Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico.

Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 4659470, 15 pages http://dx.doi.org/10.1155/2016/4659470

Research Article Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico Fernando Bastida-González,1,2 Dolores G. Ramírez-Hernández,2 Erika Chavira-Suárez,3 Eleazar Lara-Padilla,1 and Paola Zárate-Segura1 1

Laboratorio de Medicina Traslacional, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y D´ıaz Mirón s/n, Santo Tomás, Miguel Hidalgo, 11340 Ciudad de México, CDMX, Mexico 2 Laboratorio de Biolog´ıa Molecular, Laboratorio Estatal de Salud Pública del Estado de México, Paseo Tollocan s/n, La Moderna de la Cruz, 50180 Toluca, MEX, Mexico 3 Departamento de Bioqu´ımica, Facultad de Medicina, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Copilco Universidad, 04510 Coyoacán, CDMX, Mexico Correspondence should be addressed to Fernando Bastida-González; [email protected] Received 23 February 2016; Revised 25 April 2016; Accepted 18 May 2016 Academic Editor: Muhammad Abubakar Copyright © 2016 Fernando Bastida-González et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing.

1. Introduction Despite the significant progress for prevention of the rabies disease and its control in the developing countries, this disease still causes over 60 thousand human deaths every year. Rabies disease is caused by infection with viruses of the family Rhabdoviridae, genus Lyssavirus [1]. Until now, fourteen species of Lyssavirus have been described in the world. Actually, the rabies virus (RABV) is the only one present in the American continent [2–6]. Although all mammals are susceptible to lyssaviruses, bats and carnivores are the major Lyssavirus reservoirs. In the Americas, distinct RABV variants are associated with different animals, such as foxes, coyotes, raccoons, skunks, and multiple species of nonhematophagous (frugivorous, insectivorous) and hematophagous bats [7–12]. In Mexico, we have been faced with less than ideal surveillance in animal populations. The reduced resources available are prioritized for

diseases with overwhelming human morbidity and mortality. Accurate diagnosis and determination of RABV variants are paramount components of surveillance system and frequently are important from the perspective of veterinary and public health, when the source of exposure needs to be determined and relevant control strategies need to be implemented [13]. The direct fluorescent antibody test (FAT) is the “gold standard” for rabies diagnosis [14]; the modern conjugates used in FAT are able to detect antigens of all lyssaviruses described to date [15, 16]. Virus variants associated with certain host species can be distinguished by application of antinucleocapsid monoclonal antibodies (MAbs) via indirect IFA. The MAbs are still commonly used in Latin American countries, particularly in the laboratories lacking established molecular techniques [17]; these have been applied to Mexican rabies virus samples and provide data regarding the most likely reservoir species involved in rabies transmission and

2 dissemination. Even though there has been a decrease in dog rabies, as a result of massive dog vaccination in Mexico, there is a high risk of an increase of human rabies cases transmitted from wild reservoirs as well as the simultaneous presence of more than one reservoir and more than one virus variant [18– 20]. The reactivity of certain viral isolates does not match the reactivity patterns in some cases [8, 17–19]. Other molecular assays like the restriction analysis of RT-PCR amplified fragments of RABV genes were suggested for the differentiation of two major RABV variants but suffered from low specificity. Amplification and sequencing of viral genes followed by their phylogenetic analysis have provided more robust characterization. However, this approach requires expensive equipment and experienced laboratory staff, and it takes a relatively long processing time (typically at least 10–12 hours) [20–23]. So, in this study, we designed eight pairs of primers of the RABV associated variant, which were used in a nested endpoint RTPCR (four external and four internal primers) for the realtime RT-PCR assay, in order to detect and type the major RABV variants present in Mexico.

2. Materials and Methods 2.1. Primer Design. Primer design was based on the alignment constructed with ClustalW using complete RABV N gene sequences available in GeneBank associated variant; these were designed in consensus region. Two pairs of the primers (external and internal) were designed for each of the RABV variants associated, and the maximum average entropy (Hx) and the maximum entropy of each position were calculated using Bio Edit v7.2.5. Two external primers and two internal primers were designed for dogs variant; 36 N gene sequences were obtained from different Mexican states; for the vampire bats variant, 18 N gene sequences were considered from Mexican states; for the nonhematophagous bat variant, the primer design comprised 50 N gene complete sequences from hosts Eptesicus, Myotis, and Nycticeius genera, distributed close to Mexico [21]; these genera are distributed from North America to Central America and have high diversity; in the case of skunks variant, 4 Mexican RABV sequences were considered; 34 RABV sequences were from USA and 13 CASK RABV sequences were from USA related to Mexican skunk rabies virus; previous studies consider two variants circulating in Mexico, MEXSK-2 and MEXSK-1 [22], located in South Baja California (SBC skunk) and Central Mexico; these are closely related and circulate predominantly in spotted skunks [23] (Table 1). 2.2. Samples. Twenty-three brain samples collected in Mexico were used as follows: nine brain samples tested negative by FAT and fourteen tested positive by FAT and typed by MAbs: these RABV isolated consisted of six samples of dog brain, one sample of skunk, two samples of cow, two samples of vampire bat, and three samples of nonhematophagous bat (Table 2).

BioMed Research International 2.3. Nucleic Acid Extraction. The brain tissues (approximately 3 mm3 ) were homogenized in 200 𝜇L of lysis/binding buffer using MagNA Lyser Green Beads (Roche, Germany) and MagNA Lyser (Roche Applied Science, Germany). Total RNA was extracted from the homogenates using MagNA Pure LC Total Nucleic Acid Isolation kit (Roche, Germany) and MagNA Pure LC 2.0 (Roche Applied Science, Germany) following the manufacturer’s instructions. Total RNA was eluted in 200 𝜇L buffer elution and quantified into Nanodrop (Invitrogen); RNA concentration was calculated considering 1 UAb𝜆260 nm = 50 ng/𝜇L, and all samples were adjusted at 20 ng/L of final concentration with elution buffer. 2.4. Nested RT-PCR Amplification and Sequence Determination. The reverse transcription reaction was performed using four singleplex reactions with external primers and SuperScriptIII Platinum One-Step qRT-PCR kit (Invitrogen) in 50 𝜇L of reaction mixture containing 25 𝜇L of 2x reaction mix, 1 𝜇L of forward sense primer (10 𝜇M), 1 𝜇L of reverse sense primer (10 𝜇M), 1 𝜇L SuperScript III RT/Platinum TaqMix, 17 𝜇L of DEPC-treated water, and 5 𝜇L RNA extracted (20 ng/𝜇L). Amplification was performed in C1000 Thermal Cycler (Bio-Rad, USA) using the following program: one cycle of RT at 50∘ C for 30 min, followed by denaturation at 92∘ C for 3 min, 35 cycles with denaturation at 92∘ C for 30 s, annealing at primer-specific temperature (Table 3) for 30 s, and elongation at 72∘ C for 1 min, with the final extension at 72∘ C for 4 min. For nested PCR, 1 𝜇L of the primary amplification products was added to a new singleplex PCR reaction using internal primers and Taq DNA polymerase kit; in a 50 𝜇L total volume, add 5 𝜇L 10x PCR buffer, 1 𝜇L of 1x dNTP mix (200 𝜇M of each dNTP), 1 𝜇L internal forward primer, 1 𝜇L internal reverse primer, 0.25 𝜇L Taq DNA polymerase (1.25 units/reaction), 1 𝜇L of primary amplification, and 40.75 𝜇L RNase-free water. The thermal program consisted of a first cycle of 2 min at 94∘ C, followed by 35 repetitive cycles of denaturation of 1 min at 93∘ C, 1 min of annealing at the primer-specific temperature (Table 3), 1 min of elongation at 72∘ C, and the final elongation at 72∘ C for 4 min. The four singleplex RT-PCR and four nested PCR products were analyzed in 1-2% agarose gel. Bands of the expected size were excised, purified, and cloned in TOPO-TA vector (Invitrogen, Carlsbad, USA). The resulting plasmids were purified from E. coli colonies using Pure Link Quick Plasmid Miniprep kit (Invitrogen), sequenced with the universal M13 primers (Macrogen, Korea), and analyzed with MEGA 6.06 [24]. 2.5. Real-Time RT-PCR with SYBR Green. The one-step realtime PCR was performed using internal primers and LCFastStart RNA Master SYBR Green I kit (Roche, Germany) in 20 𝜇L of total volume, four singleplex reactions including 100 ng total of total RNA and 0.01 𝜇M of each internal pair of primers for RABV associated variant (Table 3). Amplification was performed in LightCycler 2.0 (Roche, Germany) using the following program: one cycle of RT at 55∘ C for 30 min, followed by denaturation at 95∘ C for 30 s, 40 cycles with denaturation at 95∘ C for 10 s, annealing at 60∘ C for 15 s,

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Table 1: RABV N gene sequence for external and internal primer design for different rabies variant.

GI AF351832.1 GU644667.1 GU644664.1 GU644662.1 AY039229.1 AY039228.1 GU644676.1 GU644668.1 AF351862.1 GU644655.1 GU644695.1 GU644661.1 AY039227.1 GU644677.1 GU644670.1 GU644666.1 GU644660.1 GU644656.1 GU644654.1 GU644690.1 GU644669.1 AF351861.1 GU644689.1 GU644684.1 GU644663.1 GU644659.1 GU644657.1 AF351833.1 AF351828.1 GU644665.1 GU644671.1 GU644658.1 GU644652.1 AF351831.1 AF351855.1 GU644754.1 AF351854.1 AF394868.1 AY039225.1 AF351829.1 AF394871.1 AF351859.1 AF351860.1 GU644673.1 AY039226.1 AF351839.1 AF351853.1 HQ341796.1 AF351827.1 GU644675.1

Nonhematophagous bat Host Country Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Adams County, Pennsylvania Eptesicus fuscus (big brown bat) USA: El Paso County, Colorado Eptesicus fuscus (big brown bat) USA: Virginia Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: Iowa Eptesicus fuscus (big brown bat) USA: Washington Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Washington Eptesicus fuscus (big brown bat) USA: Virginia Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Iowa Eptesicus fuscus (big brown bat) USA: Iowa Eptesicus fuscus (big brown bat) USA: Washington Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: Washington Eptesicus fuscus (big brown bat) USA: Washington Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: New Jersey Eptesicus fuscus (big brown bat) USA: Michigan Eptesicus fuscus (big brown bat) USA: Georgia Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) Nycticeius humeralis (evening bat ) USA: Florida Eptesicus fuscus (big brown bat) Antrozous pallidus (pallid bat) USA: Monterey, California Myotis austroriparius (southeastern myotis bat) USA: Highlands County, Florida Eptesicus fuscus (big brown bat) Myotis californicus (California bat) USA: Plumas County, California Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: New Jersey Eptesicus fuscus (big brown bat) USA: Perry County, Pennsylvania Myotis sp. (bat) Eptesicus fuscus (big brown bat) Myotis chiloensis (bat) Chile Eptesicus fuscus (big brown bat) Eptesicus fuscus (big brown bat) USA: New Jersey

Collection date 2005 2003 2003 1984 1985 2004 2005 2005 2005 2003 1987 2004 2005 2005 2003 2005 2005 2004 2005 2004 2003 2003 2003 2003

2005 2005 2003 2004

2001 1991 1988 1987

2005 1984

2009 2005

4

BioMed Research International Table 1: Continued. Skunk

GI JQ513553 JQ513552 JQ513548 JQ513547 JQ513551 JQ513541 JQ513546 JQ513542 JQ513545 JQ513549 JQ513539 JQ513544 JQ513540 JQ513550 FJ228485 FJ228484 FJ228483 JX856026.1 JX856036.1 JX856035.1 JX855972.1 JX856024.1 JX856023.1 JX856017.1 JX856016.1 JX856015.1 JX856014.1 JX856006.1 JX856004.1 JX856001.1 JX855993.1 JX855990.1 JX855980.1 JX855988.1 JX855987.1 JX855986.1 JX855985.1 JX855984.1 JX855981.1 JX855979.1 JX855973.1 JX855976.1 JX855975.1 JX855974.1 JX855970.1 JX855968.1 JX855967.1 JX855966.1 JX855965.1 JX855963.1 JX855962.1

Host Skunk V854 Skunk V658 Skunk V652 Skunk V651 Fox V657 Dog V640 Skunk Mountain lion Skunk Skunk Skunk Skunk Skunk Skunk Cow Spilogale putorius leucoparia, skunk Spilogale putorius leucoparia, skunk Mephitis mephitis (striped skunk) Bovine (cow) Mephitis mephitis (striped skunk) Felis silvestris (bobcat) Mephitis mephitis (striped skunk) Bovine (cow) Bovine (cow) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Felis catus (cat) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Felis catus (cat) Mephitis mephitis (striped skunk) Canis lupus familiaris (dog) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk) Equus caballus (horse) Mephitis mephitis (striped skunk) Mephitis mephitis (striped skunk)

Country Mexico: San Luis Potos´ı USA: Mariposa County, California USA: Mariposa County, California USA: Mariposa County, California USA: Mariposa County, California USA: Sonoma County, California USA: Trinity County, California USA: Yolo County, California USA: Mendocino County, California USA: Amador County, California USA: Glenn County, California USA: Colusa County, California USA: Sutter County, California USA: Glenn County, California Mexico: Chihuahua Mexico: San Luis Potos´ı Mexico: Zacatecas USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA USA

Collection date 2002 1997 1997 1997 1997 1994 1997 1994 1997 1997 1994 1994 1994 1997 1999 2002 2001 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009


5 Table 1: Continued. Dog

GI FJ228513.1 FJ228512.1 FJ228507.1 FJ228532.1 FJ228506.1 FJ228505.1 FJ228504.1 FJ228503.1 FJ228502.1 KJ001535.1 KJ001525.1 KJ001524.1 KJ001518.1 KJ001517.1 KJ001516.1 KJ001515.1 KJ001509.1 KJ001502.1 KJ001500.1 KJ001499.1 KJ001492.1 KJ001490.1 KJ001488.1 FJ228525.1 FJ228523.1 FJ228521.1 FJ228518.1 FJ228511.1 FJ228510.1 FJ228509.1 FJ228508.1 FJ228522.1 FJ228519.1 AY854591.1 AY854589.1 FJ228526.1

Host Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Bos taurus (cow) Sus scrofa domesticus (pig) Mustela putorius furo (ferret) Homo sapiens (human) Bos taurus (cow) Felis catus (cat) Canis lupus familiaris (dog) Canis lupus familiaris (dog) Canis latrans (coyote)

Country Mexico: Estado de México Mexico: Estado de México Mexico: Estado de México Mexico: Puebla Mexico: Guerrero Mexico: Tlaxcala Mexico: Puebla Mexico: Tlaxcala Mexico: Distrito Federal Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico Mexico: Yucatán Mexico: Yucatán Mexico: Durango Mexico: Chiapas Mexico: Puebla Mexico: Distrito Federal Mexico: Distrito Federal Mexico: Distrito Federal Mexico: Chihuahua Mexico: Michoacán Mexico Mexico Mexico: Coahuila

Collection date 2000 2002 1999 1995 1999 2002 2001 2000 1999 2005 2009 2005 2011 2011 2011 2011 2009 2006 2006 2006 2005 2005 2005 2002 1998 1991 2002 1994 1991 1990 1991 1994 1990

Mexico: East Mexico Mexico: East Mexico Mexico: East Mexico Mexico: East Mexico East Mexico Mexico Mexico Mexico Mexico Mexico Mexico: Tamaulipas Mexico: Veracruz Mexico: Hidalgo Mexico: San Luis Potos´ı

1999 1999 2004 2002 2003 1988

2001

Vampire bat GU991828.1 GU991827.1 GU991826.1 GU991825.1 GU991824.1 GU991823.1 KP202393.1 AY854592.1 AY854587.1 AY854595.1 AY854594.1 FJ228491.1 FJ228490.1 FJ228489.1 FJ228488.1 AY877435.1 AY877434.1 AY877433.1

Desmodontinae (vampire bat V3) Desmodontinae (vampire bat V3) Desmodontinae (vampire bat V3) Desmodontinae (vampire bat V3) Desmodontinae (vampire bat V11) Desmodontinae (vampire bat V11) Desmodontinae (vampire bat) Desmodontinae (vampire bat) Desmodontinae (vampire bat) Desmodontinae (vampire bat) Desmodontinae (vampire bat) Bos taurus (cow) Bos taurus (cow) Ovis aries (sheep) Bos taurus (cow) Desmodontinae (vampire bat) Desmodontinae (vampire bat) Desmodontinae (vampire bat)

2003 2003 2003 2004

6


Table 2: RABV isolated typed by MAbs used in the present study. Sample 1 2

Sample name 68EDOMEXDOG05 647EDOMEXDOG05

Host species Dog 068 Dog 647

Antigenic variant V1 V1

3 4 5

659EDOMEXDOG05 748EDOMEXDOG05 2293EDOMEXDOG05

Dog 659 Dog 748 Dog 2293

V1 V1 V1

6 7

885EDOMEXDOG05 658EDOMEXCOW05

Dog 885 Cow 658

V1 V8

8 9 10

460EDOMEXCOW11 757EDOMEXMUR06 1594EDOMEXVAM07

Cow 460 Bat nonhematophagous 757 Bat nonhematophagous 1594

V8 A V5

11 12

1079EDOMEXMUR08 3919EDOMEXVAM05

Bat nonhematophagous 1079 Vampire bat 3919

None None

13 14 15

110EDOMEXVAM06 1369EDOMEXSK06 65EDOMEXDOG05

Vampire bat 110 Skunk 1369 Dog

Atypical V8 NA

16 17

543EDOMEXDOG05 642EDOMEXDOG05

Dog Dog

NA NA

18 19 20

223EDOMEXDOG05 1001EDOMEXDOG05 455EDOMEXDOG05

Skunk Skunk Cow

NA NA NA

21 22

755EDOMEXDOG05 2187EDOMEXDOG05

Bat nonhematophagous Vampire bat

NA NA

23

Negative control

CN

NA

All samples are from Mexican state. Varian antigenic test: V1, dog; V5, Tadarida brasiliensis; V8, skunk; V11, vampire bat; A, atypical; NA, not applicable; AC Number: sequences refer to NCBI accession number (http://www.ncbi.nlm.nih.gov/).

Table 3: Primer details for host mammals specific rabies virus variants detection in Mexico. Primer name E I E I E I E I

VAMPIROF VAMPIRORC FBVFQ FBVRCQ PERROF PERRORC FBPFQ PERRORC

Sense Rabies variant detection TTCAAGGTCAATAATCAGGTGGTCTCTC F AGACTGCTGTTCCTCATTCCTATTT R Vampire bat ATTGGGCTCTAACAGGGGGCAT F ATAGAGCAGATTTTCGAGACAGCCCCCT R Sequence (3󸀠 to 5󸀠 )

TTCAAAGTCAATAATCAGGTGGTC AATCATCAAGCCCGTCCAAACT CAAGAATATGAGGCGGCTGAACT AATCATCAAGCCCGTCCAAACT

MURCIELAGOF GACCCTGATGATGTATGCTCTTAT MURCIELAGORC GTTCCTCACTCYTATTTCATCCA FBMFQ GCTTGACCCTGATGATGTATGCTCTTAT FBMRCQ TGGGCTCTAACAGGGGGTATGG ZORRILLOF ZORRILLORC ZORRILLOF FBZRCQ

ATAGAACAGATTTTTGAGACGGC TGTCTCAGTTAGTTCCAATCATCAAGC ATAGAACAGATTTTTGAGACGGC GTTCCTCACTCCTATTTCATCCA

F R F R F R F R F R F R

Fragment size Tm (∘ C) Length∗ (pb) (pb) 59 22–49 836 53.8 833–857 59.6 356–377 177 62.5 575–602 1288

Dog 212 668 Bat 184 794 Skunk 359

51.9 56.4 55 56.4

22–45 1288–1309 1099–1120 1288–1309

51 50.6 59 58.7

196–219 742–764 192–219 358–379

51.4 56.9 51.4 51.7

505–527 1272–1298 506–527 742–764

Nested endpoint PCR and real-time RT-PCR primers designed. E: external; I: internal; F: forward; R: reverse. ∗ According to RABV strain SAD VA1 sequence.

BioMed Research International and elongation at 72∘ C for 25 s. The measurement of the fluorescent signal was carried out during the extension phase at 530 nm. By the end of the amplification test, an analysis of the dissociation curves from the product was made to ensure the absence of hairpin and dimer formation. Hybridization temperatures and primer concentrations were optimized for each reaction based on the preliminary standardization experiments.

3. Results The set of primers for specific RABV variants was designed aligning the sequence of N gene region. Four regions highly conserved were selected for two external primers and two internal primers designed, with more than 90% of conservation, for each variant and high variability between variants. Two mismatches were permitted for the primers design, and less was possible for the internal primers located at the primers beginning or end. As the maximum entropy values increased, the number of identified conserved regions, their length, the coverage of conserved regions, and the average length of single conserved regions also increased. Two external primers and two internal primers were designed for dog-associated variant; the alignment presented a high conservation level (Figure 1). The maximum average entropy (Hx) was 0.04 and the maximum entropy of each position was 0.97. In the case of the set of primers for skunk-associated variant, the alignment presented high conservation level (Figure 2). The maximum average entropy (Hx) was 0.19 and the maximum entropy of each position was 0.99. The alignment of the set of primers for vampire bat-associated variant showed a high conservation level (Figure 3). The maximum average entropy (Hx) was 0.04 and the maximum entropy of each position was 0.98. Finally, the alignment of the set of primers for nonhematophagous bat-associated variant presented high conservation level (Figure 4). The maximum average entropy (Hx) was 0.07 and the maximum entropy of each position was 0.97 on average; this means that, at the same position of every base, a few sequences of alignment of the associated variant differed from the others and thus were considered conservative; with respect to the maximum average entropy, the variant associated with more differences was the nonhematophagous bat-associated variant (Figure 4). The sequences and locations of the two pairs of variantspecific primers are listed in Table 3. Even when the melting temperature is similar between them, the sequence is dependent on the specific host variant. All brain samples from Mexican host mammals were diagnosed as negative or positive in FAT, with a corresponding signal in the nested RT-PCR assay. A positive control for each variant was performed using a positive example previously MAbs tested. In the first step, the external amplification produced a single band of 608–1187 bp, while the second amplification of the primary PCR products with the internal primer showed products of 200–400 bp. To complement the nested information, one-step RT-PCR as well as the second

7 nested RT-PCR was performed with external primers and the same samples (Figure 5). The optimal annealing temperature for external RT-PCR was in the range 48–55∘ C and was 56∘ C in the case of nested RT-PCR. Optimal concentration of Mg2+ was in the order of 2.5–3 mM for both RT-PCR and nested RT-PCR reaction mixtures. 3.1. RT-PCR SYBR Green. For real-time RT-PCR assay, we used the internal primers (Table 3). This technique had an optimal annealing temperature of 60∘ C from four pairs of primers, and the dissociation temperature curves were as follows: 85.50∘ C for skunk specific variant, 80.19∘ C for dogassociated variant, 83.96∘ C for bat-associated variant, and 85.23∘ C for vampire bat specific variant (Figure 6). 3.2. Comparison of Diagnostic Methods. A total of twentythree samples were assessed as follows: nine negative-control samples performed by nested or real-time RT-PCR assays showed no positive detection with the internal and external primers; previously, fourteen positive-RABV variants samples were tested by FAT; eleven of them were categorized with monoclonal antibodies resulting in six positive to variant 1 (V1), three to V8, one to V5, two atypical variants, and two undetected. The RABV specific variant characterizations to dog, vampire bat, nonhematophagous bat, and skunk were determined by real-time RT-PCR using external primers. However, to prevent cross-reaction and to increase sensitivity in the nested PCR, internal primers were used to confirm thus the abovementioned variants. In addition, real-time PCR detection using internal primers confirmed the reservoir variant with dissociation temperatures of 60∘ C. The amplified fragments were sequenced with a subsequent analysis by BLAST; this analysis confirmed the reservoir for nested PCR and real-time RT-PCR (Table 4). 3.3. Sensitivity of nRT-PCR and SYBR Green. Twenty-three brain samples were analyzed as follows: nine negative-control samples and fourteen positive samples were confirmed by nucleotide sequencing. Regarding the results of the nested PCR and real-time PCR assays of the brain samples, they showed 100% sensitivity (100% CI: 76.84% to 100.00%) and 100% specificity (100% CI: 66.37% to 100%).

4. Discussion In some studies of antigenic characterization of rabies virus, a panel of eight anti-N protein monoclonal antibodies (MAbs) has been used, which can differentiate between eleven distinct variants harbored by a variety of terrestrial and chiropteran hosts [25, 26]. Application of this panel to rabies virus collections from many Latin American countries has identified two major variants, associated with dog and vampire bat (Desmodus rotundus), as well as other variants associated with several insectivorous bats, including the free-tailed bat (Tadarida brasiliensis) and the hoary bat (Lasiurus cinereus) [27].

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20

PERROF KJ001518.1 KJ001517.1 KJ001516.1 KJ001515.1 KJ001500.1 KJ001492.1 KJ001490.1 KJ001535.1 KJ001502.1 KJ001499.1 AY854591.1 KJ001488.1 FJ228513.1 KJ001525.1 FJ228512.1 FJ228506.1 FJ228504.1 KJ001524.1 FJ228502.1 FJ228505.1 FJ228503.1 FJ228509.1 FJ228508.1 FJ228510.1 FJ228507.1 FJ228511.1 FJ228521.1 FJ228522.1 FJ228519.1 FJ228518.1 KJ001509.1 FJ228525.1 FJ228523.1 FJ228532.1 AY854589.1 FJ228526.1 Consensus 100% Conservation 0%

(a) 1,080 FBPFQ FJ2285 13 .1 FJ2285 12.1 FJ2285 07 .1 FJ2285 32.1 FJ2285 06 .1 FJ2285 05 .1 FJ2285 04 .1 FJ2285 03 .1 FJ2285 02.1 KJ0015 35.1 KJ0015 25 .1 KJ0015 24 .1 KJ0015 18.1 KJ0015 17 .1 KJ0015 16 .1 KJ0015 15 .1 KJ0015 09 .1 KJ0015 02.1 KJ0015 00.1 KJ0014 99.1 KJ0014 92.1 KJ0014 90.1 KJ0014 88.1 FJ2285 25 .1 FJ2285 23 .1 FJ2285 21.1 FJ2285 18.1 FJ2285 11.1 FJ2285 10.1 FJ2285 09 .1 FJ2285 08.1 FJ2285 22.1 FJ2285 19 .1 AY 85 4591.1 AY 85 4589 .1 FJ2285 26 .1 Consensus 100% Conservation 0%

1,280

60

TTCAAAGTCAATAATCAGGTGGTC-----------------------------------. . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . T TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C CG G AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC TC TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC TC TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG . . . . . . . . . . . . . . . . . . . . . . . . TC T T TG A AG C C TG AG A T TA TCG TG G A C CA A TA TG AG TTCAAAG TCAA TAA TCAGG TGG TC TC TTTG AAG CC TG AG A TTA TCG TGG A CCAA TA TG AG

24 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60

1,300

PERRORC - - - - - - A A T C A T C A A G C C C G T C C A A A C T - - - - - - - - - - - - - - 22 KJ001518.1 KJ001517.1 KJ001516.1 KJ001515.1 KJ001500.1 KJ001492.1 KJ001490.1 KJ001535.1 KJ001502.1 KJ001499.1 AY854591.1 KJ001488.1 FJ228513.1 KJ001525.1 FJ228512.1 FJ228506.1 FJ228504.1 KJ001524.1 FJ228502.1 FJ228505.1 FJ228503.1 FJ228509.1 FJ228508.1 FJ228510.1 FJ228507.1 FJ228511.1 FJ228521.1 FJ228522.1 FJ228519.1 FJ228518.1 KJ001509.1 FJ228525.1 FJ228523.1 FJ228532.1 AY854589.1 FJ228526.1 Consensus A G T T C C A A T C A T C A A G C C C G T C C A A A C T C A T T C G C A G A G T T T 100% Conservation 0%

(b) 1,100

-------------CA AGAATATGAGGCGGCTGAACT-------------------TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CCC CG AG AAAG AA C TT...........A...........G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT...........A...........G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT...........A...........G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT...........A...........G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT...........A...........G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TAG CG C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TAG CG C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TAG CCC TG AG AAAG AA C TT.......................G A CAAAG A C TG A TG TGG CA C TG AG AAAG AA C TT............A..........G A CAAAG A C TG A TG TGG CA C TG AG AAGG AA C TT.......................G A C TAAG A C TG A CG TGG CCC TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CAAAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CAAAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TAAG AAAG AA C TT.......................G A CG AAG A C TG A TG TGG CA C TG AG AAAG AA C TTCAAG AA TA TG AGG CGG C TG AA C TG A CG AAG A C TG A TG TGG CA C

(c)

1,280

1,120 23 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120 1120

1,300

PERRORC - - - - - - A A T C A T C A A G C C C G T C C A A A C T - - - - - - - - - - - - - - 22 KJ001518.1 KJ001517.1 KJ001516.1 KJ001515.1 KJ001500.1 KJ001492.1 KJ001490.1 KJ001535.1 KJ001502.1 KJ001499.1 AY854591.1 KJ001488.1 FJ228513.1 KJ001525.1 FJ228512.1 FJ228506.1 FJ228504.1 KJ001524.1 FJ228502.1 FJ228505.1 FJ228503.1 FJ228509.1 FJ228508.1 FJ228510.1 FJ228507.1 FJ228511.1 FJ228521.1 FJ228522.1 FJ228519.1 FJ228518.1 KJ001509.1 FJ228525.1 FJ228523.1 FJ228532.1 AY854589.1 FJ228526.1 Consensus A G T T C C A A T C A T C A A G C C C G T C C A A A C T C A T T C G C A G A G T T T 100% Conservation 0%

(d)

Figure 1: External and internal primer alignments for dog specific RABV variant detection. (a) Forward sequence of the external primer named PERROF. (b) Reverse sequence of the external primer named reverse PERRORC. (c) Forward sequence of the internal primer named FBPFQ. (d) Reverse sequence of the internal primer named PERRORC.

Real-time RT-PCR techniques have been used for diagnosis and genotyping of all the Lyssavirus genus including the RABV [28]. The nested RT-PCR assay, which requires both multiple transfers of material and substantial time, is sufficient to detect virus from each virus-positive brain sample [29] and therefore still offers a useful tool for variants rabies diagnosis where conventional PCR technology exists.

The real-time RT-PCR detection with SYBR Green, where the specificity is being given by the primers, is an easy-to-use assay to detect infected brain material in a single tube test and, consequently, is an attractive option for laboratory use as a screening surveillance tool. In the present study, these latest technologies for typing the RABV variants depending on the host (vampire bat, skunk, dog, and bat) were used.


9

460

440

480

500

520

540

ZORRILLOF - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A T A G A A C A G A T T T T T G A G A C G G C - - - - - - - - - - - - - - - - - - 23 JQ513553.1 G T C T G T A C A G A T T G A G C A A A A T A T C T G G T C A G A A C A C T G G A A A T T A C A A G A C A A A T A T C G C A G A T C G T . . . . . G . . . . . C . . C . . . . . . . . C C C C T T T G T G A A A G T C G T 545 JQ513552.1 G T C T G T A T A G G T T G A G C A A A A T A T C A G G A C A A A A C A C A G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . A . . A . . C C C T T T T G T T A A A A T T G T 545 JQ513548.1 G T C T G T A T A G G T T G A G C A A A A T A T C A G G A C A A A A C A C A G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . A . . A . . C C C T T T T G T T A A A A T T G T 545 JQ513547.1 G T C T G T A T A G G T T G A G C A A A A T A T C A G G A C A A A A C A C A G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . A . . A . . C C C T T T T G T T A A A A T T G T 545 JQ513551.1 G T C T G T A T A G G T T G A G C A A A A T A T C A G G A C A A A A C A C A G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . A . . A . . C C C T T T T G T T A A A A T T G T 545 JQ513541.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G A C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . . . . A . . C C C T T T T G T T A A A A T C G T 545 JQ513546.1 G T T T G T A T A G G C T G A G C A A A A T A T C A G G A C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A A A T C G T 545 JQ513542.1 G T T T G T A T A G G C T G A G C A A A A T A T C A G G A C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . . . . A . . C C C T T T T G T T A A A A T C G T 545 JQ513545.1 G T T T G T A T A G G C T G A G C A A A A T A T C A G G A C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A T A G G . . . . . G . . . . . . . . . . . . . . A . . C C C T T T T G T T A A A A T C G T 545 JQ513549.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G G C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A C A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A A A T C G T 545 JQ513539.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G G C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A C A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A G A T C G T 545 JX856026.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856035.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856000.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855972.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856024.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856023.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856017.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856016.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX856015.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C T G G C A A T T A C A A G A C A A A C A T T G C C G A T C G C . . . . . . . . . . . . . . . . . . . . . . . C C C C T T T G T G A A G A T C A T 545 JX856014.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C T G G C A A T T A C A A G A C A A A C A T T G C C G A T C G C . . . . . . . . . . . . . . . . . . . . . . . C C C C T T T G T G A A G A T C A T 545 JX856007.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C T G G C A A T T A C A A G A C A A A C A T T G C C G A T C G C . . . . . . . . . . . . . . . . . . . . . . . C C C C T T T G T G A A G A T C A T 545 JX856006.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C T G G C A A T T A C A A G A C A A A C A T T G C C G A T C G C . . . . . . . . . . . . . . . . . . . . . . . C C C C T T T G T G A A G A T C A T 545 JX856004.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855999.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855993.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855990.1 G T C T G T A T C G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855980.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855988.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855987.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855984.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855969.1 G T C T G T A T C G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855981.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855979.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855973.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855976.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C C T T T G T G A A G A T C A T 545 JX855975.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855974.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855970.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855968.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855967.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855966.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855965.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855963.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JX855962.1 G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T . . . . . . . . . . . . . . . . . . . . . . . C C C T T T T G T G A A G A T C A T 545 JQ513544.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G G C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A C A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A G A T C G T 545 JQ513540.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G G C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A C A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A G A T C G T 545 JQ513550.1 G T T T G T A T A G G T T G A G C A A A A T A T C A G G G C A A A A C A C C G G T A A C T A T A A G A C A A A C A T T G C A G A C A G G . . . . . G . . . . . C . . . . . . . . A . . C C C T T T T G T T A A G A T C G T 545 FJ228484.1 G T C T G T A C A G A T T G A G C A A A A T A T C T A G T C A G A A C A C C G G A A A T T A C A A G A C A A A T A T C G C A G A T C G T . . . . . G . . . . . C . . C . . . . . . . . C C C C T T T G T G A A A G T C G T 545 FJ228483.1 G T C T G T A C A G A T T G A G C A A A A T A T C T G G T C A G A A C A C T G G G A A T T A T A A G A C A A A T A T T G C A G A T C G C . . . . . G . . . . . C . . C . . . . . . . . C C C C T T T G T G A A A G T C A T 545 FJ228485.1 G T C T G T A C A G A T T G A G C A A A A T A T C T G G T C A G A A C A C C G G A A A T T A C A A G A C A A A T A T T G C A G A T C G T . . . . . G . . . . . C . . C . . . . . . . . C C C C T T T G T G A A A G T C G T 545 Consensus G T C T G T A T A G A C T T A G C A A A A T A T C T G G A C A A A A C A C C G G C A A T T A C A A G A C A A A C A T T G C C G A T C G T A T A G A A C A G A T T T T T G A G A C G G C C C C T T T T G T G A A G A T C A T 100% Conservation 0%

1,280 1,300 1,320 ZORRILLORC -----------TGTCTCAGTTAGTTCCAATCATCAAGC---------------------- 27 JQ513553.1 ATAAGAAGATA.........C.................CCGCCCCAACTCGTTCGCTGAG 1320 JQ513552.1 ATACGGAGATA......C..C.................CCGTCCAAACTCCTTCGCCGAG 1320 JQ513548.1 ATACGGAGATA......C..C.................CCGTCCAAACTCCTTCGCCGAG 1320 JQ513547.1 ATACGGAGATA......C..C.................CCGTCCAAACTCCTTCGCCGAG 1320 JQ513551.1 ATACGGAGATA......C..C.................CCGTCCAAACTCCTTCGCCGAG 1320 JQ513541.1 ATACGAAGATA.........C.................TCGTCCAAACTCATTCGCCGAG 1320 JQ513546.1 ATACGAAGATA.........C.................TCGTCCGAACTCATTCGCAGAG 1320 JQ513542.1 ATACGAAGATA.........C.................TCGTCCGAACTCATTCGCAGAG 1320 JQ513545.1 ATACGAAGATA.........C.................TCGTCCGAACTCATTCGCAGAG 1320 JQ513549.1 ATACGGAGATA.........C.................TCGTCCAAACTCATTCGCCGAG 1320 JQ513539.1 ATACGGAGATA.........C.................TCGTCCAAATTCATTCGCCGAG 1320 JX856026.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856036.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856035.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855972.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856024.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856023.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856017.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856016.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX856015.1 ATAAGGAGATA..........G................TCGCCCCAATTCATTCGCTGAG 1320 JX856014.1 ATAAGGAGATA..........G................TCGCCCCAATTCATTCGCTGAG 1320 JX856006.1 ATAAGGAGATA..........G................TCGCCCCAATTCATTCGCTGAG 1320 JX856004.1 ATAAGGAGATA.........C...........C..G..TCGCCCTAATTCATTCGCTGAG 1320 JX856001.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855993.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855990.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855980.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855988.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855987.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855986.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855985.1 ATAAGGAGATA.........C.....T.....C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855984.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855981.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855979.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855973.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855976.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855975.1 ATAAGAAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855974.1 ATAAGGAGATA.........C...........C..G..TCGCCCTAATTCATTCGCTGAG 1320 JX855970.1 ATAAAGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855968.1 ATAAGGAGATA.........C...........C.....TCGCCCCAATTCATTCGCTGAG 1320 JX855967.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855966.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855965.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855963.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JX855962.1 ATAAGGAGATA.........C...........C.....TCGCCCTAATTCATTCGCTGAG 1320 JQ513544.1 ATACGGAGATA.........C.................TCGTCCAAATTCATTCGCCGAG 1320 JQ513540.1 ATACGGAGATA.........C.................TCGTCCAAATTCATTCGCCGAG 1320 JQ513550.1 ATACGGAGATA.........C..C..............TCGTCCGAATTCATTCGCCGAG 1320 FJ228484.1 ATAAGAAGATA.........C.................CCGCCCCAACTCGTTCGCTGAG 1320 FJ228483.1 ATAAGAAGATA.........C.................CCGCCCCAACTCATTCGCTGAG 1320 FJ228485.1 ATAAGAAGATA.........C.................CCGCCCCAACTCGTTCGCTGAG 1320 Consensus ATAAGGAGATATGTCTCAGTCAGTTCCAATCACCAAGCTCGCCCTAATTCATTCGCTGAG 100% Conservation 0%

(a)

(b) 520

500 ZORRILLOF JQ513553.1 JQ513552.1 JQ513548.1 JQ513547.1 JQ513551.1 JQ513541.1 JQ513546.1 JQ513542.1 JQ513545.1 JQ513549.1 JQ513539.1 JX856026.1 JX856036.1 JX856035.1 JX855972.1 JX856024.1 JX856023.1 JX856017.1 JX856016.1 JX856015.1 JX856014.1 JX856006.1 JX856004.1 JX856001.1 JX855993.1 JX855990.1 JX855980.1 JX855988.1 JX855987.1 JX855986.1 JX855985.1 JX855984.1 JX855981.1 JX855979.1 JX855973.1 JX855976.1 JX855975.1 JX855974.1 JX855970.1 JX855968.1 JX855967.1 JX855966.1 JX855965.1 JX855963.1 JX855962.1 JQ513544.1 JQ513540.1 JQ513550.1 FJ228484.1 FJ228483.1 FJ228485.1

T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A

C T T T T T T T T T T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T C T C



G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G


C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C




T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T



C T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C T T



A A A A A A A A A A A C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C A A A A A A



T T T T T T T T T C C T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C C C T T T

C A A A A A A A A A A C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C A A A C C C


T G G G G G G G G G G T T T T T T T T C C C T T T T T T T T T T T T T T T T T T T T T T T G G G T C T

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A G G G G G G G G G G G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G G G G G G

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T C . . . . . C . . C C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C C C C C C

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C C C

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

860

540 G . A A A A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . A A A A A A A A A A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A A A . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .




C T T T T T T T T T T T T T T T T T T C C C T T T T T T T T T T T T T C T T T T T T T T T T T T C C C






G T T T T T T T T T T G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G T T T G G G



A A A A A A A A A A G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A A A

23 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540

Consensus T A C A A G A C A A A C A T T G C C G A T C G T A T A G A A C A G A T T T T T G A G A C G G C C C C T T T T G T G A A G 100% Conservation 0%

(c)

FBZRCQ JQ513553.1 JQ513552.1 JQ513548.1 JQ513547.1 JQ513551.1 JQ513541.1 JQ513546.1 JQ513542.1 JQ513545.1 JQ513549.1 JQ513539.1 JX856026.1 JX856036.1 JX856035.1 JX855972.1 JX856024.1 JX856023.1 JX856017.1 JX856016.1 JX856015.1 JX856014.1 JX856006.1 JX856004.1 JX856001.1 JX855993.1 JX855990.1 JX855980.1 JX855988.1 JX855987.1 JX855986.1 JX855985.1 JX855984.1 JX855981.1 JX855979.1 JX855973.1 JX855976.1 JX855975.1 JX855974.1 JX855970.1 JX855968.1 JX855967.1 JX855966.1 JX855965.1 JX855963.1 JX855962.1 JQ513544.1 JQ513540.1 JQ513550.1 FJ228484.1 FJ228483.1 FJ228485.1 Consensus 100% Conservation 0%

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T T C C T C A C T C T T A T T T C A T C C A. . . . . . . . . . C . . . . . . . . . . .C . . . . . . . . . . . . . . . . . . . T . .C . . . . . . . . . . . . . . . . . . . T . .C . . . . . . . . . . . . . . . . . . . T . .C . . . . . . . . . . . . . . . . . . . T . .C . . . . . . . . . . . . . C . . . . . T . .T . . . . . . . . . . . . . C . . . . . T . .C . . . . . . . . . . . . . C . . . . . T . .C . . . . . . . . . . . . . C . . . . . T . .C . . . . . . . . . . . . . C . . . . . T . .C . C . . . . . . . . . . . C . . . . . T . .C . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . . . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T . . . . . . . . . . C . . . . . . . . . . .T ..........C...........T ......................T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ......................T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T ..........C...........T .C...........C.....T..C .C...........C.....T..C .C...........C.....T..C ..........C...........C ..........A...........C .C.................T..T

800 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T


C C C C C C C C C C C T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C C C C T C



G T T T T T T T T T T G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G T T T G G T



T A A A A A A A A A A T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T A A A T G A

T C C C C C C C C C C C T C T C T T C T T T T T C C C T T T T C C C T T T T C T T T C T T C C C T T C


G A A A A A A A A A A G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A A A G A A




C T T T T T T T T T T C C C C C T C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T C C T


900 G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A A G G G G A G



T T T T T T T T T T T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T T C T



G G G G G G G G G G G A G A G A G G A G G G G G A A A G G G G A A A G G G G A G G G A G G G G G G G G



A G G G G G G G G G G A G A A A A G A A G A A A A A A G G G G A A A G A G A A G G G A G G G G G A A G






A T T T T T T T T T T G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G T T T A A T



T T T T T T T T T T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T T T

23 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900 900

GTTCCTCACTCCTATTTCATCCATTTTCGGTCTCTGGGCCTGAGCGGGAAGTCTCCGTAC

(d)

Figure 2: The external and the internal primer alignments for skunk specific RABV variant detection. (a) Forward sequence of the external primer named ZORRILLOF. (b) Reverse sequence of the external primer named ZORRILLORC. (c) Internal primer named ZORRILLOF. (d) Reverse sequence of the internal primer named FBZRCQ.

The real-time RT-PCR detection with SYBR Green, whose specificity is given by the primers, is an easy-to-use assay to detect infected brain material in a single tube test and, consequently, is an attractive option for laboratory use as a screening surveillance tool. In the present study, these latest

technologies for typing the RABV associated variants on the host (vampire bat, skunk, dog, and bat) were used. In the design with highly specific primers from the conserved region from the nucleoprotein of RABV, the maximum average entropy (Hx) was in the order of 0.03–0.19 and the

10

BioMed Research International 20 VAMPIROF AY854595.1 AY854594.1 AY854592.1 GU991823.1 FJ228490.1 FJ228489.1 FJ228488.1 GU991824.1 FJ228491.1 GU991825.1 AY877434.1 KP202393.1 AY877435.1 GU991826.1 GU991827.1 AY854587.1 AY877433.1 GU991828.1 Consensus 100% Conservation 0%

A A A A A A A A A A A A A A A A A A A

T T T T T T T T T T T T T T T T T T T

G G G G G G G G G G G G G G G G G G G

G G G G G A G G G G G G G G G G G G G

A A A A A G A A A A A A A A A A A A A

T T T T T C T T T T T T T T T T T T T

G G G G G C G G G G G G G G G G G G G

C C C C C C C C C C C C C C C C C C C






A A A A G A A A A A A A A A A A A A A







40 A A A A A A A A A A A A A A A A A A A

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

A . . . . . . . . . . . . . . . . . . A

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . A . . . . G

G . . . . . . . . . . . . . . . . . . G

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

A . . . . . . . . . . . . . . . . . . A

A . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . T

A . . . . . . . . . . . . . . . . . . A

A . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . . . . G

G . . . . . . . . . . . . . . . . . . G

60 T . . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . G

G . . . . . . . . . . . . . . . . . . G

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

T C C C C C C C . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C


G G G G G G G G G G G G G A G G G G G










840 28 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60 60

VAMPIRORC AY854595.1 AY854594.1 AY854592.1 GU991823.1 FJ228490.1 FJ228489.1 FJ228488.1 GU991824.1 FJ228491.1 GU991825.1 AY877434.1 KP202393.1 AY877435.1 GU991826.1 GU991827.1 AY854587.1 AY877433.1 GU991828.1 Consensus 100% Conservation 0%



G G G G G G G G G G G G G G G A A A G





A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . A

C . . . . . . . . . . . . . . . . . . C

T . . . . . A . G G . . . . C . . . . T

G . . . . . . . . . . . . . . . . . . G

C . . . . . . . . . . . . . . . . . . C

T . . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . G

860 T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

C . . . . . . . . . . . . . . . . . . C

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

A . . . . . . . . . . . . . . . . . . A

(a)

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . C

C . . . . . . . . . . . . . . . T . T C

T . . . . . . . . . . . . . . . . . . T

A . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

C C C C C C C C C C C C C C C T T T C


340 T T T T T T T T T T T T T T T T T T

A A A A A A A A A A A A A A A A A A


G G G G G G G G G A G G G G G A A A


C C C C C C C C C C C C C C C C C C

G G G G G G G G G G G G G G G G G G

T T T T T T T T T T T T T T T T T T


C C C C C C C C C C C C C C C C C C

360 T T T T T T T T T T T T T T T T T T



C C T T T T T T T T T T T T T T T T



G G G G G G G G G G G G G A G G G G








A . . . . . . . . . . . . . . . . . .

T . . . . . . . . . C . . . . . . . .

T . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . A A A G

A . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . A . . . . . . T

T . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . T . . . . C

T T T T T T T C C T T T T T T C C C T

880 C C C C C C C C C C C C C C C C C C C








C C C C C C C C C C C C C C C G T G C






G G G G G G G G G G G G G G G G G A G




G . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . A

C . . . . . . . . . . . . . . . . . . C

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . . . . G

C . . . . . . . . . . . . . . . . . . C

C . . . . . . . T T T T . . . . . T . C

C . . . . . . . . . . . . . . . . . . C

C . . . . . . . . . . . . . . . . . . C

C . . . . . . . . . . . . . . . . . . C

T . . . . . . . . . . . . . . . . . . T


T T T T T C C T T C T T T T T T T T T



A A A A A A A A A A A A A A A A A C A

A A A A A A A A A A A A A A A A A C A


T T T T T T T T T T T T T T T C T T T

26 880 880 880 880 880 880 880 880 880 880 880 880 880 880 880 880 880 880

(b) 500

FBVFQ AY854595.1 A AY854594.1 A AY854592.1 A GU991823.1 A FJ228490.1 A FJ228489.1 A FJ228488.1 A GU991824.1 A FJ228491.1 A GU991825.1 A AY877434.1 A KP202393.1 A AY877435.1 A GU991826.1 A GU991827.1 A AY854587.1 A AY877433.1 A GU991828.1 A 100% Conservation 0%


G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

380 C . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . C . . .

C . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . .

A . . . . . G . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . A . . . . .

A . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . .








- 24 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385 A 385

(c)

FBVRCQ AY854595.1 T AY854594.1 T AY854592.1 T GU991823.1 T FJ228490.1 T FJ228489.1 T FJ228488.1 T GU991824.1 T FJ228491.1 T GU991825.1 T AY877434.1 T KP202393.1 T AY877435.1 T GU991826.1 T GU991827.1 T AY854587.1 T AY877433.1 T GU991828.1 T Consensus T 100% Conservation 0%














C C C C C C C C C C C C C C C T C C C






T T T T T T T T T T T T T T T T C T T

520 A A A A A A A A A A A A A A A A A A A



A . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . T

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . A

G . . . . . . . . . . . . . . . . . . G

C . . . . . . . . . . . . . . . . . . C

G . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . A

540 G G G G G G G G G G G G G G G G G G G

29 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540 540

(d)

Figure 3: The external and the internal primer alignments for vampire bat specific RABV variant detection. (a) Forward sequence of the external primer named VAMPIRORC. (b) Reverse sequence of the external primer named VAMPIROF. (c) Forward sequence of the internal primer named FBVFQ. (d) Reverse sequence of the internal primer named FBVRCQ.

maximum entropy of each position was 0.97–0.99. In addition, the positions of different primers in N gene sequence are close but different for variant host, increasing the specificity. The current gold standard test has been and is the fluorescent antibody test (FAT), which uses a conjugated monoclonal antibody against the RABV nucleoprotein. Although it is cheaper, some laboratories have no access to MAbs but have PCR and/or real-time technology. In the characterization of the antigenic variants (AgV) with MAbs in the dog samples, the dog variant-specific primers identified the dog variant (V1). This result matched both the nRT-PCR and SYBR Green primers at 100%. Similarly, the skunk samples matched the same percentage with skunk variant-specific primers. Furthermore, in the bovine samples where the MAbs detection identified the skunk variant (V8), the determined host by nRT-PCR and SYBR Green was diagnosed as positive with the vampire primers, with this last result confirmed by sequences and AC Numbers JQ037818 to JQ037831 (Table 1). The real-time RT-PCR result coincides with some other studies where rabies transmission from vampire bats to bovines has been described. The MAbs detection in nonhematophagous bat was V5 bat, a result which coincides with both nRT-PCR and SYBR Green with the bat primer. The vampire bat 110 sample was determined as atypical and the vampire bat 3919 was not determined with the MABs; however, both samples were diagnosed as positive with the bat primers for nRT-PCR and SYBR Green.

In some cases, the classification of certain rabies virus isolates by monoclonal panel can obtain nontypical reactivity patterns and is not assigned to any known variant, as found in certain Argentinian rabies viruses [12]. The application of molecular genetic techniques for characterization of viral collections can assist in resolving such typing difficulties. In the hematophagous bat samples determined as atypical and the one not determined with the MAbs, it was concluded that the host was a vampire bat by nRT-PCR and SYBR Green detection. This may have occurred due to the high sensitivity of the RT-PCR molecular technique, as it has been shown in studies where positive results in brains analysis were demonstrated by nRT-PCR and negative results by FAT [30, 31]. In all results, the host was confirmed by the amplicon sequencing. The access numbers are shown in Table 4. According to the RABV variant detection, the external primers and internal primers detect a specific variant and do not present cross-reaction between them, and the final result is given for the internal primer reaction in nested and/or RTPCR real time, as they were obtained in different samples (Table 5). In addition, this study showed 100% sensitivity and 100% specificity assessed by nRT-PCR and real-time RT-PCR with SYBR Green. These findings are an early estimate by what is required of a greater number of related studies, increasing the number of samples to obtain better sensitivity and specificity evaluation. However, this assay could be useful, for


11

200 MURCIELAGOF AF351832.1 GU644667.1 GU644664.1 GU644662.1 AY039229.1 AY039228.1 GU644676.1 GU644668.1 AF351862.1 GU644655.1 GU644695.1 GU644661.1 AY039227.1 GU644677.1 GU644670.1 GU644666.1 GU644660.1 GU644656.1 GU644654.1 GU644690.1 GU644669.1 AF351861.1 GU644689.1 GU644684.1 GU644663.1 GU644659.1 GU644657.1 AF351833.1 GU644665.1 GU644671.1 GU644658.1 GU644652.1 AF351831.1 AF351855.1 GU644754.1 AF351854.1 AF394868.1 AY039225.1 AF351829.1 AF394871.1 AF351859.1 AF351860.1 GU644673.1 AY039226.1 AF351839.1 AF351853.1 AF351828.1 HQ341796.1 AF351827.1 GU644675.1 Consensus 100% Conservation 0%

220

820

240

---------------GACCCTGATGATGTATGCTCTTAT--------------------AATGCAGCCAAGCTT........................TTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........C...............CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT....................A...CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........C...............CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........C...............CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAATTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT...........C............CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT..T.....................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT..T.....................CTGGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT.......................CCTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT..............G........CCTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT.................T......CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................TTGGCGGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCTGCAATGCAGTTC AATGCAGCCAAGCTT........................CTGGCAGCCGCAATGCAGTTC AATGCGGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTT........................CTAGCAGCCGCAATGCAGTTC AATGCAGCCAAGCTTGACCCTGATGATGTATGCTCTTATCTGGCAGCTGCAATGCAGTTC

24 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240

MURCIELAGORC AF351832.1 GU644667.1 GU644664.1 GU644662.1 AY039229.1 AY039228.1 GU644676.1 GU644668.1 AF351862.1 GU644655.1 GU644695.1 GU644661.1 AY039227.1 GU644677.1 GU644670.1 GU644666.1 GU644660.1 GU644656.1 GU644654.1 GU644690.1 GU644669.1 AF351861.1 GU644689.1 GU644684.1 GU644663.1 GU644659.1 GU644657.1 AF351833.1 AF351828.1 GU644665.1 GU644671.1 GU644658.1 GU644652.1 AF351831.1 AF351855.1 GU644754.1 AF351854.1 AF394868.1 AY039225.1 AF351829.1 AF394871.1 AF351859.1 AF351860.1 GU644673.1 AY039226.1 AF351839.1 AF351853.1 HQ341796.1 AF351827.1 GU644675.1 Consensus 100% Conservation 0%

840

(a)






A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A G A A









T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . T . . . . . . . . . . . C

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

220 G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

T . . . . . . . C . . . C . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

23 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870 870

(b)

200 FBMFQ AF351832.1 GU644667.1 GU644664.1 GU644662.1 AY039229.1 AY039228.1 GU644676.1 GU644668.1 AF351862.1 GU644655.1 GU644695.1 GU644661.1 AY039227.1 GU644677.1 GU644670.1 GU644666.1 GU644660.1 GU644656.1 GU644654.1 GU644690.1 GU644669.1 AF351861.1 GU644689.1 GU644684.1 GU644663.1 GU644659.1 GU644657.1 AF351833.1 AF351828.1 GU644665.1 GU644671.1 GU644658.1 GU644652.1 AF351831.1 AF351855.1 GU644754.1 AF351854.1 AF394868.1 AY039225.1 AF351829.1 AF394871.1 AF351859.1 AF351860.1 GU644673.1 AY039226.1 AF351839.1 AF351853.1 HQ341796.1 AF351827.1 GU644675.1 Consensus 100% Conservation 0%

860

----------------------------GTTCCTCACTCYTATTTCATCCA------AATGTTTGAGCCGGGGCAGGAAACCGCT.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCG.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCG.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAACCTGGGCAGGAAACCGCA.......................TTTTCGT GATGTTTGAGCCAGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCG.......................TTTCCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCG.......................CTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGACAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA....................T..TTTTCGT AATGTTTGAGCCGGGGCAGGAGACTGCA....................A..TTTTCGT AATGTTTGAACCAGGGCAGGAAACCGCA.................T.....TTTTCGT AATGTTTGAGCCGGGGCAGGAAACCGCA.......................TTTTCGT AATGTTCGAGCCGGGGCAGGAGACTGCA....................A..TTTTCGT AATGTTTGAGCCTGGGCAAGAAACGGCA.......................TTTCCGT AATGTTCGAGCCGGGGCAGGAGACTGCA.................T..A..TTTTCGT AATGTTCGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTCGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTTGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCA........T..............TTTTCGT AATGTTCGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTTGAGCCAGGGCAGGAAACCGCGA.C..C.................TTTTCGT AATGTTCGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTCGAGCCGGGGCAGGAGACGGCA....................A..TTTTCGT AATGTTTGAGCCTGGGCAGGAAACCGCAGTTCCTCACTCNTATTTCATCCATTTTCGT

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

(c)

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . A

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . C

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C

T . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . C . . . . . . . . T

T C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T C C C C C


360

240 G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A G G A G A A A A G A G A A G



A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A G A A A A A



C T T T T T T T T T T T C T T T T T T T T C T T T T T T T T T T T C T C C C C C T C C C C T C C C C T









G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A G G G G G G G G G G G G G G G G G G G G




25 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240 240

FBMRCQ AF351832.1 GU644667.1 GU644664.1 GU644662.1 AY039229.1 AY039228.1 GU644676.1 GU644668.1 AF351862.1 GU644655.1 GU644695.1 GU644661.1 AY039227.1 GU644677.1 GU644670.1 GU644666.1 GU644660.1 GU644656.1 GU644654.1 GU644690.1 GU644669.1 AF351861.1 GU644689.1 GU644684.1 GU644663.1 GU644659.1 GU644657.1 AF351833.1 AF351828.1 GU644665.1 GU644671.1 GU644658.1 GU644652.1 AF351831.1 AF351855.1 GU644754.1 AF351854.1 AF394868.1 AY039225.1 AF351829.1 AF394871.1 AF351859.1 AF351860.1 GU644673.1 AY039226.1 AF351839.1 AF351853.1 HQ341796.1 AF351827.1 GU644675.1 Consensus 100% Conservation 0%

G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A T A A A A A A A A A A A A A A A

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A G


G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A G G G G G G G G G G G G G G G


A A A A A A A A A A A A A A A A A A A A A A A A A A A C A A A A A C A A A A A A A A A A A A A A A A

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A

C C C C C C C T C C C C C C C C C C C C C C C C C C C C C C C C C C T C C T C C T C C C C T C C C C

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

380 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . T . . . T . . T . . . . . . . . . . . . . . . T . . . . . T . . . . . . T T . T . T T

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . G G . G G . G G G G . G . G G

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . T . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C C . . . . . . . . .

A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



400 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T

T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T

G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G A G G A G G G G G G G G G G G


C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A G G G G G G G G G G G A G G G



A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A G A A G A A A A G A A A A



C C C C T C T C C C C C T T C C C C C C C T C C C C C C C C C C T T C C C T C C C C C C C C C C C C



C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T C C C C C C C C C C C C C C C C C



C C C C C C C C C C C C C C C T T C T C T C C C T T C C C T C T C C C C A C C A C A A A A C A T A A


T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C T T T T T T T T T T T T T T T T T

T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C T T T T T T T T T T T T

T C C C C C C C C C C C T C C C C C C C C C C C C C C C C C C C C T C C T T C T C T T T T T G T T T


G G G G G G G G G G G G G G A G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G


22 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406 406

GAAGGGAACTGGGCTCTAACAGGGGGTATGGAGTTGACAAGAGACCCCACCGTTCCGG

(d)

Figure 4: The external and the internal primer alignments for nonhematophagous bat specific RABV variant detection. (a) Forward sequence of the external primer named MURCIELAGOF. (b) Reverse sequence of the external primer named MURCIELAGORC. (c) Forward sequence of the internal primer named FBMQ. (d) Reverse sequence of the internal primer named FBMRCQ.

12


Table 4: Comparison between different methods of rabies virus variants detection. Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

FAT

Host Dog 0068 Dog 647 Dog 659 Dog 748 Dog 2293 Dog 885 Cow 658 Cow 2688 Bat nonhematophagous 757 Bat nonhematophagous 1594 Bat nonhematophagous 1079 Vampire bat 3919 Vampire bat 110 Skunk 1369 65EDOMEXDOG05 543EDOMEXDOG05 642EDOMEXDOG05 223EDOMEXDOG05 1001EDOMEXDOG05 455EDOMEXDOG05 755EDOMEXDOG05 2187EDOMEXDOG05 Negative control

Antigenic variant

External RT-PCR D V B

S

Internal PCR d v b s

d

v

b

s

+ + + + + + − −

− − − − − − + +

GenBank sequence AC number (N gene)

+ + + + + + + +

V1 V1 V1 V1 V1 V1 V8 V8

+ + + + + + − −

− − − − − − + +

− − − − − − − −

− − − − − − − −

− − − − − − − −

+ + + + + + − −

− − − − − − + +

− − − − − − − −

− − − − − − − −

JQ037820 JQ037819 JQ037823 JQ037821 JQ037824 JQ037822 JQ037825 JQ037826

+

A

−

−

+

− − − + −

−

−

+

−

JQ037830

+

V5

−

−

+

− − − + −

−

−

+

−

JQ037829

+

None

−

−

+

− − − + −

−

−

+

−

JQ037831

+ + + − − − − − − − − −

None Atypical V8 NA NA NA NA NA NA NA NA NA

− − − − − − − − − − − −

− − − − − − − − − − − −

+ + − − − − − − − − − −

− − + − − − − − − − − −

− − − − − − − − − − − −

+ + − − − − − − − − − −

− − − − − − − − − − − −

− − + − − − − − − − − −

JQ037827 JQ037828 JQ037818 — — — — — — — — —

− − − − − − − − − − − −

− − − − − − − −

SYBR Green

+ + − − − − − − − − − −

− − − − − − − − − − − −

− − + − − − − − − − − −

FAT: fluorescent antibody test; Varian antigenic test: V1, dog; V5, Tadarida brasiliensis; V8, skunk; V11, vampire bat; A, atypical; + positive diagnosis. Capital letter: positive external RT-PCR; lowercased letter: positive internal PCR; italic letter: positive SYBR Green. Variants were represented in the following sense: D, dog; V, vampire; B, bat; and S, skunk. Sequences refer to NCBI accession number (http://www.ncbi.nlm.nih.gov/).

1

2

3

1500 1000

4

5

6

1

7

2

3

4

5

6

7

8

9

1500

800 600 400 200

500 200

(a)

(b)

Figure 5: Mammals specific rabies virus variants detected by nested endpoint PCR assays. (a) Detection of dog specific variant (lines 2–4) and bat specific variant (lines 5–7). Lines 2 and 5: brain negative sample; lines 3 and 6: external amplification by RT-PCR (1187 and 668 pb, resp.); lines 4 and 7: internal amplification by nested PCR. (b) Detection of vampire bat specific variant (lines 2–4) and skunk specific variant (lines 7–9). Lines 2 and 7: brain negative sample; lines 3 and 8: external amplification by RT-PCR (835 and 795 pb, resp.); lines 4 and 9: internal amplification by nested PCR; line 5: empty. Lines 1 in (a) and 1 and 6 in (b) correspond to 100 pb DNA-ladder.

FBVFQFBVRCQ∗,∗∗ + − − −

VAMPIROFVAMPIRORC

+ − − −

− + − −

PERROFPERRORC − + − −

FBPFQPERRORC∗,∗∗ − − + −

MURCIELAGOFMURCIELAGORC

− − + −

FBMFQFBMRCQ∗,∗∗

− − − +

ZORRILLOFZORRILLORC

Only a sample can be considered positive if the result with the internal primers is positive regardless of the outcome of the external primers, ∗ for nested and/or ∗∗ real-time RT-PCR.

Rabies variant detection/primer name Vampire bat Dog Bat Skunk

Table 5: Test results interpretation of nested RT-PCR.

− − − +

ZORRILLOFFBZRCQ∗,∗∗

BioMed Research International 13

BioMed Research International 1.196 1.096 0.996 0.896 0.796 0.696 0.596 0.496 0.396 0.296 0.196 0.096 −0.004

−(d/dT) fluorescence (530)


14 0.698 0.628 0.558 0.488 0.418 0.348 0.278 0.208 0.138 0.068 −0.002

66 68 70 72 74 76 78 80 82 84 86 88 90 92 94

66 68 70 72 74 76 78 80 82 84 86 88 90 92 94

Temperature (∘ C)

Temperature (∘ C)

1.12 1.02 0.92 0.82 0.72 0.62 0.52 0.42 0.32 0.22 0.12 0.02

(b) −(d/dT) fluorescence (530)


(a)

1.149 1.049 0.949 0.849 0.749 0.649 0.549 0.449 0.349 0.249 0.149 0.049

66 68 70 72 74 76 78 80 82 84 86 88 90 92 94

66 68 70 72 74 76 78 80 82 84 86 88 90 92 94

Temperature (∘ C)

Temperature (∘ C)

(c)

(d)

Figure 6: Specific rabies virus variants detected by real-time RT-PCR assays. (a) Bat specific variant. (b) Dog specific variant. (c) Vampire bat specific variant. (d) Skunk specific variant.

institutions without access to MAbs and those that have PCR and/or real-time technology as an alternative. The relevance of the present study falls in the rabies virus typing from original host-brains samples and the association with the variant-specific host performed by nested endpoint PCR or real-time RT-PCR assays. Previous studies report the detection in decomposed brains from dogs and humans samples [32]; in humans exhumed between 8 and 30 days after burial [27]; in wolves by nested RT-PCR [33]; in mice previously infected by heminested RT-PCR [29]; and in bats and herbivores by RT-PCR. The sequence obtained for this study, a splitting between the urban rabies (dog) and the sylvan rabies (bat, vampire bat, and skunk), was shown in Tables 4 and 5; the results were according to the primers designed associated variant for the dog (urban rabies) and bat, skunk, and vampire bat (sylvan rabies).

5. Conclusion This study describes the development of an alternative tool for RABV typifying in real-time RT-PCR and/or nested RT-PCR, considering dog, skunk, vampire bat, and nonhematophagous bat specific variants.

Competing Interests The authors declare that they have no competing interests.

Acknowledgments The authors would like to acknowledge Lizdah Ivette Garc´ıa Rodr´ıguez and José A. Valdes-Zún˜ iga, Unidad de Enseñanza, Investigación y Calidad, for supporting the administrative project permission and formats, IPN: COFAA SIP.

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