Figure 1

fAPN is efficiently expressed on BHK-21 cell surface via transient or constitutive expression. A) BHK-21 cells were transfected with fAPN/pcDNA3.1D/TOPO for 24 h and fAPN expression was stained with R-G-4 antibody post transfection. Transfection frequency was determined by counting 300 cells. B & C) BHK cell lines constitutively expressing fAPN and pCiNeo (vector only) were labeled with anti-fAPN monoclonal antibody R-G-4. fAPN expression frequency was determined by counting >300 cells.

Figure 2

fAPN expression rescues FIPV-1146 and TGEV infection in non-permissive BHK-21 cells. A) CRFK, BHK, or BHK cells transfected with fAPN/pcDNA3.1D/TOPO were separately infected with FIPV-1146 for 9 h. Monolayers were stained with 17B71 monoclonal antibody to detect FIPV-1146 infections. B) A72, BHK, or BHK cells transfected with fAPN/pcDNA3.1D/TOPO were separated infected with TGEV for 8 h. Monolayers were labeled with rabbit polyclonal antibody 367 to detect TGEV infections. C) BHK and BHK transfected with fAPN/pcDNA3.1D/TOPO cells were separately infected with FIPV-1146 or TGEV. Viral infectivity was determined by counting >300 cells in three separate experiments. Error bars represent the standard deviation from the mean.

Figure 3

IBV strain Mass41 can infect feline cells at low levels. A & B) Primary CK or CRFK, cells were infected with IBV_Mass41 for 12 h and labeled with monoclonal α-S1(15:88) antibody to detect viral infection in three independent experiments. Viral infectivity was determined by counting >300 cells. Error bars represent the standard deviation from the mean.

Figure 4

IBV strain Mass41 does not utilize fAPN as an entry receptor. A & B) Primary CK cells, wild type BHK cells, BHK cells transfected with fAPN/pcDNA3.1D/TOPO, BHK cells constitutively expressing fAPN cells, or control BHK cell lines, (BHK exp.fAPN and BHK exp.pCiNeo) were separately infected with IBV_Mass41 for 12 h and labeled with monoclonal α-S1(15:88) antibody to detect viral infection in three independent experiments. Viral infectivity was determined by counting >300 cells. Error bars represent the standard deviation from the mean.

Figure 5

IBV field isolates efficiently infect primary chicken kidney cells. Monolayers of CK cells were separately infected with IBV strain Ark99, Ark_DPI, CA99, Conn46, H52, Iowa97, and Mass41 for 12 h. Cell monolayers were labeled with monoclonal α-S1 or M (15:88, 13:18, or 9:19) antibodies to detect viral infection.

Figure 6

fAPN is not a functional receptor for field strains of IBV. BHKexp.pCiNeo (A) or BHKexp.fAPN (B) cells were independently infected with IBV strain Ark99, Ark_DPI, CA99, Iowa97, Conn46, H52, and Mass41 for 12 h. Cell monolayers were labeled with monoclonal α-S1 or M (15:88, 13:18, or 9:19) antibodies to detect and determine viral infections. Viral infectivity was determined by counting approximately 10⁶ cells in three separate experiments.