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Consensus nested PCR amplification and sequencing of diverse

reptilian, avian, and mammalian orthoreoviruses

James F.X. Wellehan Jr.a,*, April L. Childress a, Rachel E. Marschang c,April J. Johnson a,1, Elaine W. Lamirande d,2, John F. Roberts b, Mary L. Vickers e,

Jack M. Gaskin b, Elliott R. Jacobson a

a Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida,

Gainesville, FL 32610, USAb Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida,

Gainesville, FL 32610, USAc Institut fur Umwelt- und Tierhygiene, Hohenheim University, Garbenstr. 30, 70599 Stuttgart, Germany

d Department of Pathology, National Zoological Park, Smithsonian Institution, Washington, DC 20008, USAe Livestock Disease Diagnostic Center, University of Kentucky, Lexington, KY 40512, USA

Received 17 March 2008; received in revised form 9 June 2008; accepted 12 June 2008

Abstract

The orthoreoviruses are segmented RNA viruses that infect diverse vertebrate host species. While the most common human

orthoreovirus, Mammalian Reovirus, is not typically associated with significant disease, the majority of Orthoreovirus species

have been shown to cause significant and often fatal disease in reptiles, birds, and primates. There is significant potential for

jumping species. A consensus nested-PCR method was designed for investigation of the RNA-dependent RNA polymerase gene

of Orthoreovirus and Aquareovirus. This protocol was used to obtain sequencing template from reoviruses of three different

vertebrate classes. Bayesian and maximum likelihood phylogenetic analysis found that all viruses analyzed clustered in the

genus Orthoreovirus, that reptile reoviruses formed three distinct clusters, and that an African grey parrot reovirus clustered with

Nelson Bay virus from bats. This PCR method may be useful for obtaining templates for initial sequencing of novel

orthoreoviruses from diverse vertebrate hosts.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Reovirus; Orthoreovirus; Reptiles; Birds; Reptilian orthoreovirus; Nelson Bay virus; Consensus PCR; Polymerase

www.elsevier.com/locate/vetmic

Available online at www.sciencedirect.com

Veterinary Microbiology 133 (2009) 34–42

* Corresponding author at: Zoological Medicine Service, Department of Small Animal Clinical Sciences, College of Veterinary Medicine,

University of Florida, P.O. Box 100126, Gainesville, FL 32610, USA. Tel.: +1 352 392 2226; fax: +1 352 392 4877.

E-mail address: wellehanj@vetmed.ufl.edu (J.F.X. Wellehan Jr.).1 Current address: Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.2 Current address: Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, USA.

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2008.06.011

Author's personal copy

1. Introduction

Reoviruses are medium-sized nonenveloped seg-

mented double stranded RNA viruses. Due to lack of

proofreading by their polymerases and constant

selection by the host immune system, RNA viruses

have the fastest mutating genomes found in nature,

and tracking evolution of these viruses is highly

clinically significant. As segmented viruses, reo-

viruses are also capable of gene segment reassortment,

further increasing their ability to change. One recent

meta-analysis found that of the 20 virus families

infecting humans, four families (Reoviridae, Bunya-

viridae, Flaviviridae, and Togaviridae) accounted for

more than half of emerging and reemerging viruses

(Woolhouse and Gowtage-Sequeria, 2005). Emerging

disease is also frequently associated with host

switches. The aforementioned meta-analysis of

human diseases found that 816 of 1407 (58%) are

zoonotic, and of human diseases, zoonotic diseases are

significantly more likely to be emerging (Woolhouse

and Gowtage-Sequeria, 2005). Characterization and

understanding of reoviruses of nonhuman animals is

therefore important to human health.

The family Reoviridae contains 12 genera, which

can infect plants, fungi, arthropods, and vertebrates,

illustrating their ability to work as a family with diverse

host cells. Of the 38 reoviruses known to infect

vertebrates that were recognized in the VIII report of the

International Committee on the Taxonomy of Viruses,

14 have been shown to infect both humans and other

animals, and aquareoviruses are capable of infecting

human cell lines but are temperature intolerant (Samal

et al., 1998). Medicine has traditionally waited for

viruses to cause epidemics or epizootics before

significant surveillance occurs. With our increased

understanding of virus ecology and evolution, it

becomes more feasible to identify probable candidates

for future novel disease outbreaks, and increase

surveillance. Throughout biology, hybridization is a

factor allowing rapid non-detrimental change, allowing

species to invade novel habitats (Nolte et al., 2005;

Rieseberg et al., 2007). Segmentation of viruses,

allowing reassortment, may provide a hybrid advantage

for crossing host species; this has been best studied in

the Orthomyxoviridae (Macken et al., 2006). One

recent study scored the viruses infecting mammals for

biological properties that were considered advanta-

geous to host switching, and found that Reoviridae

scored highest (Pulliam, 2008).

Within Reoviridae, Aquareovirus and Orthoreo-

virus are the only two genera that have been shown to

form a clearly monophyletic cluster (Attoui et al.,

2002). These two genera together have a very broad

host range, and together are known to infect bony fish

and all tetrapod classes with the exception of the

Amphibia. The gene encoding RNA-dependent RNA

polymerase, known in the genus Orthoreovirus as the

lambda3 protein and in Aquareovirus as VP2, has been

found to be the most conserved gene throughout

known members of the family Reoviridae, and

therefore most useful for long range phylogeny

(Attoui et al., 2002).

In reptiles, evidence of reovirus infection has been

found in 8 lizard species (Ahne et al., 1987; Drury

et al., 2002; Gravendyck et al., 1998; Marschang et al.,

2002; Raynaud and Adrian, 1976), 10 snake species

(Ahne et al., 1987; Blahak and Gobel, 1991; Blahak

et al., 1995; Jacobson, 1986; Lamirande et al., 1999;

Vieler et al., 1994), and 2 tortoise species (Drury et al.,

2002; Marschang and Chitty, 2004). The clinical

picture in a reptile with reoviral disease typically

presents as pneumonia and neurologic signs, and is

very similar to the clinical picture seen with

paramyxovirus infection. Experimental infection of

snakes with a snake reovirus has been shown to cause

fatal proliferative interstitial pneumonia and subacute

tracheitis, fulfilling Koch’s postulates (Lamirande

et al., 1999). Diagnosis is challenging, as there are no

inclusions, and histologic lesions resemble paramyx-

oviral disease. Reptile reoviruses cultured to date are

fusogenic, meaning that they form syncytia when

grown in cell culture, much like paramyxoviruses.

There are several serologically distinct reptile reovirus

isolates, indicating significant diversity is present

(Blahak et al., 1995). The degree of cross reactivity of

neutralizing antibodies to reptile reoviruses is not

known, limiting the utility of serologic diagnosis.

Furthermore, serologic diagnosis has been limited to

virus neutralization (Marschang et al., 2002), which is

labor intensive and requires that a virus be previously

cultured, making this a poor method for novel virus

discovery. As reptile reoviruses do not hemaggluti-

nate, hemagglutination inhibition is not an option.

Viral taxonomy is now primarily based upon gene

content and gene sequence homology. No published

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 133 (2009) 34–42 35

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sequence for reptile reoviruses has been available until

recently (Duncan et al., 2004), and that sequence was

obtained from a relatively non-conserved region

through virus isolation and labor-intensive cloning.

In birds, evidence of Orthoreovirus infection has

been found worldwide in diverse host species (Docherty

et al., 1994; van den Brand et al., 2007). Serologic

diversity amongst avian orthoreoviruses has been

shown (van den Brand et al., 2007). Avian Orthor-

eovirus infections are associated with disease syn-

dromes including acute hepatic necrosis, arthritis/

tenosynovitis, enteric disease, respiratory disease, and

immunosuppression (van den Brand et al., 2007). Avian

reoviruses are also fusogenic (Conzo et al., 2001; Day

et al., 2007). Experimental infection of African grey

parrots (Psittacus erithacus) with an African grey

parrot reovirus has been shown to cause fatal hepatic

necrosis, and Koch’s postulates were fulfilled (Graham,

1987). Published sequence for avian orthoreoviruses is

all from galliform birds, and psittacine Orthoreovirus

sequence has not been available. Available avian

Orthoreovirus sequence does not include RNA-

dependent RNA polymerase, the most conserved gene

in the genera Orthoreovirus and Aquareovirus.

In mammals, orthoreoviruses have been shown to

have a diverse host range (Leary et al., 2002). To date,

most human Orthoreovirus infections are not clini-

cally significant, although they may be associated with

mild respiratory or enteric signs that resolve rapidly.

Unlike aquareoviruses, or orthoreoviruses found in

birds and reptiles, the common mammal orthoreo-

virus, Mammalian Orthoreovirus (MRV, consisting of

strains Mammalian Reovirus 1, 2, 3, and Ndelle virus)

does not form syncytia. However, there are two known

exceptional mammalian reoviruses. Nelson Bay virus,

first isolated from a grey-headed flying fox (Pteropus

poliocephalus) (Gard and Compans, 1970), and

Baboon orthoreovirus are both fusogenic. These

viruses have also been associated with much more

significant pathology than is typical with nonfuso-

genic mammalian orthoreoviruses. A strain of Nelson

Bay virus, Melaka virus, has recently been associated

with acute respiratory disease and high fever in

humans (Chua et al., 2007). Baboon orthoreovirus was

found to cause meningoencephalomyelitis in yellow

baboons (Papio cynocephalus) (Leland et al., 2000).

Many important human respiratory viruses are

fusogenic, and this characteristic may be pathologi-

cally relevant. While the phylogeny of nonfusogenic

mammalian orthoreoviruses has been examined using

polymerase sequence (Leary et al., 2002), polymerase

sequence has not been available for the fusogenic

mammalian orthoreoviruses.

Consensus PCR represents a rapid way to obtain

DNA template from clinical samples of novel viruses

suitable for sequencing (VanDevanter et al., 1996;

Wellehan et al., 2004). There was a clear need for a

PCR technique that can be utilized for identifying for

novel reoviruses of diverse hosts. This study was

initiated to develop methods for rapid identification

and characterization of known and novel orthoreo-

viruses from diverse hosts.

2. Materials and methods

2.1. Viruses

Reptile reoviruses from a Mojave rattlesnake

(Crotalus scutulatus), brown tree snake (Boiga irregu-

laris), 2 variable bush vipers (Atheris squamigera), red

rat snake (Elaphe guttata), carpet python (Morelia

spilota), 4 Boa constrictors (Boa constrictor) and a

Mediterranean spur-thighed tortoise (Testudo graeca)

were collected for this study. An avian reovirus was

isolated from an African grey parrot (Psittacus

erithacus) with hepatic necrosis. The type strain of

Nelson Bay virus, originally from a grey-headed flying

fox (Pteropus poliocephalus), was kindly provided by

Dr. Richard Compans from Emory University. Infor-

mation on these isolates may be found in Table 1.

2.2. PCR amplification and sequencing

RNA-dependent RNA polymerase sequences from

mammalian orthoreoviruses and piscine aquareo-

viruses were obtained from GenBank (National Center

for Biotechnology Information, Bethesda, MD) and

aligned. Degenerate primers were designed targeting

conserved regions. RNA was extracted from samples

using the RNeasy Mini Kit (Qiagen, Valencia, CA).

Reverse transcription PCR was performed using the

OneStep RT-PCR Kit (Qiagen) according to standard

protocol using forward primer 1607F (50-CARMGNCGNSCHMGHTCHATHATGCC-30) and

reverse primer 2608R (50-TAVAYRAAVGWC-

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 133 (2009) 34–4236

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CASMHNGGRTAYTG-30). For the second round of

PCR amplification, 2 ml of product from the first

reaction was used with forward primer 2090F (50-GGBTCMACNGCYACYTCBACYGAGCA-30) and

reverse primer 2334R (50-CDATGTCRTAHWYC-

CANCCRAA-30). These primers lie within the central

enzymatically active region of the polymerase (Kim

et al., 2004). The mixtures were amplified in a thermal

cycler (PCR Sprint, Thermo Hybaid, Franklin, MA)

with an initial denaturation at 95 8C for 1 min, then 45

cycles of denaturation at 95 8C for 60 s; annealing at

47 8C for 60 s, and DNA extension at 72 8C for 30 s,

followed by a final extension step at 72 8C for 8 min.

The PCR products were resolved in 1% agarose gels.

The bands were excised and purified using the

QIAquick gel extraction kit (Qiagen). Direct sequen-

cing was performed using the Big-Dye Terminator Kit

(PerkinElmer, Branchburg, NJ) and analyzed on ABI

377 automated DNA sequencers at the University of

Florida Center for Mammalian Genetics DNA

Sequencing Facilities. All products were sequenced

in both directions. Primer sequences were edited out

prior to further analyses.

2.3. Phylogenetic analysis

The sequences were compared to those in

GenBank, EMBL (Cambridge, United Kingdom),

and Data Bank of Japan (Mishima, Shizuoka, Japan)

databases using TBLASTX (Altschul et al., 1997).

Predicted homologous 75 amino acid sequences of

reoviral RNA-dependent RNA polymerase were

aligned using three methods; ClustalW (Thompson

et al., 1994), T-Coffee (Notredame et al., 2000), and

MUSCLE (Edgar, 2004).

Bayesian analyses of amino acid alignments were

performed using MrBayes 3.1 (Ronquist and Huelsen-

beck, 2003) with gamma distributed rate variation and a

proportion of invariant sites, and mixed amino acid

substitution models. Four chains were run and statistical

convergence was assessed by looking at the standard

deviation of split frequencies as well as potential scale

reduction factors of parameters. The first 10% of

1,000,000 iterations were discarded as a burn in.

Maximum likelihood (ML) analyses of amino acid

alignments were performed using PHYLIP (Phylo-

geny Inference Package, Version 3.66) (Felsenstein,

1989), running each alignment in proml with amino

acid substitution models JTT (Jones et al., 1992), PMB

(Veerassamy et al., 2003), and PAM (Kosiol and

Goldman, 2005) further set with global rearrange-

ments, 5 replications of random input order, less

rough, gamma plus invariant rate distributions, and

unrooted. The values for the gamma distribution were

taken from the Bayesian analysis. Striped bass

reovirus (GenBank accession number AAM93410)

was designated as the outgroup. The alignment

producing the most likely tree was then used to create

data subsets for bootstrap analysis to test the strength

of the tree topology (200 re-samplings) (Felsenstein,

1985), which was analyzed using the amino acid

substitution model producing the most likely tree.

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 133 (2009) 34–42 37

Table 1

Reoviruses amplified by consensus PCR in this study

Isolate Year Host species Organ Origin

Common name Scientific name

KYMojave 2002 Mojave rattlesnake Crotalus scutulatus Multiple tissues USA

CH1197/96 1996 Mediterranean spur-thighed tortoise Testudo graeca Tongue Switzerland

112-99 1999 Boa constrictor Boa constrictor Pancreas Germany

200-99 1999 Boa constrictor Boa constrictor Heart Germany

968-1-00 2000 Boa constrictor Boa constrictor Blood Germany

IBD26-00 2000 Boa constrictor Boa constrictor Liver Germany

47-02 2002 Variable bush viper Atheris squamigera Intestine Germany

55-02 2002 Carpet python Morelia spilota Trachea Germany

LCV2 1996 Red rat snake Elaphe guttata Multiple tissues USA

VBV 2000 Variable bush viper Atheris squamigera Lung USA

BTS984 2001 Brown tree snake Boiga irregularis Liver USA

AGPReo 1983 African grey parrot Psittacus erithacus Liver and spleen USA

Nelson Bay virus 1968 Grey-headed flying fox Pteropus poliocephalus Blood Australia

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3. Results

3.1. PCR amplification

PCR amplification of all reovirus samples resulted in

a 226 base pair product when primer sequences were

edited out. Sequence from Boa constrictor isolate

200-99 was identical to that from Boa constrictor

isolate 112-99, and sequence from Boa constrictor

isolate IBD26-00 was identical to that from Boa

constrictor isolate 968-1-00.

Sequences were submitted to GenBank under

accession numbers EU309694-EU309706.

3.2. Phylogenetic analysis

TBLASTX results for African grey parrot reovirus

showed equally highest scores with Mammalian

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 133 (2009) 34–4238

Fig. 1. Bayesian phylogenetic tree of predicted 75 amino acid partial reoviral RNA-dependent RNA polymerase sequences based on MUSCLE

alignment. Bayesian posterior probabilities of branchings as percentages are in bold, and ML bootstrap values for branchings based on 200 re-

samplings are given below. Striped bass reovirus (GenBank accession number AAM93410) was designated as the outgroup. Thin brackets delineate

host classes, and thick brackets delineate virus genera. Branches with Bayesian posterior probabilities less than 50 are collapsed, and arcs mark areas

of trifurcation. Reovirus sequences determined in this study are bolded. Sequences retrieved from GenBank include chum salmon reovirus (GenBank

accession # AAL31497), golden ide reovirus (AAM93415), grass carp reovirus (AAG10436), mammalian orthoreovirus 1 (NP694626), mammalian

orthoreovirus 2 (NP694627), mammalian orthoreovirus 3 (ABP48913), Ndelle virus (AAL36027), and striped bass reovirus (AAM93410).

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orthoreovirus 3 RNA polymerase (GenBank accession

number EF494435) and Mammalian orthoreovirus 1

RNA polymerase (GenBank accession number

DQ167425). TBLASTX results for Nelson Bay virus

showed the highest score with Mammalian orthor-

eovirus 2 RNA polymerase (GenBank accession

number M31057). TBLASTX results for all reptile

reoviruses in this study showed the highest score with

Ndelle virus RNA polymerase (GenBank accession

number AF368033).

All three alignment algorithms found no indels

present, resulting in identical alignments with no gaps

(supplemental data).

Bayesian phylogenetic analysis showed the Wag

model of amino acid substitution was most probable

with a posterior probability of 0.949 (Whelan and

Goldman, 2001). The Bayesian tree is shown (Fig. 1).

ML analysis found the most likely tree from the JTT

model of amino acid substitution, and this model was

used for bootstrap analysis. ML bootstrap values are

shown on the Bayesian tree (Fig. 1).

4. Discussion

Viral polymerases are usually good choices for

long-range phylogeny (Attoui et al., 2002; Gonzalez

et al., 2003; Knopf, 1998). When looking at

evolutionary relationships of more distantly related

organisms, the continued accrual of mutations

resulting in homoplasy can diminish the ability to

correctly resolve phylogeny, making a rapidly mutat-

ing gene a poor choice. Genes for which there is strong

negative selection will have fewer nucleotides with a

history of multiple changes, making them a better

choice for resolving phylogeny over greater distances.

Genes that are critical for basic organismal functions

and are not under heavy immune selection are often

highly conserved. Reoviral RNA-dependent RNA

polymerase is found on the internal aspect of the viral

capsid, and is thus less exposed to the immune

response. Polymerases are essential for viral function

and generally tend to be highly conserved, and are

therefore more likely to reflect the history of the virus.

The primary disadvantage of using this gene is the

paucity of reference information for comparison;

the outer capsid protein has more available sequence.

The availability of a more complete representation of

existing species for comparison results in greater

phylogenetic resolution (Flynn et al., 2005). However,

the total number of available Orthoreovirus sequences

from any gene is still relatively small, and techniques

developed in this study could lead to rapid accumula-

tion of sequence data from the polymerase gene of

additional orthoreoviruses.

All reptile reoviruses in this study are well supported

as members of the genus Orthoreovirus. The reptile

reoviruses in this study are monophyletic, and appear to

form three distinct clusters. It remains to be determined

whether these represent different species. The distance

between these clusters is greater than the distance

between striped bass reovirus and chum salmon

reovirus, which are both members of the species

Aquareovirus A, but less than the distance between

Nelson Bay virus and African grey parrot reovirus,

which can reasonably be expected to be different

species. Conclusive species classification of orthor-

eoviruses requires the direct demonstration of exchange

of genetic material via reassortment of genome

segments (Chappell et al., 2005). Analysis of sequence

data from the major outer capsid protein found weak

support for clustering of reptilian reovirus with Baboon

orthoreovirus (Chua et al., 2007). Analysis of the more

conserved polymerase gene may more strongly support

or refute this finding. Unfortunately, we were unable to

obtain samples of Baboon orthoreovirus for this study.

Further study is indicated to determine the relationship

between the reptile reoviruses and this pathogen known

to cause significant disease in primates.

Nonfusogenic mammalian orthoreoviruses form one

fairly tight monophyletic cluster, which is consistent

with their grouping into a single species. This small

cluster of nonfusogenic viruses differs from most of the

genus, and may be of recent origin. Formation of

syncytia is a common finding in some significant

respiratory diseases, including paramyxoviruses and

herpesviruses, and ability to form syncytia could be a

feature associated with pathogenicity. If this is the case,

the low pathogenicity seen with the nonfusogenic

mammalian orthoreoviruses could be the exception in

this genus rather than the rule.

Nelson Bay virus and African grey parrot reovirus

form a monophyletic group that is strongly supported.

Previous studies of the more variable sigma protein

genes have also supported clustering of Nelson Bay

virus with avian orthoreoviruses (Day et al., 2007;

J.F.X. Wellehan Jr. et al. / Veterinary Microbiology 133 (2009) 34–42 39

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Duncan, 1999), although less strongly. In this study,

the distance between these viruses is less than that

seen between any other pair of viruses from different

host classes. This is suggestive of a viral switch

between host taxa. With the association of Melaka

virus with human disease, further study is indicated on

the relationship between Nelson Bay virus and avian

orthoreoviruses, and the ability to switch hosts.

The genera Aquareovirus and Orthoreovirus are the

two most closely related genera in the family

Reoviridae, and genetic distances between these

two genera are less than those seen within the single

genus Rotavirus (Attoui et al., 2002). However,

members of Aquareovirus have 11 segments, whereas

members of Orthoreovirus have 10 segments. Taken

together with host range differences, this has been

considered sufficient to keep these genera separate.

All members of Orthoreovirus form a monophyletic

cluster in this analysis, which is consistent with these

two phyla being distinct.

It has been suggested that orthoreoviruses and

aquareoviruses may have codiverged with their hosts

(Attoui et al., 2002). While our analysis does find that

these viruses generally tend to cluster according to

host taxa, the deeper level phylogeny does not reflect

that of the host species. Host taxa/virus congruence on

shorter branches with lack of congruence at deeper

branchings may be more consistent with preferential

host switching than host/virus codivergence (Jackson

and Charleston, 2004). Preferential host switching

between more closely related hosts appears to be a

more plausible explanation than codivergence in this

case for congruence of host and virus phylogeny.

These primers can be of use for obtaining initial

sequence data from novel reoviruses, and primers from

this study have already been used for characterization of

orthoreoviruses from clinical samples (Wellehan,

unpublished data). Although short, sequence from this

region provides good genus-scale phylogenetic resolu-

tion. Analysis of sequence data will enable design of

diagnostic PCR testing for specific viruses, and provide

valuable phylogenetic and epidemiologic information.

Acknowledgments

We thank Dr. Richard Compans from Emory

University for kindly providing Nelson Bay virus. The

authors have no competing interests, financial or

otherwise, and no external funding was used for this

project.

Appendix A. Supplementary data

Supplementary data associated with this article can

be found, in the online version, at doi:10.1016/

j.vetmic.2008.06.011.

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