Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Tavakoli NP, Wang H, Dupuis M, et al. Fatal case of deer tick virus encephalitis. N Engl J Med 2009;360:

2 Supplemental Methods, Tables and Figures. Supplemental Methods Viruses and controls. Strains POW LB (representing POW virus lineage I) and DT-SPO (representing POW virus lineage II) were obtained from the Arbovirus Laboratory at the Wadsworth Center and were used as positive controls for the POW PCR assays. Controls for the encephalitis PCR assays (HSV 1 and 2, varicella zoster virus, Epstein Barr virus, human herpes virus 6, enterovirus, West Nile virus, eastern equine encephalitis virus, California serogroup viruses (La Crosse virus), Cache Valley virus and Saint Louis encephalitis virus) were obtained from the Arbovirus or Viral Proficiency Testing Laboratories at Wadsworth Center. Cytomegalovirus DNA was purchased from Sigma-Aldrich (St. Louis, MO) and human herpes virus 6 and adenovirus were obtained from ATCC (Manassas, VA). Conventional PCR. Following RT-PCR, the product was run on a 2% agarose gel. The firstround PCR should generate a product of approximately 250 bp, and the second round PCR should generate a product of approximately 220 bp. A number of other PCR assays, some published previously (1, 4), were subsequently performed with the aim of obtaining PCR products for sequencing reactions. Sequence information was sought for the envelope coding region, NS5, and 3 untranslated region (3 UTR) of the virus. Bands of the expected size were cut from the gel and purified using an Ultrafree-DA Centrifugal Filter Unit (Millipore, Billerica, MA). Sequencing reactions of the PCR products were performed, with the same primers that had been used for the PCR reaction, at the Wadsworth Center Molecular Genetics Core facility on an automated DNA sequencer Model 3100 (Applied Biosystems, Foster City, CA).

3 Sequence Analysis. The obtained sequences were assembled with MEGA4 (13) and were subjected to a BLAST search against the GenBank database. The phylogenetic analyses were performed by the neighbor-joining method (14) within the MEGA4 analytical package. The evolutionary distances were computed by the Maximum Composite Likelihood method (15). Sequences from the envelope coding region (accession number EU338402), and NS5 and 3 UTR (accession number EU338403) were deposited in GenBank. Histology and Immunohistochemistry. Paraffin sections (4 μm thick) were stained with Hematoxylin and Eosin (H&E) for standard histopathologic evaluation. For immunohistochemistry, sections were pretreated for antigen retrieval by boiling in 10 mm citrate buffer at ph6 for 15 minutes. Commercially available antibodies (DAKO, Carpinteria, CA) to glial fibrillary acidic protein (GFAP; 1:200), CD3 (1:200), CD4 (1:200), CD8 (1:50), CD79a (1:500), and PGM (macrophage marker; 1:100) were applied to sections using a DAKO AutostainerPlus according to manufacturer s protocols. Polyclonal antisera directed against the whole DTV (wdtv) or DTV envelope (E) protein were generated in mice. The wdtv antibody was generated by single intraperitoneal injection of ~10 3 PFU of the Spooner strain of DTV. Recombinant DTV E ectodomain was produced in E. coli and purified as described elsewhere (8). The BALB/c mice were primed and boosted at two week intervals with 100 ug of recombinant E protein (re) suspended in Freund s Incomplete Adjuvant (Sigma-Aldrich, St. Louis, MO). Serum was harvested two weeks after the last boost and tested for reactivity to DTV re by ELISA as described (8), except that two-fold serial dilutions of mouse serum were used to determine ELISA endpoint titers. Sera from uninoculated mice failed to react with re, while sera from immunized mice reacted at 1:1600-1:3200. These re reactive sera (redtv) were pooled for immunohistochemical staining.

4 After antigen retrieval, sections were incubated for 30 minutes in 1% H 2 O 2, washed in phosphate buffered saline ph7.4 (PBS), and then blocked at room temperature in 10% normal goat serum, 1% bovine serum albumin, 0.5% Triton-X100 in PBS, ph 7.4. The redtv or wdtv antiserum (1:200) was incubated overnight at 4 o C in diluent (1% bovine serum albumin, 0.5% Triton-X100 in PBS). Sections were washed in PBS and incubated with biotinylated goat anti-mouse antibody (1:400) followed by streptavidin-peroxidase (1:500) (Jackson ImmunoResearch, West Grove, PA). Antigen complexes were detected with diaminobenzidine as chromogen. Control of antibody specificity was by omission of the primary antibody. Negative controls were provided by parallel immunostaining of a cerebellar surgical biopsy adjacent to a vascular malformation and an autopsy case from a patient of similar age but without neurologic disease or histopathologic changes; these specimens showed no immunoreactivity with either DTV sera (not shown). Images were obtained by brightfield microscopy with a Zeiss Axioplan 2 microscope fitted with an Axiocam HR digital camera (Carl Zeiss, Thornwood, NY) and compiled in Adobe Photoshop CS TM, with image adjustment for brightness and contrast.

5 Supplemental Table A. Primers targeting the envelope coding region, NS5, and 3 UTR of the deer tick virus genome. Forward Primer Sequence (5 3 ) Reverse Primer Sequence (5 3 ) Envelope region DT928-F ACGAGATGCACACACCTTGA DT1235-R TTTCCCCATCCACGGTCA DT1113-F CGCCAAGCTCTCCAACA DT1415-R GCCGTTTTCCTGTTCTCGTT DT-Env-F6 TGGTGTGCAAGAGAGACCAG DT-Env-R6 AGGTCTTCGAACCAGTCACG DT1511-F CCCAGACTGTGGTGATGTC DT1905-R CACTGTGTCATGGCCACT DT1827-F AGGCACAACTTACTCCATGTG DT2213-R CCGCCCACTGAACCAAA NS5 and 3 UTR region DT7981-F AGCCTTGGATGGAACCTCAT DT8280-R TGTGGAATTCCGTGAGAAAGG DT8156-F CAGACGCTGCTGCTGTT DT8418-R GGTCCCGGTTCCTAGATCA DT-F4 GGGTGGAGGACTTGTGAGAG DT-R4 TCCACCAGGTTCCAAAACTC DT8772-F GCTACTTGAGCACCTTCACA DT9092-R TCAAACTCAAGGAAACGGCT DT8920-F GAGTTTTGGAACCTGGTGGA DT9092-R TCAAACTCAAGGAAACGGCT MAMD a AACATGATGGGRAARAGRGARAA CFD2 a GTGTCCCAGCCGGCGGTGTCATCAGC FS778 a AARGGHAGYMCDGCHATHTGGT CFD2 a GTGTCCCAGCCGGCGGTGTCATCAGC DT9178-F GGTTGGTTGCTCAGGGAGCT DT9498-R GCGGATTAGTTGGACTTTCATGTT DT9397-F TGTGTGATGGATGTGATCACG DT9666-R CAGGGCCTTTCCAAACCT DT9448-F GTCACGTATGCCCTCAATACC DT-R3 CATGGAAATGGTGTGAGCAG DT9695-F GTAAGGATGTGGGCGAGT DT10105-R CCATGTGAGGGTTGTCCAAA DT9731-F TCACAGCCTGGGAAGAAGTG DT10105-R CCATGTGAGGGTTGTCCAAA DT10011-F CACCTGGAGCATCCATGCGA POW-2 b GCTCTCTAGCTTGAGCTCCCA POW-1 b TGGATGACAACAGAAGACATG POW-2 b GCTCTCTAGCTTGAGCTCCCA DT10220-F TCTGGGGCTCAGTGGAA DT10507-R GCCTTCTTACTACACGGCTG utr-f c GCACTGGGAGCTCAAGCTAGAGAGC utr-r c AGCGGGTGTTTTTCCGAGTCACACA

6 utr-f c GCACTGGGAGCTCAAGCTAGAGAGC DT10637-R GGCTCTAGAATGGCCTAACT DT-UTR-F AGTAAGAAGGCCCCGACCTA DT-UTR-R GAAAAATCCCGGGGAAGAT a (11); b, (1); c, (4).

7 Supplemental Figure 1. Histologic and immunohistochemical characterization of the surgical cerebellar biopsy. Paraffin sections were stained with H&E (A-C) or with antibodies to T-cell markers CD4 (D) and CD8 (E, J), B-cell marker CD79a (F), macrophage marker PGM (G, I) or astrocyte marker GFAP (H). (A-C) There was a dense leptomeningeal infiltrate, increased cellularity in the molecular layer (ML), diffuse loss of Purkinje cells (PCL, Purkinje cell layer) and a preserved internal granule cell layer (IGL). The meningitis was associated with angioinvasion/angiodestruction of veins (asterisk in B). A microglial nodule was seen in the PCL (arrow in C). (D-G) The leptomeningeal infiltrate consisted predominantly of reactive CD4+ helper T-cells (D). The minor population of B-cells in this infiltrate (F) were also PAX5-positive

but CD23 negative (not shown); while this may indicate that some CLL/SLL cells were present, these were highly likely to be bystanders of the true pathologic process ongoing in the cerebellum.

(I, J) The increased cellularity in the ML was due to macrophage (I) and CD8+ T cell (J) infiltration. These inflammatory cells also cluster within the destroyed PCL. Supplemental Figure 2.

8 but CD23 negative (not shown); while this may indicate that some CLL/SLL cells were present, these were highly likely to be bystanders of the true pathologic process ongoing in the cerebellum. (H) GFAP immunostain revealed marked Bergmann gliosis, with immunoreactive cell bodies in the PCL. (I, J) The increased cellularity in the ML was due to macrophage (I) and CD8+ T cell (J) infiltration. These inflammatory cells also cluster within the destroyed PCL. Supplemental Figure 2. MRI scan on hospital day 7. There was marked interval increase since admission in cerebellar swelling on T2 sequence (A), a new T2 FLAIR hyperintensity in the right thalamus (arrow in B) and persistent hydrocephalus despite shunt placement with marked transependymal cerebrospinal fluid flow (arrowheads, B).

9 Supplemental Figure 3. Additional histologic findings in the brain autopsy. Paraffin sections were stained with H&E or immunostained with an antibody to microglia/macrophage marker PGM (A, lower panel in E, right panel in H, upper panel in K), cytotoxic T-cell marker CD8 (lower panel in J) or T cell marker CD3 (lower panel in K). (A) In the pons, microglial nodules and diffuse microglial activation involved basal pontine nuclei (asterisks), with less prominent

10 involvement of descending fiber tracts (arrows) and pontocerebellar fibers (arrowhead). (B) Microglial/lymphocytic infiltrates surrounding atrophic/dying neurons in the pontine tegmentum. (C) Dense perivascular lymphocytic infiltrate in the midbrain. (D) Medulla, inferior olivary nucleus with a microglial nodule and neuronophagia (arrow). (E) Cerebellar dentate nucleus with a necrotic focus (upper panel); PGM immunostain (lower panel) showed diffuse microglial activation/macrophage infiltration. (F-I) Basal ganglia and thalamic region. A focal microglial/lymphocytic nodule in the lateral putamen (F) and extreme capsule (G). PGM immunostain of globus pallidus (H, right panel) identified diffuse microglial activation and microglial nodules. The pallidal (H, left panel) and lateral right thalamic (I, upper panel) parenchyma had significant edema and many atrophic/dying neurons apparently unassociated with inflammatory infiltrates. The medial right thalamic parenchyma was better preserved but a few neuronophagic microglial nodules were identified (I, lower panel). (J-K) Spinal cord. Marked depletion of anterior horn motor neurons throughout the cord (J, upper panel; cervical cord), with only rare surviving neurons (arrow) and atrophic/dying neurons (arrowheads); a prominent CD8+ cytotoxic T cell lymphocytic infiltrate was evident (J, lower panel; lumbar cord). Ventral nerve roots (K) were also infiltrated by macrophages (upper panel) and T lymphocytes (lower panel, CD3 immunostain); dorsal nerve roots were more focally involved (not shown). Note also the adjacent spinal leptomeningeal infiltrate containing many T cells.

inflammatory infiltrates in the autopsy brain.

antibodies to the T cell markers CD8 (A,C,E)

In the third nerve nucleus (A,B), midbrain

cytotoxic lymphocytes are identified in the

adjacent to surviving motor neurons (arrows

11 Supplemental Figure 4. Immunohistochemical characterization of inflammatory infiltrates in the autopsy brain. Paraffin sections were stained with antibodies to the T cell markers CD8 (A,C,E) or CD4 (B,D). In the third nerve nucleus (A,B), midbrain (C,D) and substantia nigra (E) many CD8+ cytotoxic lymphocytes are identified in the parenchyma, some of which are closely adjacent to surviving motor neurons (arrows in A,E). CD4+ helper lymphocytes are present predominantly around blood vessels with a smaller parenchymal component (D). CD4+ T cells are less frequently seen in close association with surviving motor neurons (arrows in B).

Paraffin sections from the surgical cerebellar biopsy (A) and

thalamus) were stained with the wdtv antibody (A-C, F) or redtv

(A) In the cerebellar biopsy, the wdtv serum recognized surviving

molecular layer and occasional identification of axons in the

12 Supplemental Figure 5. Immunohistochemistry with deer tick virus antisera. Paraffin sections from the surgical cerebellar biopsy (A) and autopsy brain (B, cerebellum; C-D, pons; E, spinal cord; F, right thalamus) were stained with the wdtv antibody (A-C, F) or redtv antibody (D-E). (A) In the cerebellar biopsy, the wdtv serum recognized surviving Purkinje cells, with prominent filling of their dendrites in the molecular layer and occasional identification of axons in the granule cell layer (arrow). (B) In the autopsy cerebellum, the DTV sera identified rare clusters of surviving/degenerating Purkinje cells, some of which had characteristic filling of

13 dendrites (inset). (C-D) Many surviving neurons in the basis pontis were immunolabeled by both sera. The wdtv antibody also recognized viral antigen engulfed in macrophages (arrow in C). The same focus was imaged in C and at lower power in panel D (lower panel), demonstrating a somewhat greater neuronal labeling with the wdtv antibody and absence of reaction product in macrophages with the redtv antibody (arrow in D). (E) Spinal cord anterior horn with two motor neurons labeled by the redtv antibody, one of which had granular cytoplasmic staining (arrow), shown at higher magnification in inset (arrowhead). (F) In the right thalamus, a large number of immunoreactive neurons were identified in both lateral (left panel) and medial (right panel) regions.

This serum immunolabeled these rounded tubular structures in large neurons from the midbrain tegmentum (A)

14 Supplemental Figure 6. DTV antibody immunolabeled tubular structures in infected neurons. Paraffin sections from the midbrain were stained with the redtv antibody. This serum immunolabeled these rounded tubular structures in large neurons from the midbrain tegmentum (A) and third cranial nerve nucleus (B). Particulary in (B), the perinuclear distribution of these structures resembled the cellular distribution of the golgi apparatus.

Explain the laboratory diagnosis of Rabies?

Explain the laboratory diagnosis of Rabies? The standard test for rabies testing is dfa. This test has been thoroughly evaluated for more than 40 years, and is recognized as the most rapid and reliable