A novel nonhuman primate model for influenza transmission

Abstract

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.

Figure 1. The human influenza virus isolate A/California/07/2009 replicates to high titers in common marmosets.

Log vRNA copy number was determined by QRT-PCR in (A) the lower respiratory tract using bronchoalveolar lavage (BAL) and (B) in the upper respiratory tract using nasal washes (NW). (C) Viral RNA was also detected in the lower (solid traces) and upper (dashed traces) respiratory tracts of one contact animal, CJ1721.

Figure 2. Body weight did not change during the study period in index (A) and contact (B) animals.

No statistically significant difference in body weight lost was observed between infected and uninfected animals (unpaired t-test with Welch's correction, p = 0.8355).

Figure 3. Symptom scores of infected animals reflect development of human-like symptoms.

Symptoms were scored as follows: nasal discharge, 1 point; sneezing, 1 point; labored breathing, 3 points. Each animal was observed at least once daily throughout the experiment; symptoms were recorded for the days shown. The graphs show the summed symptom scores for each index (A) and contact (B) animal on each day on which symptoms were recorded.

Figure 4. Protein is secreted into BAL fluid as influenza infection progresses.

Bradford assay was used to detect secretion of proteins into the lungs as an indicator of damage to lung tissue. Values were normalized to protein levels detected on the first day BALs were collected. Data on subsequent days are expressed as fold increase over this baseline level; therefore negative values on this scale indicate decreased protein concentration in BAL with respect to baseline. Blue bars represent index animals; green bars represent contact animals.

Figure 5. The NP D53E mutation is rapidly fixed in all infected animals.

(A) D53E quickly rises in frequency in all index animals. (B) D53E is transmitted to CJ1721. Frequencies in the upper and lower respiratory tracts in CJ1721 right after transmission closely resemble frequencies in the index animal CJ1450 at the time of transmission.

Figure 6. Variants present at both low and high frequencies are transmissible.

(A) PA E327K was present in less than 10% of the viruses replicating in the index animal but was present at a high frequency in both the lung and nasal secretions of CJ1721 shortly after transmission. This variant frequency declined in CJ1721 after transmission. (B) PA E18G was present at a frequency around 80% in CJ1721 directly after transmission, but declined over the remainder of the study.