Novel mass spectrometry based detection and identification of variants of rabies virus nucleoprotein in infected brain tissues

Abstract

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms.

Fig 1. MS analysis of purified RABV ERA virus.

(A) RABV ERA virus variant purified from culture supernatants by sucrose density gradient centrifugation was denatured by 1X LDS sample buffer, separated on 4%– 12% Bis-Tris NuPAGE gels and twelve 1-mm slices were analyzed by MS. Molecular weight of protein standards in kilodalton (kDa) is indicated. M–molecular weight marker. The gel slices in which RABV proteins detected are indicated. (B) The peptides identified by MS/MS fragmentation is highlighted in red. The predicted glycosylation sites are highlighted in yellow. (C) One of the MS/MS peptide fragments derived from N protein provided in (B) is enlarged and presented.

Fig 2. MS analysis of uninfected and RABV CVS-11 infected cell lysates.

Mock and RABV CVS-11 infected MNA cells were harvested by centrifugation 24 h post infection. The sample was denatured, separated by gel and analyzed by MS. The slices corresponding to the RABV encoded proteins detected in infected cell lysate lanes by MS are indicated. C and I indicate MNA cells either mock or RABV CVS-11 virus infected cell lysates, respectively. M–protein molecular weight standards in kDa.

Fig 3. MS analysis of CNS tissues.

(A) DFA negative and positive brain tissues homogenates were separated on protein gels and the position of the gel corresponding to the size of N and G proteins based on RABV ERA purified virus were analyzed by MS (red line). M–molecular weight marker in kDa. U–uninfected cell lysate; ERA–purified RABV ERA virus. (B) MS results for the samples analyzed are tabulated. The nature and condition of DFA negative and positive samples, the RABV variant determined by antigenic typing and MS results are provided. (C) The amino acid sequence of peptides determined by MS/MS fragmentation and the unique amino acid used for RABV variant identification are shown in red.

Fig 4. The limit of N protein detection by MS.

(A) Different volume of purified RABV ERA virus was added to 1 μl of uninfected cell lysate. The position of the gel corresponding to N protein (marked by red lines) was sliced, in-gel tryptic digested and analyzed by MS. U–uninfected cell lysate, ERA–purified RABV ERA virus. (B) MS results for different gel slices. Height target above 2000 is considered as the cut-off for N protein detection. ERA–denotes to the samples in which N protein variant was identified by MS/MS fragmentation based amino acid sequencing.

Fig 5. Clustal alignment of N protein amino acid sequences from different RABV variants used in this study.

N protein amino acid sequence from representative RABV variants circulating in different hosts are compared using Clustal Omega program. N protein from E Raccoon (GenBank U27221.1), NC Skunk (AF461045.1), SC skunk (ADF80603.1), AZ gray fox (JQ685899.1), Arctic fox (AEV22313.1), T. brasiliensis (bat, ACN51665.1), CVS-11 (Q8JXF6.1) and ERA (P0DOF3.1) are aligned by Clustal analysis. The most frequently observed peptide fragments in MS are underlined and boxed to demonstrate the conservation or differences in RABV variants.

Fig 6. Percentage identity matrix for N protein variants determined by Clustal.

The percentage identity of different N protein from RABV variants are tabulated based on the values obtained from Clustal analysis.

Fig 7. Peptide view.

MS/MS fragmentation of peptide DPTIPEHASLVGLLLSYLYR derived from N protein corresponding to E. Raccoon RABV variant. The position and mass of various N- and C- terminal fragments (denoted by b_n and y_n, respectively) are indicated in the graph.