Fatal neurological respiratory insufficiency is common among viral encephalitides

Abstract

Background: Neurological respiratory insufficiency strongly correlates with mortality among rodents infected with West Nile virus (WNV), which suggests that this is a primary mechanism of death in rodents and possibly fatal West Nile neurological disease in human patients.

Methods: To explore the possibility that neurological respiratory insufficiency is a broad mechanism of death in cases of viral encephalitis, plethysmography was evaluated in mice infected with 3 flaviviruses and 2 alphaviruses. Pathology was investigated by challenging the diaphragm, using electromyography with hypercapnia and optogenetic photoactivation.

Results: Among infections due to all but 1 alphavirus, death was strongly associated with a suppressed minute volume. Virally infected mice with a very low minute volume did not neurologically respond to hypercapnia or optogenetic photoactivation of the C4 cervical cord. Neurons with the orexin 1 receptor protein in the ventral C3-5 cervical cord were statistically diminished in WNV-infected mice with a low minute volume as compared to WNV-infected or sham-infected mice without respiratory insufficiency. Also, WNV-infected cells were adjacent to neurons with respiratory functions in the medulla.

Conclusions: Detection of a common neurological mechanism of death among viral encephalitides creates opportunities to create broad-spectrum therapies that target relevant neurological cells in patients with types of viral encephalitis that have not been treatable in the past.

Keywords: West Nile virus; alphavirus; electromyography; encephalitis; flavivirus; neurons; optogenetic; respiratory.

Figure 1.

Association of mortality with suppressed minute volume in mice infected with West Nile virus (WNV), Japanese encephalitis virus (JEV), neuroadapted Sindbis virus (NSV), Powassan virus (POWV), and western equine encephalitis virus (WEEV). To calculate the daily normal mean minute volume (MV) for determining the percentage of the normal mean value for each measurement, 10 normal mice for WNV, 10 sham-infected mice for JEV, 10 sham-infected mice for NSV, 8 sham-infected mice for POWV, and 10 sham-infected mice for WEEV were used. The horizontal dotted lines were 2 SDs from the normal mean values across all days.

Figure 2.

Association of viral titers with suppressed minute volume in mice infected with West Nile virus (WNV), neuroadapted Sindbis virus (NSV), and Powassan virus (POWV) in brain, spinal cord, diaphragm, and lung tissues. Viral infected mice were necropsied when the minute volume was >2 SDs below the normal mean value (vertical dotted lines). A corresponding mouse with an MV of >2 SDs above the normal mean value was also necropsied. Tissues were homogenized and processed for infectious virus titers. Percentages of the normal mean MVs were calculated as described in Figure 1. The horizontal lines were the limits of detection. *P ≤ .05 and **P ≤ .01, by the nonparametric Mann–Whitney 2-tailed test.

Figure 3.

Effect of viral infections on diaphragmatic electromyographic (EMG) activities for mice challenged with hypercapnia. C57BL/6 and BALB/c mice underwent sham infection; C57BL/6 mice underwent West Nile virus (WNV), neuroadapted Sindbis virus (NSV), or western equine encephalitis virus (WEEV) infection; and BALB/c mice underwent Powassan virus (POWV) infection. Plethysmography was performed daily to detect the virally infected mice with minute volumes (MVs) of >2 SDs below the normal mean MVs, as calculated in Figure 1. The diaphragmatic EMG activities of these mice with respiratory insufficiencies were then measured in mice before (top EMG readings) and after intubation and vagotomy (middle EMG readings). The mice were challenged with 7% CO₂, and the diaphragm EMGs were measured (bottom EMG readings). Mouse identification numbers, MVs, and root mean squares (RMSs) reflecting the amplitudes are all listed above the EMG readings.

Figure 4.

Effect of viral infections on electromyographic (EMG) activities in diaphragms of ChAT-mhChR2-YFP transgenic mice challenged with optogenetic photoactivation of C4 cervical cord containing motor neurons innervating the diaphragm. Mice underwent sham infection or were infected with neuroadapted Sindbis virus (NSV) or West Nile virus (WNV). Plethysmography was performed daily to detect the virally infected mice with MVs of >2 SDs below the normal mean MV, as calculated in Figure 1. The diaphragmatic EMG activities for these mice with respiratory insufficiencies were then measured in mice before (top EMG readings) and after intubation and vagotomy (data not shown) to confirm the absence of EMG readings. An optical fiber was inserted into the C4 vertebra by laminectomy [20]. Diaphragmatic EMG was used to measure photoactivation of the phrenic neurons. The EMG activities in the diaphragm (blue) were directly aligned with the photoactivation signals (red). Mouse identification numbers, MVs, and root mean squares (RMSs) reflecting the amplitudes are listed above the EMG readings.

Figure 5.

Orexin 1 receptor (Ox1R)–stained cells in the ventral horn of cervical cords (C3–C5) of West Nile virus (WNV)–infected mice with respiratory insufficiency. A, Quantification of Ox1R-stained cells, as measured by minute volume (MV), compared with controls. Each value represents a mean number (±SD) of positive cells per coronal section of the cervical cord. Every third section (12 µm) was collected, for a total of 40 sections per cervical cord. A normal MV was defined as an MV of >1 SD above the normal mean value (ie, 151 mL/min) from 1315 readings of C57BL/6 mice from prior experiments during the past half year. An abnormal MV was defined as an MV of >3 SDs below the normal mean value (ie, 73 mL/min). One-way analysis with the Newman-Keuls multiple comparison test (*P < .05, compared with the sham-infected group). B, Immunohistochemistry findings for 1 representative of 4 animals in each of 3 the groups (ie, mouse 310 from the group of WNV-infected mice with abnormal MVs, mouse 387 from the group of WNV-infected mice with normal MVs, and mouse 377 from the group of sham-infected mice with normal MVs). Mice were injected subcutaneously with either WNV or sham, and the MVs were monitored. Mice with abnormal MVs of >3 SDs below the normal mean value (ie, 73 mL/min) were euthanized for immunohistochemical staining of Ox1R and WNV envelope. For each WNV-infected mouse, another WNV-infected mouse with a normal MV of >1 SD above the normal mean value (ie, 151 mL/min) and a sham-infected mouse were selected for immunohistochemistry analysis.

Figure 6.

Somatostatin- and paired-like homeobox 2b (Phox2b)–stained cells in the medulla of West Nile virus (WNV)–infected mice. Examples of immunohistochemistry findings for WNV-infected mice with reduced minute volumes (MVs) as compared to sham-infected mice. A, Somatostatin and WNV staining. B, Phox2b and WNV staining. Separate mice were used in panels A and B. Scale bar, 50 µm.