Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018

Abstract

ABSTRACTLow pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Keywords: Ferret; LPAI; avian; influenza; mouse; transmission.

Figure 1.

Replication kinetics of LPAI and HPAI H7 influenza viruses in Calu-3 cells. Calu-3 cells were infected apically at an MOI of 0.01 or 1, and cultured at 37°C or 33°C, as indicated with LPAI (A, B, D, E) or HPAI (C, F) viruses. Supernatants were collected at 2, 24, 48, and 72 hrs p.i. and serially titered in eggs for detection of infectious virus. (G), comparison of viral titres 24hrs p.i. from cells infected at a MOI of 0.01 and cultured at either 37°C or 33°C; * denotes p < 0.05 between culture temperatures for each virus. The limit of virus detection was 10^1.5 EID₅₀/ml. The mean from triplicate independent cultures per virus plus standard deviation is shown.

Figure 2.

Replication of LPAI H7 viruses from 2018 in mice. BALB/c mice were inoculated with 10⁶ or 10³ EID₅₀ in 50 µl of ck/TX (H7N1) or tky/CA (H7N3) virus by the intranasal route (n = 6 per virus dose) and euthanized on day 3 or 6 p.i. (n = 3 per day) for collection of lung (black bars) and nose (grey bars) tissues. Homogenates (n = 3 per group) were titered in eggs to measure infectious virus and are reported as mean viral titre plus standard deviation. The limit of virus detection was 10^1.5 EID₅₀/ml. 1/3, one mouse from the group was positive for virus detection.

Figure 3.

Replication of LPAI H7 viruses from 2018 in ferrets. Three ferrets were inoculated with 10⁶ EID₅₀ in 1 ml of ck/TX (H7N1) or tky/CA (H7N3) virus and euthanized day 3 p.i. for collection of systemic tissues. Clarified tissue homogenates were titered in eggs. Bars represent individual ferrets. The limit of virus detection was 10^1.5 EID₅₀/ml. NW, nasal wash; NT, nasal turbinate; Tr, trachea; Lg, lung; OB, olfactory bulb; Bn, pooled anterior and posterior brain; Int, intestine (pooled duodenum, jejunoileum, and descending colon); RS, rectal swab; Lv, liver; Kd, kidney; Sp, spleen; Bd, blood; Eye, pooled right and left eyes; Conj, pooled right and left conjunctival tissue. All tissue titres are expressed per g of tissue with the exception of NW, NT, Bd, Eye, and Conj which are expressed per ml of tissue.

Figure 4.

Transmissibility of LPAI H7 viruses from 2018 in ferrets. Three ferrets were inoculated with 10⁶ EID₅₀ in 1 ml of ck/TX (H7N1) or tky/CA (H7N3) virus, and nasal washes were collected on alternate days p.i. (left set of bars). Twenty-four hours p.i., a naïve ferret was placed in the same cage as each inoculated ferret and remained in direct contact for the duration of the experiment. Nasal washes were similarly collected (right set of bars). All nasal washes were titered in eggs with a limit of virus detection was 10^1.5 EID₅₀/ml.