Supplementary data on virus-like particles in the brainstem of Parkinson’s disease patients and controls

In this study, we present 77 transmission electron microscopy (TEM) images of human brainstem tissue from 11 cases of late onset Parkinson’s disease (PD). The tissues were fixed, embedded, sectioned, and stained for TEM application. In addition, we present 11 images from autopsy specimens of 1 case of human poliomyelitis infection as positive controls and 12 images from 8 cases of autopsy specimens of other conditions as negative controls. In the TEM images of the PD cases there were cytoplasmic inclusion bodies consisting of virus-like particles (VLP) 30 nm in diameter that were associated with endoplasmic reticulum membranes. In the nuclei of the PD neurons there were VLP ranging from 40 nm to 50 nm in diameter. In the poliomyelitis cases, similar particles as were observed in PD which were interpreted to be poliomyelitis virus particles. In the negative controls one case was identified which showed similar VLP (Figure 1, controls). A Lewy body was found in this “control” case (Figure 10) suggesting that this was an undiagnosed case of PD. Cytoplasmic ribosomes measuring approximately 17 nm were observed in the control neurons.


Introduction
In our previous communication, we presented a number of TEM images selected from our work on PD. We also presented our immunohistochemical results in which we detected enterovirus antigen in PD brainstem tissue 1 . In order to assist in the interpretation of our TEM results, we now present a larger number of TEM images from PD archives together with relevant controls. We also include TEM images of Lewy bodies from the cases of PD, the diagnostic sign of PD (see Underlying data) 2 .

Methods
The methods for TEM were the same as those employed in our previous communication 1 . Included were 8 control cases, 1 case of poliomyelitis and 11 cases of Parkinson's disease. The tissue samples from the control cases were obtained from the John Radcliffe Hospital, Department of Neuropathology. The tissue samples from the PD cases and from the poliomyelitis case were from the Armed Forces Institute of Pathology, in Washington DC, now closed. Ethical clearance Study of brain specimens had been cleared for ethical agreement by the National Ethics Committee for Oxford UK, rec. no. 07/H0606/85. The brain samples from human autopsy material were obtained from the pathology collections of the John Radcliffe Hospital, Oxford UK and the former Armed Forces Institute of Pathology, Washington DC USA. These institutions approved the use of the tissues for research and they were satisfied that no further ethical approval was required. In the case of the material from the UK, the principal author releases the "tissue disclaimer" from the Thomas Willis Oxford collection, at the Neuropathology Department.

Conclusions
We note the role α-synuclein plays in the pathogenesis of PD 3 . It is conceivable that a virus is involved with α-synuclein in the pathogenesis of PD. We suggest that virologists undertake the isolation of infectious virus from diseased PD This project contains the following underlying data: Legends for TEM images of PD brain. Legends of TEM images of control brain.
• Control Figure 4. Control case; a group of ribosomes in a neuron in the basal ganglia. Measurements, image 10.09015.
• Control Figure 5. Control case; a group of ribosomes in a Nissl body in the same area as above. Image 10a09009.
• Control Figure 6. Measurements. Control case, as above; endoplasmic reticulum in the cytoplasm of a neuron. Image 10c.09011.
• Control Figure 7. A case that was reported as being a control case. VLP characteristic of PD are shown (arrows). Image 11.09b001.
• Control Figure 8. In a same case as in Figure 9, an intracellular Lewy body. Image 11.09006.
• Control Figure 9. A control case, showing cytoplasm and nucleus. Image 12.09015.

Marie-Eve Tremblay
Division of Medical Sciences, University of Victoria, Victoria, British Columbia, Canada This supplemental material provides additional transmission electron microscopy pictures in support of the presence of viral-like particles in the brain stem of post-mortem Parkinson's patient samples (compared with controls).
The rationale for providing additional pictures should be explained a little further, considering that these additional pictures do not provide more support to the original evidence published by the authors. In particular, it would have been useful to perform immunostaining or use additional validation methods to confirm that some of the viral-like particles are indeed viral-like particles and not something else (e.g. glycogen granules, which have a similar size and can also associate with the endoplasmic reticulum).
If presented as supplemental material, the pictures should have more annotations (e.g. arrows, pseudo-colours, etc.) to help readers visualize the structures of relevance. I would also recommend removing pictures showing glial processes, myelin, neurons (of note, sometimes neurons looked like astrocytes, it would be important to verify the ultrastructural identification criteria for the dataset), focusing on the topic of investigation: the viral-like particles.
In addition, the ultrastructural preservation of the samples is generally suboptimal, which could be accounted for by the most-mortem interval or the pH of the cerebrospinal fluid, known to affect brain ultrastructure. More information regarding the samples could be useful to help interpret the dataset.

Is the rationale for creating the dataset(s) clearly described? Partly
Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of methods and materials provided to allow replication by others? Overall, need to improve labelling of figure legends by use of arrows pointing to key features.  Fig. 7., Fig. 8, Fig. 9-need arrows.  Fig. 66, Fig. 67. Fig. 68-difficult to see cytoplasmic membrane-need arrows.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound? Yes

Are sufficient details of methods and materials provided to allow replication by others? Partly
Are the datasets clearly presented in a useable and accessible format? Partly Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Virology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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