Adaptation of chicken H7N7/03 virus to mice

Previously, we had observed that the A/ch/Netherlands/621557/03 isolate (designated as chH7N7 virus) was not lethal for mice, even at a dose of 106 TCID 50 (unpublished observations). To increase its virulence in mice (BALB/c), the chH7N7 virus was repeatedly passaged in mouse lungs. On day 4 of each passage, the lungs were harvested and virus titers determined in pooled lung homogenates. The initial infection with chH7N7 virus yielded a titer of 106.5 TCID 50 /g tissue. Virus titers increased during subsequent passages to 108.0 TCID 50 /g at the second passage (P2) and 108.5 TCID 50 /g at the third passage (P3). Following inoculation of the P3 lung suspension, all 3 extra mice tested became ill and died 7 days after infection (data not shown).

Virulence of mouse-adapted H7N7/03 virus in mice and chickens

The virus present in pooled lung homogenate after 3 passages was propagated in embryonated chicken eggs to prepare the maH7N7 virus stock, which was subsequently used to assess the virulence of the mouse-adapted variant in mice. For this purpose, groups of 10 BALB/c mice were inoculated with increasing doses of maH7N7 virus. For comparison, two groups of 10 mice were inoculated with different doses of chH7N7 virus. The percentages of survival and mean body weights per group observed after infection are shown in Figure 1. Mice inoculated with 106 TCID 50 of chH7N7 virus showed considerable weight loss, but all survived. When a dose of 103 TCID 50 was given, one mouse died on day 13 post infection (p.i.), but all other mice survived without showing weight loss or other signs of disease. In contrast, mice inoculated with the same dose of maH7N7 virus began to lose weight soon after infection and all died 7 to 9 days later. Even with a dose as low as 50 TCID 50 of maH7N7, 50% of the mice died. Thus, three mouse passages selected a virus with a MLD 50 titer that was at least 4.3 logs lower than that of the chH7N7 virus (assuming that the MLD 50 of the chH7N7 virus was higher than 106 TCID 50 ).

Figure 1 Virulence of H7N7/03 viruses in mice. Groups of 10 BALB/c mice were inoculated i.n. with maH7N7 or chH7N7 virus at the indicated dose (TCID 50 ). Clinical signs and body weights were monitored daily until day 14 p.i. A) Kaplan-Meier survival curves, indicating the percentage of survivors over time. B) Body weight changes relative to the initial weight on day 0 (day of inoculation). Error bars indicate the standard deviations. Full size image

Based on the standard OIE test, avian influenza viruses with an intravenous pathogenicity index (IVPI) in 6-week-old chickens greater than 1.2 (out of a possible maximum of 3.0) are considered highly pathogenic. The chH7N7 virus had an IVPI of 2.9[32]. We examined the pathogenicity of the maH7N7 virus in chickens following respiratory tract exposure by inoculating virus via the combined intranasal and intratracheal (IN/IT) route. The results indicated that the PB2 E627K mutant was lethal for chickens. Similar to what was previously observed after IN/IT administration of the chH7N7 virus[32], inoculation of maH7N7 virus resulted in rapid death of the birds; one chicken died on day 2 and the other 4 chickens died on day 3 p.i., corresponding with an IN/IT pathogenicity index of 2.6.

Sequence analysis

We sequenced the entire genome of both viruses and found that the maH7N7 virus differed from the chH7N7 virus by only one amino acid at position 627 of PB2, where glutamic acid was changed into a lysine (E627K). Subsequent sequence analysis of the PB2 gene of virus present in P3 lung homogenates confirmed the presence of PB2 E627K. The PB2 sequence of virus from the P2 lung homogenate revealed that 627E was substituted already after the second mouse passage. Table 1 shows the amino acid sequence of the chicken virus selected for this study compared to that of other human and avian H7N7/03 viruses. The chH7N7 virus differed from the A/ch/Netherlands/1/03 prototype at only one position in NA (T164N). The maH7N7 and A/Netherlands/219/03 viruses had 14 amino acid differences, but shared the 627K in PB2.

Table 1 Comparison of amino acid sequences of different H7N7/03 viruses Full size table

Growth of H7N7/03 viruses in cell culture

In vitro growth of maH7N7 and its parental virus was assessed by analysis of plaque formation in cell cultures. As shown in Figure 2A and B, infection of MDCK cells with maH7N7 virus resulted in plaques that were 2.7-fold larger than those of the chH7N7 virus. The maH7N7 virus produced larger plaques (2.2-fold) also in monkey kidney-derived VERO cells. No difference in plaque size between the maH7N7 and chH7N7 viruses was observed in QM5 cells, a quail myogenic cell line[33]. When the replication kinetics of both viruses were examined, we found that the maH7N7 virus replicated faster than the chH7N7 virus in MDCK cells, yielding titers that were 1.6 logs higher at 24 and 32 h p.i. (Figure 2C). However, the maH7N7 and chH7N7 viruses produced similar growth curves in QM5 cells (Figure 2D), which was consistent with their plaque phenotypes. These results indicate that the maH7N7 virus was more efficient at replicating in mammalian cells than the chH7N7 virus.

Figure 2 Growth of H7N7/ 03 viruses in cell culture. A) Plaque formation. MDCK, VERO or QM5 cells were infected with ~50 PFU of virus and stained with amido black after 7 days of incubation. B) Quantification of plaque size in MDCK, VERO and QM5 cells. The mean plaque size (measured in mm2) of chH7N7 is plotted relative to that of maH7N7 (set at 100%). Error bars represent the standard deviations. The asterisk indicates significant difference in mean plaque size (P < 0.05). C, D) Growth kinetics of H7N7/03 viruses. MDCK (C) or QM5 cells (D) were infected with chH7N7 or maH7N7 virus at an m.o.i. of 0.001 TCID 50 per cell. Culture supernatants were harvested at 8, 24, 32, 48 and 72 h p.i. and virus titers were determined by end-point titration in MDCK cells. Shown are means from duplicate experiments with error bars indicating standard deviations. The dotted line indicates the lower limit of virus detection. Undetectable titers were assigned a value of 1.3. Full size image

Pathogenicity of H7N7/03 viruses in ferrets

Next, we examined whether the PB2 E627K mutation in the chicken virus also had an effect on its pathogenicity in ferrets. To this end, two groups of 7 ferrets each were inoculated with 107.4 TCID 50 of maH7N7 or chH7N7 virus and one group was mock-infected. In each group, 3 animals were euthanized on day 5 p.i. for post mortem examination; the other 4 animals were monitored for clinical signs and body weight during 14 days after infection. From day 3 onwards, clinical signs were observed in maH7N7-infected ferrets, including lethargy, sneezing and respiratory distress. These symptoms were also observed in ferrets infected with chH7N7 virus, but were generally milder, had a later onset and the animals recovered earlier (Figure 3A and B). Furthermore, ferrets in the maH7N7-infected group displayed loss of appetite from day 6 to 9 p.i., whereas reduction of food intake was reported only on day 7 p.i. for the chH7N7-infected group. All chH7N7-infected ferrets survived (Figure 3C). Of the ferrets infected with maH7N7 virus, two needed to be euthanized (one on day 11 and 12 each) because of severe illness; one animal exhibited severe lethargy (a score of 3), weakness and trembling, while the other was persistently lethargic (a score of 2 for 8 days). Ferrets in the chH7N7 group lost maximal ~5% of their initial body weight, whereas those in the maH7N7 group lost maximal ~9% of their weight (Figure 3D). On day 14 p.i., one chH7N7-infected animal showed a sudden drop in body weight, but no severe signs of disease or pathological changes in the lungs. Mock-infected animals did not show any signs of disease and gained weight over time. All chH7N7 and maH7N7 virus-infected ferrets exhibited H7 hemagglutination inhibiting (HI) antibody titers (geometric mean titers of 420 and 680, respectively) on day 14 p.i.

Figure 3 Pathogenicity of H7N7/ 03 viruses in ferrets. Ferrets were inoculated i.n. with 107.4 TCID 50 of either maH7N7 or chH7N7 virus. As negative control, one group of ferrets was mock-infected. On day 5 p.i., 3 of the 7 animals per group were euthanized for post mortem examination. The remaining animals were monitored for clinical signs and body weight changes during 14 days p.i. A) Median scores for lethargy. B) Median scores for sneezing (if a ferret was euthanized, the last assigned score was carried forward). C) Kaplan-Meier survival curves. D) Mean body weight changes relative to baseline weights at day 0. Only 2 ferrets of the maH7N7 group remained on day 13 p.i. Error bars represent standard deviations. E, F) Semi-quantitative scoring of histopathological changes in lung tissues. Microscopic lung changes were scored on a scale of 0 to 5 as detailed in the Methods section. Each point represents the histology score of an individual animal and horizontal bars represent the median group scores. Lungs were examined E) on day 5 and F) on day 14 p.i. or when an animal needed to be euthanized before the end of the study period (one on day 11 and 12 each, shown with open circles). Full size image

Pathological examination of the lungs at day 5 p.i. revealed foci of consolidation in two chH7N7-infected ferrets in 1 or 2 lobes affecting ~1 or 3% of lung tissue (Table 2). Comparable lesions were observed in all three maH7N7-infected animals in 1 to 3 lobes affecting ~3 to 5% of lung tissue. Histopathological changes in the lungs were similar in character in both infection groups; however, the lesions were more severe and affected a greater percentage of tissue in lungs infected with maH7N7 virus than in lungs infected with chH7N7 virus (Figure 3E). The lungs of maH7N7-infected ferrets showed alveolar hemorrhages, infiltrations of granulocytes, lymphocytes and histiocytes in the alveoli, and necrotizing bronchiolitis with obliteration of bronchioli. No histopathological changes were found in other organs, except in one chH7N7-infected animal, which had foci of mononuclear cell infiltrates in the liver.

Table 2 Gross pathology in lungs of ferrets infected with H7N7/03 virus Full size table

Gross pathology at the end of the study revealed that, in the lungs of three of four animals infected with chH7N7 virus, foci of consolidation were present in 1 or 2 lobes, involving ~1 to 3% of lung tissue (Table 2). Multifocal to coalescing consolidated areas were observed in 5 or 6 lobes in total affecting ~25 to 30% of the lungs of the two fatal cases of maH7N7 infection and in 3 lobes with ~10% lung tissue affected in one of the two surviving animals of this group. Histopathology of lungs of ferrets infected with chH7N7 virus revealed only a few, small areas with atelectasis or slight interstitial pneumonia, sometimes with mild, focal bronchitis. Lesions were more severe and extended to larger areas in the lungs of ferrets infected with maH7N7 virus (Figure 3F). In these lungs, we observed multifocal, interstitial pneumonia, alveolar hyperplasia, peribronchitis and, particularly in the two fatal cases, fibrinous alveolar exudate with many mononuclear cells. There was slight to moderate infiltration of lymphocytes and histiocytes in liver tissues from animals of both infection groups. Focal lymphohistiocytic encephalitis was seen in both fatal cases of maH7N7 infection. No pathological changes were detected in organs of the mock-infected controls. In conclusion, the maH7N7 virus generally induced more severe disease with more extensive lung pathology than did the chH7N7 virus.

Replication of H7N7/03 viruses in ferrets

Nasal washes were collected from ferrets on alternate days from day 1 to 9 p.i. and tested for the presence of virus. The nasal wash titers of the maH7N7 and chH7N7 viruses were comparable until day 5 p.i. (range, 4.3 - 5.9 TCID 50 /ml; Figure 4A). The chH7N7 virus was no longer detected or shed only at low level by day 7 p.i. At this time point, maH7N7 virus continued being shed with a mean titer that was 2.4 logs higher than that of the chH7N7 virus (range, 3.3 - 5.8 TCID 50 /ml).

Figure 4 Replication of H7N7/ 03 viruses in the upper and lower respiratory tract of ferrets. A) Ferrets were i.n. inoculated with 107.4 TCID 50 of virus and nasal washes collected at 1, 3, 5, 7 and 9 days p.i. were titrated in MDCK cells. Mean virus titers (n=4) are shown with error bars indicating standard deviations. The dotted line indicates the lower limit of detection. B) Mean virus titers in nasal turbinate (NT), trachea and lung tissues collected from ferrets (n=3) on day 5 p.i. Error bars represent the standard deviations. Full size image

After necropsy on day 5 p.i., viral loads were determined in respiratory tissues and other organs. The results are shown in Figure 4B and Table 3. Virus was detected in all nasal turbinate samples with similar mean titers for both viruses, although only two samples could be tested in the chH7N7 group. Viral titers in the trachea and lungs of ferrets infected with maH7N7 virus tended to be higher (2 and 2.5 logs on average, respectively) than those measured after infection with chH7N7 virus. In two of three maH7N7-infected ferrets, virus was detected in multiple organs, including the intestines, kidney, spleen, liver, heart and brain, indicating systemic infection. Infectious chH7N7 virus was recovered only from the respiratory tract, except in one animal, which had a virus titer in the brain. The recovery of chH7N7 from the brain, and not from other extra-respiratory organs, of one animal is consistent with previous observations[21, 22] and was suggested to be due to virus ascending via sensory nerves directly from the nose to the brain and not the result of systemic virus distribution[34, 35]. Since olfactory bulb tissues were not sampled separately, this could not be confirmed.