TITLE: Rabies Virus Bat-Clade Sequencing
AUTHORS: Fernanda Godinho, Aline Campos, rosana-huff, Amanda Ruivo, Milena Bauermann, Thales Bermann, Gabriel Luz Wallau, Paulo Michel Roehe, Richard Salvato

[DESCRIPTION]
Join us in advancing global genomic surveillance of the rabies virus.

We are actively engaged in pioneering a pan-clade whole genome amplicon sequencing protocol for the rabies virus. If you're interested in collaborating on this crucial endeavor and wish to obtain a primer aliquot from our established protocol or the one currently in development, please do not hesitate to contact us at richardssalvato@gmail.com. 

Background

Rabies virus (RABV) causes fatal encephalitis in domestic animals and humans. This high-impact zoonotic virus is poorly studied from the genomic perspective so establishing genomic surveillance protocols for RABV is crucial to track their evolution and monitor spillover events and wildlife reservoirs. 

Despite the importance of RABV zoonotic cycle, there is limited complete genome sequence data of RABVs from some hosts as bats. We developed a novel rapidly deployable, cost-effective, and flexible amplicon-based sequencing approach usable with protocols widely established during the COVID-19 pandemic and suitable to different hosts based on a one-health context. We used PrimalScheme to generate a primers panel and then aligned them with a RABV sequences dataset from different species and manually degenerated the primers to cover a wider diversity of hosts. 

The set of 47 primers is compatible and ready to use with COVIDSeq sequencing protocol and Illumina DNA Prep, and allows to sequence up to 384 samples per run on the Illumina MiSeq system or to accommodate in libraries with other sample types. Additionally, we included primers for amplification of a fragment of the mitochondrial gene COI to host species identification. 
COI

Initial Validation 

In the initial validation, we sequenced 160 complete RABV genomes from different species from five distinct families (Bovine, Equine, Caprine, Felines, Microchiroptera) with an average coverage of 98%, most of them recovering the whole genome (88/160). 

Conclusions

Here, we introduced a cost-effective and easy-to-use sequencing protocol for RABV bat-clade in order to support genomic surveillance of a re-emerging zoonotic disease allowing targeting viral control programs and adequate public health policies.

Acknowledgements

We would like to thank Instituto de Pesquisas Veterinárias Desidério Finamor for sample processing and initial rabies virus diagnosis. 





[STEPS]
SECTION: Primer Preparation
1. Reconstitute each primer shown in Table 1 (See Materials section), using nuclease-free water to get a 100 µM stock solution.


SECTION: Primer Preparation
2. Prepare RABV-BAT primer pools A and B as described here. 
SECTION: Primer Preparation
2.1. Separate all primers at 100 µM into two separate boxes labeled as Pool A and Pool B, according to Table 1. 
SECTION: Primer Preparation
2.2. Label a 2.0 ml microtube as  Pool A and another as Pool B.
SECTION: Primer Preparation
2.3. Vortex and spin down all the primers tubes.

Add 5 µL of each 100 µM primer from Pool A into the tube labeled as Pool A.
Add 5 µL of each 100 µM primer from Pool B into the tube labeled as Pool B.

SECTION: Primer Preparation
2.4. Add 1080 µL of nuclease-free water into the tube with Pool A.
Add 1215 µL of nuclease-free water into the tube with Pool B.

Now you have Pool A and B at a concentration of 10 µM and ready to use.
SECTION: Primer Preparation
2.5. Pooled Primers Can Be Stored at -20 °C

 

SECTION: Sample Extraction and Cycle threshold (Ct) determination
3. Samples were extracted as described below and we used a previously published Real-time RT-PCR on all samples to determine viral load with Cycle threshold (Ct) value. Samples with a Ct value <28 are recommended for optimal results. 

SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.1. A total of200 µL of homogenate samples, containing 50 mgof brain tissue fragments macerated in Basal Medium Eagle (BME), were added to 800 µL of TRIzolTM Reagent (Invitrogen, Grand Island, NY, USA) in 2.0 ml microtubes.
SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.2. The tissue was disrupted using a vortex at maximum speed for 15 s, followed by Room temperature incubation for 5 min and then  180 µL of chloroform was added. 
SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.3. The tissue was disrupted using a vortex at maximum speed for 15 s, followed by Room temperature incubation for 5 min and then  180 µL of chloroform was added. 
SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.4. The mixture was mixed for 15 s, incubated at Room temperature for 3 min, and centrifuged at 12000 rcf  at 4 °C for 15 min. 

SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.5. Viral RNA extraction was carried out using 200 µL of supernatant using a commercially available Extracta Kit Fast – DNA e RNA Viral (MVXA-PV96-B FAST) and the Loccus Extracta‱ 96  equipment (Loccus, Sao Paulo, Brazil) following the manufacturer’s instructions. Extracted RNA samples were stored at −80◦C until tested by RT-qPCR.
SECTION: Sample Extraction and Cycle threshold (Ct) determination
3.6. For Cycle threshold (Ct) determination TaqMan single-step RT-PCR assay was conducted according previously described by Crystal Gigante (See protocol below).
 

3. 
SECTION: cDNA Synthesis
4. cDNA synthesis and amplification of amplicons can be performed using the Illumina COVIDSeq Test (RUO version) or Illumina DNA Prep. Choose one of these approaches below.
SECTION: cDNA Synthesis
5. This step reverse transcribes the RNA fragments primed with random hexamers into first-strand cDNA using reverse transcriptase.

* Include a negative PCR control (NTC; nuclease-free water) for each pool.
SECTION: cDNA Synthesis
5.7. In a 1.7 ml tube, combine the following volumes to prepare First Strand cDNA Master Mix. 
*Multiply each volume by the number of samples.  

FSM 9 µL 
RVT 1 µL

Reagent overage is included to account for small pipetting errors.

SECTION: cDNA Synthesis
5.8. Add 8 µL master mix to each well of the CDNA1 plate.

SECTION: cDNA Synthesis
5.1. Label new PCR plate CDNA1.
SECTION: cDNA Synthesis
5.2. Add 8.5 µL EPH3 to each well.

SECTION: cDNA Synthesis
5.3. Add 8.5 µL eluted sample to each well.

SECTION: cDNA Synthesis
5.4. Seal and shake at   for 1 min.

SECTION: cDNA Synthesis
5.5. Centrifuge at 1000 × g for 1 minute.
SECTION: cDNA Synthesis
5.6. Place on the preprogrammed thermal cycler and run the COVIDSeq Anneal program.

 COVIDSeq Anneal program:

- Choose the preheat lid option
- Set the reaction volume to 17 µl
- 65°C for 3 minutes
- Hold at 4°C
SECTION: cDNA Synthesis
5.11. Place on the preprogrammed thermal cycler and run the COVIDSeq FSS program.

COVIDSeq FSS program:

- Choose the preheat lid option
- Set the reaction volume to 25 µl
- 25°C for 5 minutes
- 50°C for 10 minutes
- 80°C for 5 minutes
- Hold at 4°C


SECTION: cDNA Synthesis
5.10. Centrifuge at 1000 x g for 1 min.

SECTION: cDNA Synthesis
5.9. Seal and shake at   for 1 min.

SECTION: Amplicon Generation
6. This step uses two separate PCR reactions to amplify cDNA using the previously prepared primers pool A and B. 
* Include a negative PCR control (NTC; nuclease-free water) for each pool.


SECTION: Amplicon Generation
7. Label two new PCR plates POOL A and POOL B. The plates represent two separate PCR reactions.
SECTION: Amplicon Generation
8. In a 15 ml tube, combine the following volumes to prepare  PCR 1 Master Mix and PCR 2 Master Mix. Multiply each volume by the number of samples. 
*Reagent overage is included to account for small pipetting errors.

 

SECTION: Amplicon Generation
9. Add 20 µL PCR 1 Master Mix to each well of the POOL A plate corresponding to each well of
the CDNA1 plate.

SECTION: Amplicon Generation
9.1. Add 5 µL first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of the POOL A plate.

SECTION: Amplicon Generation
9.2. Add 20 µL COVIDSeq PCR 2 Master Mix to each well of the POOL B plate corresponding to each well of the CDNA1 plate.

SECTION: Amplicon Generation
9.3. Add 5 µL first strand cDNA synthesis from each well of the CDNA1 plate to the corresponding well of
the POOL B plate.

SECTION: Amplicon Generation
9.4. Seal and shake at   for 1 min.

SECTION: Amplicon Generation
9.5. Centrifuge at 1000 x g for 1 min.

SECTION: Amplicon Generation
9.6. Place in the preprogrammed thermal cycler and run the COVIDSeq PCR program.

COVIDSeq PCR program:

- Choose the preheat lid option
- Set the reaction volume to 25 µl
- 98°C for 3 minutes
- 35 cycles of:
---- 98°C for 15 seconds
---- 63°C for 5 minutes
- Hold at 4°C

SECTION: Amplicon Generation
10. *Safe Stopping Point: 

Amplicons can be stored at -20 °C until ready to use

SECTION: Amplicon checking
11. We recommend using agarose gel electrophoresis or automated electrophoresis to check the amplicons before proceeding to library construction.
SECTION: cDNA Synthesis
5. Use SuperScript‱ IV Reverse Transcriptase for cDNA synthesis

6. Heat the RNA-primer mix at 65 °C for 5 min  and then incubate on ice for at least 1 min.

SECTION: Library preparation
12. The following steps were conducted according to the standard protocol, starting from "Tagment PCR Amplicons" step on page 9. 

  

Tagment PCR Amplicons
Post Tagmentation Clean Up
Amplify Tagmented Amplicons 
Pool and Clean Up Libraries 
Quantify and Normalize Libraries 
Pool and Dilute Libraries 
Prepare for Sequencing 

SECTION: Sequencing
13. The sequencing can be performed in any Illumina platform or flowcell using 2x150 nt reads.

 

SECTION: Bioinformatics Analysis
14. Sequencing reads should be analyzed by an amplicon-based sequencing pipeline. We recommend using ViralFlow Workflow which performs several genomic analyses based on reference genome assembly.  See details at https://viralflow.github.io/

From our experience, better coverage and depth are obtained when using subclade-specific references. So, we usually assembly the sequences using the bat clade reference (JQ685956), then we run the consensus resulting sequence at http://rabv-glue.cvr.gla.ac.uk/ typing tool, and getting the nearest reference we re-assembly using this reference genome.

Reference Sequence
  

BED file
  

COI Analysis

COI amplicon analysis can be performed using ampliseq pipeline, an amplicon sequencing analysis workflow using DADA2 and QIIME2.  



6.1. RT reaction Mix
Combine the following reagents in a reaction tube. 
*Multiply each volume by the number of samples.  


 
Cap the tube or seal the plate and mix, and then briefly centrifuge the contents.
6.2. Add 7 µL from the RT reaction mix from step 6.1 to the annealed RNA from step 5.1.

6.3. Incubate at thermocycler using the following incubation settings:

23ºC ---- 10 minutes
53ºC ---- 10 minutes 
80ºC ---- 10 minutes
4ºC ---- Hold 
SECTION: cDNA Synthesis
5.1. Combine the following reagents in a reaction tube or plate well. 
*Multiply each volume by the number of samples.  

 


SECTION: Amplicon Generation
7. This step uses two separate PCR reactions to amplify cDNA using the previously prepared primers pool A and B. 
* Include a negative PCR control (NTC; nuclease-free water) for each pool.
SECTION: Amplicon Generation
8. Label two new PCR plates POOL A and POOL B. The plates represent two separate PCR reactions.
SECTION: Amplicon Generation
9. In a tube, combine the following volumes to prepare  PCR 1 Master Mix and PCR 2 Master Mix. Multiply each volume by the number of samples. 


 



SECTION: Amplicon Generation
10. Add 22.5 µL PCR 1 Master Mix to each well of the POOL A plate corresponding to each well of
the CDNA1 plate.

SECTION: Amplicon Generation
10.1. Add 2.5 µL first strand cDNA from each well of the cDNA plate to the corresponding well of the POOL A plate.

SECTION: Amplicon Generation
10.2. Add 22.5 µL PCR 2 Master Mix to each well of the POOL B plate corresponding to each well of
the cDNA plate.

SECTION: Amplicon Generation
10.3. Add 2.5 µL first strand cDNA from each well of the cDNA plate to the corresponding well of the POOL B plate.

SECTION: Amplicon Generation
10.4. Place in the preprogrammed thermal cycler and run the Taq PCR program.

Taq PCR program:

- Choose the preheat lid option
- Set the reaction volume to 25 µl

- 98ºC ---- 30 seconds
 - 45 cycles of:
---- 98°C for 15 seconds
---- 65°C for 5 minutes
- Hold at 4°C

SECTION: Amplicon Generation
11. *Safe Stopping Point: 

Amplicons can be stored at -20 °C until ready to use

SECTION: Amplicon checking
12. We recommend using agarose gel electrophoresis or automated electrophoresis to check the amplicons before proceeding to library construction.
SECTION: Library preparation
13. The following steps were conducted according to the standard Illumina DNA Prep protocol, starting from "Tagment Genomic DNA" step on page 8. 

  

Tagment Genomic DNA
Post Tagmentation Cleanup
Amplify Tagmented DNA
Clean Up Libraries 
Pool Libraries
Check Library quality 
Dilute Libraries to the starting concentration
Prepare for Sequencing 

 

SECTION: Sequencing
14. The sequencing can be performed in any Illumina platform or flowcell using 2x150 nt reads.

 

SECTION: Bioinformatics Analysis
15. Sequencing reads should be analyzed by an amplicon-based sequencing pipeline. We recommend using ViralFlow Workflow which performs several genomic analyses based on reference genome assembly.  See details at https://viralflow.github.io/

From our experience, better coverage and depth are obtained when using subclade-specific references. So, we usually assembly the sequences using the bat clade reference (JQ685956), then we run the consensus resulting sequence at http://rabv-glue.cvr.gla.ac.uk/ typing tool, and getting the nearest reference we re-assembly using this reference genome.

Reference Sequence
  
BED file
  

COI Analysis

COI amplicon analysis can be performed using ampliseq pipeline, an amplicon sequencing analysis workflow using DADA2 and QIIME2.