Salivary Blockade Protects the Lower Respiratory Tract of Mice from Lethal Influenza Virus Infection

Abstract

It is possible to model the progression of influenza virus from the upper respiratory tract to the lower respiratory tract in the mouse using viral inoculum delivered in a restricted manner to the nose. In this model, infection with the A/Udorn/307/72 (Udorn) strain of virus results ultimately in high viral titers in both the trachea and lungs. In contrast, the A/Puerto Rico/8/34 (PR8) strain causes an infection that is almost entirely limited to the nasal passages. The factors that govern the progression of virus down the respiratory tract are not well understood. Here, we show that, while PR8 virus grows to high titers in the nose, an inhibitor present in the saliva blocks further progression of infection to the trachea and lungs and renders an otherwise lethal dose of virus completely asymptomatic. In vitro, the salivary inhibitor was capable of potent neutralization of PR8 virus and an additional 20 strains of type A virus and two type B strains that were tested. The exceptions were Udorn virus and the closely related H3N2 strains A/Port Chalmers/1/73 and A/Victoria/3/75. Characterization of the salivary inhibitor showed it to be independent of sialic acid and other carbohydrates for its function. This and other biochemical properties, together with its virus strain specificity and in vivo function, indicate that the mouse salivary inhibitor is a previously undescribed innate inhibitory molecule that may have evolved to provide pulmonary protection of the species from fatal influenza virus infection.IMPORTANCE Influenza A virus occasionally jumps from aquatic birds, its natural host, into mammals to cause outbreaks of varying severity, including pandemics in humans. Despite the laboratory mouse being used as a model to study influenza virus pathogenesis, natural outbreaks of influenza have not been reported in the species. Here, we shed light on one mechanism that might allow mice to be protected from influenza in the wild. We show that virus deposited in the mouse upper respiratory tract will not progress to the lower respiratory tract due to the presence of a potent inhibitor of the virus in saliva. Containing inhibitor-sensitive virus to the upper respiratory tract renders an otherwise lethal infection subclinical. This knowledge sheds light on how natural inhibitors may have evolved to improve survival in this species.

Keywords: influenza; innate inhibitors.

FIG 1

Kinetics of viral growth in the lungs of mice following TRT and URT infection. BALB/c mice (5/group) were inoculated i.n. with 10^4.5 PFU of Udorn virus or Mem71-Bel virus or 50 PFU of PR8 virus in 50 μl of suspension in the presence of anesthetic (A) or in 10 μl in the absence of anesthetic (B). On days 1 to 8 postinfection, lungs were sampled, and infectious titers were determined by plaque formation in MDCK cells. The data are expressed as PFU per organ and represent the mean and standard deviation (SD) at each time point. The data are representative of the results of 2 independent experiments. Udorn versus PR8 virus, ****, P < 0.0001, and **, P < 0.01; Udorn versus Mem71-Bel virus, ^^^^, P < 0.0001, ^^^, P < 0.001, ^^, P < 0.01, and ^, P < 0.05 (2-way ANOVA).

FIG 2

Kinetics of viral growth in the nose, trachea, and lungs of mice following URT delivery of virus. BALB/c mice (5/group) were inoculated i.n. with 10 μl of 10^4.5 PFU of PR8 or Udorn virus in the absence of anesthetic. On days 1 to 8 postinfection, mice were killed, and infectious titers in the nose (A), trachea (B), and lungs (C) were determined by plaque assay. The data are expressed as PFU per organ and represent the mean and SD at each time point. The data are representative of the results of 3 independent experiments. ***, P < 0.001 (2-way ANOVA).

FIG 3

Viral loads in the trachea and lungs of mice following TRT and URT delivery of PR8 virus. BALB/c, CBA, C57BL/10, and SCID mice (5/group) were inoculated i.n. with 50 PFU of PR8 virus delivered to the TRT (A) or to the URT (B). On day 5 postinfection, infectious titers in the trachea (open circles) and lungs (filled circles) were determined by plaque assay. Shown are titers for individual mice, expressed as PFU per organ. Pairwise comparison of TRT versus URT, P < 0.001 for all organs and mouse strains (unpaired t test).

FIG 4

Inhibitory activity of mouse nasal washings and saliva. (A and B) PR8 or Udorn virus (30 to 100 PFU) was added to an equal volume of nasal washings (A) or saliva (B). Mock-treated virus was exposed to RPMI plus BSA (control). The final dilution of nasal washings and saliva in the assay was 1 in 8. The mixtures were incubated for 30 min at 37°C, and the amount of infectious virus recovered was determined by plaque assay on MDCK cells. The data represent the means and SD of 3 replicate samples. Saliva or nasal washings versus control, **, P < 0.01 (unpaired t test). (C) Dilutions of saliva were incubated at a 9:1 (vol/vol) ratio with either 500 PFU PR8 or Udorn virus. Virus was also incubated with RPMI plus BSA as a control. After 30 min at 37°C, the amount of infectious virus remaining was determined by plaque assay. The data are expressed as the percent reduction in virus recovered in the presence of saliva compared to that recovered when mixed with RPMI plus BSA. The error bars represent standard errors of the mean. ****, P < 0.0001; ***, P < 0.001; *, P < 0.05, comparing the two viruses (2-way ANOVA).

FIG 5

Neutralization of virus by mouse saliva. Dilutions of PR8 (A) or Udorn (B) virus were added to undiluted mouse saliva or RPMI plus BSA (control) at a virus/saliva ratio of 1:9 (vol/vol). The virus doses tested were 50, 500, 5,000, and 10^4.5 PFU. The virus-saliva mixtures were incubated for 30 min at 37°C, after which the amount of infectious virus recovered was determined by plaque assay. The data represent the means and SD of 3 replicate samples and are representative of the results of at least 5 independent experiments. Saliva versus control, ****, P < 0.0001, and **, P < 0.01 (unpaired t test).

FIG 6

Effect of RDE treatment on in vitro neutralization of virus by mouse saliva. Saliva was subjected to lyophilization and reconstitution to its original volume (A) or treated at 56°C for 30 min (B) and tested for its ability to inhibit 500 to 1,000 PFU of PR8 virus in comparison to untreated saliva, as described in the legend to Fig. 5. The data are the means and SD of 3 or 4 replicate samples and are representative of the results of 2 independent experiments. (C) Saliva was treated with V. cholerae RDE in a 1 in 10 dilution, heated at 56°C for 30 min to inactivate the RDE, and lyophilized and reconstituted to the original volume prior to testing against PR8 or Udorn virus (1,000 PFU) as for Fig. 5. The data represent the means and SD of 3 to 6 replicate samples and are representative of the results of at least 5 independent experiments. Control versus saliva, ****, P < 0.0001, and ***, P < 0.001; treated versus untreated, ^^^^, P < 0.0001; ns, not significant (1-way ANOVA).