Eurasian-Origin Gene Segments Contribute to the Transmissibility, Aerosol Release, and Morphology of the 2009 Pandemic H1N1 Influenza Virus

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

<div><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.</p></div

Histopathology in the lungs following primary infection with EMC/2012 strain of MERS-CoV.

<p>Images show H&E (left) and IHC against the MERS-CoV N protein (right) following infection with 10⁵ TCID₅₀ (A,D), 10³ TCID₅₀ (B,E), or a media only control (C,F). All images at 10x, (bar equivalent to 100μm) with 40x insets (bar equivalent to 20μm). Images shown are from day 3 post-infection for all groups.</p

Schematic of rabbit infection studies.

<p>Rabbits were inoculated intranasally with EMC/2012 strain of MERS-CoV (green arrows) and tissue samples were collected for viral titration and histopathology at necropsy (blue arrows). Three rabbits were necropsied at each time point. Numbers indicate days since virus administration for primary, (secondary), or [tertiary] infections.</p

Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.

<p>Serum ELISA and neutralizing antibody titers in rabbits following primary and secondary infection.</p

Detection of complement protein during primary infection and reinfection.

<p>ELISA against C3a protein in rabbit lung homogenates (A) show an increase in C3a levels present during reinfection compared to primary infection. Immunofluorescence images show MERS-CoV N antigen (green) and complement (red) following secondary infection (B) and primary infection (C). Images from day 3 post-infection at 40x, bar equivalent to 20μm. n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. p values *<0.05.</p

Antibody-dependent enhancement (ADE) assay using rabbit sera throughout the infection series.

<p>Sera from naïve rabbits (week 0), following primary infection (week 8), and following secondary infection (week 13) were collected from both the 10³ and 10⁵ infection schedules. Week 8 serum had no neutralizing activity while week 13 serum had neutralizing activity. Sera were tested in Vero81 cells (A), Raji cells (B) and THP-1 cells (C). None = virus only control. p values **<0.01, ***<0.001.</p